EP0562013A1 - Human retrovirus receptor and dna coding therefor - Google Patents

Abstract

Human protein molecule called H13 with strong sequence homology to receptor proteins from murine retroviruses and encoding a human retrovirus receptor. The invention also relates to DNA encoding the H13 protein, cells transformed and transfected with this DNA as well as antibodies specific for the H13 protein. The H13 protein, or its functional derivative, can be used in the prevention or treatment of retroviral infections by administration to a subject of the H13 protein or a functional derivative thereof, or alternatively of an anti- H13. Transgenic animals useful as animal models for diagnosis or therapy of human retroviral infections are obtained by transfection of embryonic cells with DNA encoding H13. A chimeric retrovirus protein comprises the H13 sequence in which the remainder of the amino acids encoding a murine retroviral receptor are substituted. Expression of the chimeric receptor in human cells allows infection or transfer of genes induced by retroviruses using murine retroviruses, which constitutes an additional safety measure for gene therapy in vivo. The invention also relates to DNA encoding the chimeric retrovirus receptor protein, cells transformed with this DNA, and methods of rendering a cell susceptible to infection by a retrovirus incapable of infecting that cell. .

Description

HUMAN RETROVIRUS RECEPTOR AND DNA CODING THEREFOR

BACKGROUND OF THE INVENTION

Field of the Invention

The invention in the field of virology and molecular genetics relates to the H13 protein molecule, which is a human protein highly homologous in sequence to a murine retrovirus receptor molecule, DNA coding therefore, methods of preparing the protein molecule, and methods of use of the protein to prevent or treat retrovirus infection. The invention also concerns substitutions in the human retrovirus receptor of amino acid residues from the murine homologue and human cells expressing this DNA which are rendered susceptible to

infection and thus gene transfer by murine retroviral

vectors.

Description of the Background Art

Viruses infect cells by first attaching to the cell. This requires specific interactions between molecules on the surface of the virus and receptor molecules on the susceptible cell. A number of virus-specific cellular receptors have been identified, and most of these receptor molecules have other known cellular functions. Human immunodeficiency virus (HIV-1) binds to the CD4 molecules (Dalgleish et al., Nature,

112:763-767 (1984)); Klatzmann, D., Nature 312:767 (1984);

Maddon et al., Cell. 42: 93-104 (1986)). The Epstein-Barr virus (EBV) binds to the complement receptor protein, CR2 (Fingeroth et al., Proc. Natl. Acad. Sci. USA, 81: 4510-4514 (1984)). Human rhinoviruses bind to the cell adhesion

molecule, ICAM-1 (Greve, J.M. et al., Cell 56:839 (1989);

Staunton, D.E. et al., Cell 56:849 (1989)). Rabies virus binds to the acetylcholine receptor (Lentz, T.L., Science 215: 182 (1082)). Reoviruses bind to beta-adrenergic receptors (Co, M.S. et al., Proc. Natl. Acad. Sci. USA 82:1494 (1985). Herpes simplex virus appears to use the fibroblast growth factor receptor as a binding site (Kaner, R.J. et al.,

Science 248: 1410-1413 (1990)).

The expression of these virus binding proteins or receptors is a strong determinant of susceptibility to virus infection. Binding is required for fusion of the virus envelope to the target cell, an event that may occur at the cell surface or within an acidified endosome after receptormediated endocytosis (White et al., Quant. Rev. Biophys. 16: 151-195 (1983)). After fusion, the virion core enters the cytoplasm and the viral replication process is initiated.

In the case of HIV, recent studies suggested that cell surface molecules other than CD4 may also be important for virus entry into human cells. First, cells lacking the CD4 molecule, including human fibroblasts and cells derived from human brain, can be infected in vitro by HIV, suggesting an alternate virus receptor. Furthermore, murine cells which have been transfected with the CD4 gene and express this molecule on their surface are often resistant to HIV,

indicating that the mere presence of CD4 is not sufficient for HIV infection. A major target cell for HIV is the CD4⁺ T lymphocyte. A majority of circulating T lymphocytes are non-dividing quiescent cells; for infection by HIV in vitro, these cells must be "activated," for example, by a mitogenic lectin. This observation-further supports the notion that the presence of the CD4 molecule on a cell is not sufficient for

susceptibility to HIV infection. Murine Retrovirus Receptors

As with HIV and EBV, susceptibility of cells to infection with ecotropic murine leukemia virus (E-MuLV) may also be determined by binding of the virus envelope to a membrane receptor. The E-MuLV envelope protein, gp70,

encoded by the env gene, binds avidly to the membranes of murine cells but poorly to those of other mammals that are not permissive for E-MuLV (DeLarco et al., Cell 8:365-371 (1976)). Furthermore, the gp70 molecule of ecotropic MuLV is

structurally distinct from the envelope proteins of other MuLV subgroup viruses with different patterns of infectivity (Levy, J.A., Science, 182:1151-1153 (1973); Elder et al.,

Nature 267:23-28 (1977)). Chimeric viruses constructed between E-MuLV and other MuLV subgroups acquire the host range of the env gene donor (Cone and Mulligan, Proc. Natl. Acad. Sci. USA. 81:6349-6353 (1984)).

Based on viral interference assays, four types of specific MuLV receptors have been postulated: (a) receptors for E-MuLV; (b) receptors for wild-type amphotropic MuLV; (c) receptors for recombinant viruses derived from E-MuLV, such as the "mink cell focus-inducing" or MCF virus; and (d) receptors for a recombinant virus derived from an amphotropic MuLV

(Rein, A. et al., Viroloσv 136:144-152 (1984)).

Hybrid cells which were created by fusion of primary mouse lymphocytes with nonpermissive Chinese hamster lung cells retained susceptibility to E-MuLV infection and bound gp70 to the membrane (Gazdar, A.F., Cell 11:949-956 (1977)). Analysis of the chromosome content of a large number of these hybrids has permitted the assignment of putative E-MuLV receptor gene(s) to the Rec-1 locus on mouse chromosome 5 (Oie et al., Nature 274: 60-62 (1978); Ruddle et al., J. Exp. Med. M8..451-465 (1978)). Purification of the protein encoded by Rec-1 has not been achieved because specific gp70 binding activity was lost upon detergent solubilization of the cell membrane (Johnson et al., J. Virol. 58-900-908 (1986)).

Assignment of a single genetic locus for

susceptibility to virus infection is consistent with the hypothesis that a single gene encodes the receptor protein. Furthermore, successful MuLV infection of the hybrid cell lines demonstrates that expression of the receptor gene in nonpermissive cells can confer MuLV susceptibility. These two observations suggest that it might be possible to transfer murine DNA into nonpermissive cells by transfection and then recover the putative receptor gene from recipient cells that had acquired susceptibility to E-MuLV infection. A similar strategy has been employed in cloning genes encoding other cell membrane proteins such as the nerve growth factor receptor (Chao et al., Science, 232:418-421 (1966)), CD8 (Littman et al., Cell 40 237-246 (1985)), and CD4 (Maddon et al., Cell 42:93-104 (1985)).

Recently, a cDNA clone (termed Wl) encoding the murine ecotropic retroviral receptor (ERR) was identified (Albritton, L.W. et al., Cell 57:659-666 (1989)). This study demonstrated that susceptibility to E-MuLV infection was acquired by the expression of a single mouse gene in human EJ cells. Furthermore, this gene appears to define Rec-1, the genetic locus on mouse chromosome 5 associated with ecotropic virus infectivity (Oie et al., Nature 274:60-62 (1978);

Ruddle et al., J. EXP. Med. 148:451-465 (1978)).

The hydropathy plot of the predicted amino acid sequence of the ERR (SEQ ID NO: 4) protein revealed an

extremely hydrophobic protein containing 14 potential

transmembrane domains (Eisenberg et al., J. Mol. Biol.

179:125-142 (1984)). Such structure strongly implies that the protein resides in the membrane. This protein may permit infection by functioning as a true receptor that binds specifically to E-MuLV gp70 in analogous fashion to HIV gp120 binding to the CD4 protein (Maddon et al., Cell 47: 333-348 (1986); McDougal et al., Science 237:382-385 (1986)).

Demonstration of a physical association between the protein and the virus envelope gp70 would strongly support its proposed role as a virus receptor.

Independent from its role in viral attachment to the cell surface, the ERR protein could also be important for virus envelope fusion to the membrane of the target cell.

Evidence for the existence of membrane proteins that mediate virus fusion comes from studies of Sendai virus (Richardson et al., Virology 131:518-532 (1983)) and HIV (Maddon et al.,

Cell 42 :93-104 (1985)), two viruses that fuse to the plasma membrane (Harris et al., Nature 205:640-646 (1965); Stein et al., Cell 4.9:659-668 (1987); Maddon et al., Cell 47:333-348 (1986)) through a mechanism that may be similar to that employed by E-MuLV (Pinter et al., J. Virol. 57: 1048-1054 (1986)). The potential role for a protein in virus fusion could be distinct from, or in addition to, the act of binding viral gp70.

Computer searches through the GenBank and NBRF databases did not reveal any sequences similar to the

predicted protein that might help classify or identify the function of the ERR protein in normal cell metabolism.

Proteins with multiple membrane-spanning domains that function as gated channels or pumps to transport ions or sugars across the lipid bilayer have been identified, but a direct

comparison of the predicted amino acid sequences of several of these proteins to the ERR protein using the BestFit algorithm (Devereux et al., Nucl. Acids Res. 12: 387-395 (1984)) also did not reveal any significant sequence similarity

((Albritton, L.W. et al., 1989, supra). T Cell Early Activation Gene Resembles Retrovirus Receptor Gene

Many genes that encode products which function in T cell development, homing, or immune responsiveness remain to be identified. In an effort to isolate novel T cell cDNA clones which identify new functions, MacLeod, C.L. et al.

(Mol. Cell. Biol. 10:3663-3674 (1990)) used two closely related T lymphoma cell clones obtained from a single

individual which differed in a limited number of

characteristics and had defined and stable phenotypes. This model system is known as the SL12 T lymphoma (Hays et al.,

Int. J. Cancer 38:597-601 (1986)); MacLeod et al., Cancer Res. 4.4:1784-1790 (1984)); JNCI 74:875-882 (1985); Proc. Natl.

Acad. Sci. USA 83:6989-6993 (1986); Cell Growth & Differ.

2:271-279 (1990)). The two cell clones derived from a single SL12 T lymphoma cell line were chosen based on their known differences in gene expression and their different capacities to cause tumors in syngeneic host animals (MacLeod, C.L. et al., 1990, supra). SL12.3 cells express very few of the genes required for T cell function, and are highly tumorigenic in syngeneic animals (MacLeod et al., 1985, 1986, supra). In contrast, the cells of sister clone SL12.4 express mRNAs for all the components of the T cell receptor (TCR)-CD3 complex except TCR-α and resemble thymocytes at an intermediate stage of development (MacLeod et al., 1986, supra; Wilkinson et al., EMBO J. 2:101-109 (1988)). SL12.4 cells are much less

tumorigenic than SL12.3 cells (MacLeod et al., 1985, supra).

A combination of subtraction hybridization-enriched probes (Hedrick et al., Nature 308:149-153 (1984); MacLeod et al., J. Biol. Chem. 1:271-279 (1990); Timberlake, Dev. Biol. 7j$:497-503 (1980)) and classical differential screening

Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989) was used by MacLeod et al. to obtain cDNA clones

representing genes which were preferentially expressed in the SL12.4 T cell clone and undetectable in sister clone SL12.3. One cDNA clone, 20.5, identified transcripts found in only a limited number of tissues. The gene expressed by this clone was designated TEA (T cell early activation, SEQ ID NO: 5).

TEA transcripts were induced in Balb/c mouse spleen cells activated in vitro with the T cell mitogen, concanavalin A (Con A). The TEA gene appears to encode a protein which traverses the membrane multiple times (SEQ ID NO:6), in contrast to the large number of known integral membrane proteins induced during T cell activation which are single¬membrane-spanning proteins (see, for review, Crabtree, G.R., Science 243:355-361 (1989)).

Seventy genes or gene products are known to increase in expression when T cells are activated in response to either antigens (in combination with self-histocompatibility

molecules) or polyclonal activators such as lectins, calcium ionophores, or antibodies to the TCR (Crabtree, 1989, supra). Some of these activation genes are involved in cell cycle progression, others encode cytokines and cytokine receptors, nuclear regulatory proteins, and still others are involved in the transport of ions and nutrients into the cells to prepare them for growth. At least 26 T cell activation gene products have been localized to the cell membrane (Crabtree, supra).

The TEA gene, as exemplified by clone 20.5 (MacLeod et al., 1990, supra), is the first example of a cloned gene or cDNA that has the potential to encode a multiple transmembrane-spanning protein which is induced during T cell activation (Crabtree, Science 243:355-361 (1989)). TEA is an early gene because TEA mRNA is virtually undetectable in normal quiescent T cells, increases to detectable levels within 6 hours, and peaks at about 24 hours after Con A stimulation of spleen cells. The function of the tea gene is not yet known; it could function to transduce signals or transport small molecules which are signal transducers, or it could function as a receptor for an unidentified ligand. The rather long carboxy terminus of the putative tea protein might function as a signal transducer. Since numerous T and B tumor cell lines do not express TEA, its expression is clearly not absolutely required for cell growth, although normal (non-tumor) T cells might require tea expression for normal

proliferation in an immune response.

The sequence of 20.5 cDNA (SEQ ID NO: 5) was found to be strikingly homologous to the murine ERR cDNA clone (SEQ ID NO: 3) discussed above (the Rec-1 gene). This finding suggests that the TEA gene product might function as a murine

retroviral receptor. In contrast to the Rec-1 gene (encoding ERR), which is ubiquitously expressed in mouse tissues

(Albritton et al., 1989, supra). expression of the TEA gene has a much more limited tissue distribution. If the TEA gene product is a retroviral receptor, this limited tissue

distribution could be responsible for the tissue specificity of retroviruses which are restricted to cells of the lymphoid lineage (Quint et al., J. Virol. 39:1-10 (1981)). Recent studies indicate that retroviruses use cell-membrane permease proteins to gain entry to target cells. The transmembrane topology of ERR is reminiscent of that of several membrane transporter proteins, the permeases for arginine, histidine and choline of yeast (Vile, R.G. et al., Nature 352:666-667 (1991). Indeed, two groups have found that the ERR protein functions as a cationic amino acids transporter (Kim, J. W. et ah., Nature 352:752-728 (1991); Wang, H. et al., Nature

352:729-731 (1991)). In fact, this was the fist mammalian amino acid transporter to be cloned and the first example of a virus exploiting a transmembrane channel protein as a receptor. Related to these findings is the fact that a human cDNA which confers susceptibility to infection by gibbon ape leukemia virus has a sequence similar to a phosphate

transporter protein in fungi (Vile et al., supra). It is not known whether Tea also encodes a permease.

HIV is an example of a virus exhibiting receptormediated tissue restriction, apparently based on its use of the CD4 protein as its primary receptor. However, cellspecific receptors are unlikely to be the sole determinant of tissue specificity. The tissue tropism of retroviruses is likely to result from a complex series of factors, such as the tissue specificity of long terminal repeats, variations in viral env proteins, cellular factors, and the expression of appropriate cell surface receptors (Kabat, Curr. TOP.

Microbiol. Immunol. 148 : 1-31 (1989)). Viral binding and infection studies are required to determine whether the TEA-encoded protein functions as a viral receptor (Rein et al., Virology 136:144-152 (1984)).

Despite the high degree of similarity between TEA and ERR, the two genes differ in chromosomal location, and their predicted protein products differ in tissue expression patterns. However, the identification of this new gene family and of regions of DNA sequence which are highly conserved between the two members of this family permits searches for new family members.

Genetically engineered chimeric receptors are known in the art (see, for example, Riedel, H. et al., Nature

324:628-670 (1986)). However, there are no known examples of genetically-engineered chimeric receptors for retroviruses which permit, for example, infection of a human cell with a murine retrovirus. Such a chimeric receptor would be useful in the gene therapy setting. With the growing interest in gene therapy, there is a constant search for more efficient and safer means for introducing exogenous genes into human cells. One relatively efficient means for achieving transfer of genes is by retrovirus-mediated gene transfer (Gllboa, E., Bio-Essays 5:252-258 (1987); Williams, D.A. et al., Nature 310:476-480 (1984); Weiss, R.A. et al., RNA Tumor Viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1985). One class of retroviruses, recombinant

amphotropic retroviruses, have been studied with greater intensity as vector for the transfer of genes into human cells (Cone, R.D. et al., Proc. Natl. Acad. Sci. USA 80,: 6349-6353 (1984); Danos, O. et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). One of the safety problems inherent in this approach, which may preclude progress in the clinic, is the fact that even retroviruses that have been rendered

replication-defective are sometimes capable of generating wild-type variants through recombinational events. Such an alteration could lead to the possibility of widespread

retroviral infection in cells and tissues which were not intended to be genetically modified. This could result in generalized disease. It is to these needs and problems that the present invention is also directed.

SUMMARY OF THE INVENTION

The present inventors have discovered and cloned a novel human DNA sequence, H13, which is highly homologous to murine ERR and TEA, and is therefore considered to encode a cell membrane protein which acts as a human retrovirus

receptor. Such a human retrovirus receptor can serve as a target for therapeutic intervention in retrovirus-induced disease such as AIDS. See Yoshimoto, T. et al., Virology 185:10-17 (1991), and Meruelo et al., U.S. Patent Application Serial No. 07/627,950, filed December 14, 1990 (which

references are hereby incorporated by reference in their entirety).

The partial sequence of the H13 gene was described in U.S. Patent Application Serial No. 07/627,950, filed

December 14, 1990, and is presented herein as SEQ ID NO:1 (and the putative amino acid sequence is SEQ ID NO: 2). This sequence was s obtained by sequencing a cDNA clone designated 7-2.

The full length sequence of H13 (SEQ ID NO: 7) was obtained by sequencing overlapping clones 1-1 and 3-2

(schematically illustrated in Figure 15). The H13 DNA bears extensive nucleotide and amino acid sequence similarity with ERR (SEQ ID NO:3) and with TEA (SEQ ID NO: 5). The cDNA sequence predicts a highly hydrophobic protein which contains several putative membrane spanning domains. The predicted amino acid sequence of the full-length H13 molecule (SEQ ID NO:8) is homologous to the amino acid sequence of ERR (SEQ ID NO:4) and TEA (SEQ ID NO: 6). The human gene maps to

chromosome 13 and appears to be conserved among mammalian and avian species. The predicted H13 protein has 629 amino acids and an expected molecular weight of about 68 kDa. This protein has 7 more amino acids than the homologous murine ERR protein.

The present invention is directed to a recombinant DNA molecule (SEQ ID NO: 7) comprising a genetic sequence which encodes the H13 molecule, or a functional derivative thereof.

The present invention is further directed to an expression vector containing the recombinant DNA molecule and a host transformed or transfected with the vector.

The present invention is also directed to a human retroviral receptor molecule termed H13, the sequence of SEQ ID NO:8 or close homology thereto, or a functional derivative thereof, substantially free from impurities of human origin with which it is natively associated.

Another embodiment of the invention relates to a method for inhibiting the infection of a cell by a retrovirus, such as HIV-1, comprising contacting the virus with an

effective amount of the H13 protein molecule or functional derivative thereof and allowing the molecule to prevent the virus from attaching to the cell thereby inhibiting infection.

The present invention includes a method for preventing, suppressing, or treating a retrovirus infection, such as HIV-1, in a subject comprising providing to that subject an effective amount of the H13 molecule or functional derivative.

The invention is further directed to an antibody specific for the H13 molecule or an epitope thereof, including polyclonal, monoclonal, and chimeric antibody. An additional embodiment involves a method for preventing, suppressing, or treating a retrovirus infection, such as HIV-1 in a subject comprising providing to that subject an effective amount of the antibody.

The present invention includes a method for producing a composition useful for preventing, suppressing, or treating a retrovirus infection in a subject comprising the steps of:

(a) providing a recombinant DNA molecule encoding the H13 protein or a functional derivative thereof in expressible form;

(b) expressing the protein or functional derivative in a host cell in culture; and

(c) obtaining the protein or functional derivative from the culture.

The method preferably also includes the additional

purification of the protein or functional derivative. This method can be carried out in bacterial or eukaryotic,

preferably mammalian, host cells.

The invention also provides a pharmaceutical

composition useful for preventing, suppressing or treating a retrovirus infection, comprising the H13 protein molecule or a functional derivative thereof or an antibody specific for the H13 protein, and a pharmaceutically acceptable carrier.

It is yet a further object of the present invention to provide a transgenic experimental animal which has been transformed by the gene carrying the DNA sequences encoding the H13 protein, alone or in combination with the CD4 gene. This transgenic animal serves as a model for human retrovirus infection and allows testing of anti-viral therapies.

The methods of the present invention which identify normal or mutant H13 genes or measure the presence or amount of H13 protein associated with a cell or tissue can serve as methods for identifying susceptibility to human retrovirus infection, as in AIDS or certain forms of leukemia.

The present invention further provides a DNA

molecule encoding a chimeric retroviral receptor protein, comprising: (a) a first nucleotide sequence which encodes retroviral receptor protein I of a first animal species; and

(b) substituted therein, a sufficient number of nucleotides from a second nucleotide sequence encoding retroviral receptor protein II of a second animal species,

wherein the substituting nucleotides confer on the chimeric retroviral receptor protein the ability to bind a retrovirus which binds to receptor protein II but not to receptor protein I, allowing the chimeric protein to function as a retroviral receptor for the retrovirus.

In the above DNA molecule, the first nucleotide sequence preferably comprises the coding portion of human H13 DNA (SEQ ID NO: 7). More preferably, the DNA molecule encodes a chimeric H13/ERR chimeric protein wherein the second

nucleotide sequence comprises the coding portion of murine ERR DNA (SEQ ID NO: 3), and the substituting ERR nucleotides are those encoding an amino acid residue selected from the group consisting of Ile214, Lys222, Asn223, Ser225, Asn227, Asn232, Val233, Tyr235, G1U237, Ile313, Asp314, Gly319, Gln324, Glu328 and any combination of the above.

The above DNA molecule may be an expression vector. The present invention also includes a host transformed or transfected with this vector. Preferably, the host is a mammalian cell.

Also provided are chimeric retroviral receptor protein molecules encoded by the above DNA molecules.

The present invention includes a method for rendering a cell of species I susceptible to infection by, and retrovirus-mediated gene transfer by, a retroviral vector normally incapable of infecting a cell of species I,

comprising the steps of:

(a) transforming a cell of species I with an expressible DNA molecule encoding a chimeric receptor as above,

(b) expressing the chimeric retroviral receptor protein on the surface of the cell in culture,

thereby rendering the cell susceptible to infection by the retroviral vector. In this method, the cell is preferably a human cell, the retrovirus is preferably a murine retrovirus, most preferably an ecotropic murine leukemia virus, and the chimeric receptor is preferably a chimeric H13/ERR protein.

In another embodiment, the present invention

provides a method for transferring a gene to a cell of species I for use in gene therapy, comprising:

(a) culturing a cell intended to receive the transferred

gene;

(b) transforming the cell with a DNA molecule encoding a

chimeric retroviral receptor as described above, thereby providing the cell with a chimeric retroviral receptor protein;

(c) infecting the cell with a retroviral vector normally

incapable of infecting a cell of species I, the

retroviral virus being capable of infecting the cell expressing the chimeric receptor, the retroviral vector further carrying the gene to be transferred; and

(d) allowing the gene carried by the retroviral vector to be expressed in the cell,

thereby transferring the gene. In this method, the cell is preferably a human cell, the retrovirus is preferably a murine retrovirus, most preferably an ecotropic murine leukemia virus, and the chimeric receptor is preferably a chimeric H13/ERR protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the H13 DNA sequence (SEQ ID NO:7), including coding and noncoding sequences, and the predicted protein sequence (SEQ ID NO: 8) of the H13 protein.

Figure 2 is a schematic diagram of the alignment of one strand of the H13 and ERR cDNA sequence (SEQ ID No: 7 and 3, respectively). The sequences were analyzed using the Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl. Acids Res. 12:387-395 (1984)).

Figure 3 shows the alignment of H13, ERR and TEA deduced amino acid sequences. Vertical lines indicate

sequence identity. Dots indicate lack of identity, with double dots representing conservative amino acid changes. The sequences were analyzed as in Figure 2. Shown in brackets are the sequences of H13 corresponding to Extracellular Domain 3 (residues 210-249) and Extracellular Domain 4 (residues 310¬337).

Figure 4 is an autoradiogram showing the

hybridization pattern of EcoRI-digested DNA of human (CCL120, CCL 119, SupTl, H9, MOLT4), hamster (CHO-Kl) and mouse (Balb/c thymocytes, BI0T6R) origin, probed with the KpnI-Kpnl fragment (390 bp) of murine ERR cDNA.

Figure 5 is an autoradiogram showing Southern blot analysis DNA from various species with H13 cDNA (SEQ ID NO:1). DNA hybridized was EcoRI-digested DNA of human (CCL120, CCL 119, SupT1, H9, MOLT4), hamster (CHO-K1) and mouse thymocytes (Balb/c or BIOT6R) origin.

Figure 6 is an autoradiogram showing H13 gene expression, RNA from the indicated human cell lines was hybridized with the H13 cDNA (SEQ ID NO:1).

Figure 7 is an autoradiogram showing the

hybridization pattern of RNA of human (CEM, H9, MOLT4 , SupT1, CCL120, CCL119), hamster (CHO K1) and mouse (RL12) origin, probed with the KpnI-KpnI fragment (390 bp) of murine ERR cDNA.

Figure 8 shows the acquisition of susceptibility to infection with murine ecotropic retrovirus by transfection of a resistant cell with ERR cDNA. After transfection of ERR cDNA into hamster CHO Kl cells, the transfectants expressing the murine retroviral receptor gene were infected with murine radiation leukemia virus (RadLV). Two weeks later, Northern blot analysis was performed using a viral probe, and reverse transcriptase (RT) activity of the cell supernatants was measured.

Figure 9 shows hydropathy plots of H13, ERR and TEA predicted proteins. The vertical axis gives the

hydropathicity values from the PEPTIDESTRUCTURE program

(Jameson et al., CABIOS 4: 181-186 (1988)).

Figure 10 is a graph indicating the antigenicity of H13 predicted protein, analyzed using the PEPTIDESTRUCTURE program. One of the highly antigenic peptides (amino acid residues 309-367) was prepared using an AccI-EcoRI fragment as shown in Figure 14. Figure 11 depicts a polyacrylamide gel electropherogram showing the synthesis of a fusion protein including the H13 protein with glutathione-S-transferase (GST). The fusion protein was prepared by ligating the 180 bp AccI-EcoRI fragment of H13 cDNA to the plasmid pGEX-2T, which expresses antigens as fusion proteins, was induced by addition of isopropyl-beta-thiogalacto-pyranoside (IPTG), and was purified using glutathione-Sepharose chromatography.

Figure 12 shows the genetic mapping of the H13 gene to human chromosome 13. The autoradiogram (Figure 12A) shows the hybridization pattern of EcoRI-digested DNA from humanhamster somatic cell hybrids probed with H13 cDNA (SEQ ID NO:l). Lane 1 and 11 contain DNA from human and hamster, respectively. Lanes 2-10 contain DNA which is derived from the chromosomes as designated in the table in Figure 12B.

Figure 13 is a schematic diagram of the genetic structure of the H13 and ERR genes, and four chimeric

constructs therebetween. The infectivity of E-MuLV on human cells transfected with the various constructs is also

indicated.

Figure 14 shows a comparison of sequences (nucleotide and amino acid) of the region of H13 and ERR termed Extracellular Domain 3 (see also SEQ ID NO: 7, SEQ ID NO:8 and Figure 1) . This region of the receptor protein is most diverse between the human and mouse sequences. The sequences were aligned using Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl. Acids Res. 12:387-395 (1984)).

Figure 15 shows a schematic illustration of several cDNA clones from which the H13 sequence was derived, and their general structural relationship to the murine ERR homologue. Clone 7-2 (H-13.7-2) represents a part of the complete H13 DNA sequence; this was the first H13 clone sequenced, yielding SEQ ID NO:1 and SEQ ID NO: 2. Clones 1-1 (H13.1-1) and 3-2 (H13.3-2) each contain parts of the H13 sequence. The

combined sequencing of these three clones resulted in the full H13 DNA and amino acid sequences (SEQ ID NO: 7 and SEQ ID NO: 8, respectively). DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a DNA molecule discovered by the inventors which is homologous to murine endogenous retrovirus receptor (ERR) and T cell early

activation antigen genes (TEA) and encodes a protein termed H13. The present inventors have conceived of a method of use of the H13 protein, or a functional derivative thereof, preferably a soluble form of the protein, to bind human retroviruses in a manner that prevents their entry into susceptible cells.

The methods of the present invention which identify normal or mutant HI3 genes or measure the presence or amount of H13 protein associated with a cell or tissue can serve as methods for identifying susceptibility to human retrovirus infection, as in AIDS or certain forms of leukemia.

in one embodiment, the invention is directed to a naturally occurring H13 protein substantially free from impurities of human origin with which it is natively

associated. In another embodiment, the invention is directed to a recombinant H13 encoded protein. "Substantially free of other proteins" indicates that the protein has been purified away from at least 90 per cent (on a weight basis), and from even at least 99 per cent, if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them. That can be achieved by subjecting the cells, tissue or fluids containing the H13 protein to protein purification techniques such as

immunoadsorbent columns bearing monoclonal antibodies reactive against the protein. Alternatively, the purification can be achieved by a combination of standard methods, such as

ammonium sulfate precipitation, molecular sieve

chromatography, and ion exchange chromatography.

It will be understood that the H13 protein of the present invention can be purified biochemically or

physicochemically from a variety of cell or tissue sources. For preparation of naturally occurring H13 protein, tissues such as human lymphatic organs and cells such as human

lymphoid cells are preferred. Alternatively, methods are well known for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support.

Because the H13 gene can be isolated or synthesized, the H13 polypeptide, or a functional derivative thereof, can be synthesized substantially free of other proteins or

glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired. As intended by the present invention, an H13 protein molecule produced by recombinant means in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells, for example, is either a naturally occurring protein sequence or a functional

derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.

A preferred use of this invention is the production by chemical synthesis or recombinant DNA technology of

fragments of the H13 molecule, preferably as small as

possible, while still retaining sufficiently high affinity in binding to HIV to inhibit infection. Preferred fragments of H13 include extracellular domain 3 and extracellular domain 4. Due to its function as a virus receptor, an extracellular fragment of the H13 protein is expected to bind to a human retrovirus. By production of smaller fragments of this peptide, one skilled in the art, using known binding and inhibition assays, will readily be able to identify the minimal peptide capable of binding a retrovirus with

sufficiently high affinity to inhibit infectivity without undue experimentation. Shorter peptides are expected to have two advantages over the larger proteins: (1) greater stability and diffusibility, and (2) less immunogenicity.

The identification of the H13 as a potential

receptor or site of entry of a retrovirus into target cells establishes a critical mechanism to explain how the retrovirus enters the cell. The availability of specific H13 receptor "mimics" or "decoys" that can prevent retrovirus uptake provides promise in controlling the spread of retrovirus infection and related pathologies. Despite the degree of similarity in amino acid sequence (87.6% identity) and structure (14 transmembrane spanning domains), between the human H13 protein of the present invention and the murine ERR, H13 fails to bind detectably to E-MuLV.

The present inventors have taken advantage of this species difference in susceptibility in infection to construct and analyze DNA molecules encoding chimeric retrovirus

receptor proteins by substituting bases encoding amino acids of murine ERR for bases encoding H13 amino acid residues.

This has resulted in the identification of critical amino acids which must be present for susceptibility of a cell to infection by E-MuLV.

The chimeric mouse-human E-MuLV receptor of the present invention can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic or non-mammalian eukaryotic cell. Preferably, however, a chimeric H13/ERR protein molecule is produced by recombinant means and expressed in mammalian cells, most preferably in human cells.

Due to the presence of critical amino acid residues from the murine ERR sequence, the chimeric receptor of the present invention endows human cells or other non-murine cells expressing the receptor with the ability to be infected by murine E-MuLV.

By appropriate substitutions of one or more of the amino acid residues of ERR in the corresponding site in H13, one skilled in the art, using known binding and inhibition assays, will be able, without undue experimentation, to identify the single or multiple amino acid substitutions which result in a chimeric retroviral receptor capable of binding E-MuLV with sufficiently high affinity to permit infection of a non-murine cell, preferably a human cell, with E-MuLV.

H13 extracellular domain 3 and H13 extracellular domain 4, appear to be the most sensitive sites for modifying virus binding. Thus, to confer E-MuLV susceptibility on a cell, it is preferred to substitute amino acid residues of these domains of H13. Domain 3 comprises residues between positions 210 and 250 (SEQ ID NO:7). Preferred substitution is with one or more amino acid residues from the corresponding domain of ERR, between amino acid residues 210 and 242 (SEQ ID NO:4). Domain 4 of H13 comprises residues 31-337 (SEQ ID NO:7). Preferred substitution is with one or more amino acid residues from the corresponding domain of ERR, between amino acid residues 303 and 330 (SEQ ID NO:4).

Substitution of between 1 and 4 residues is preferred. Substitution of as few as one amino acid may alter the virus specificity of the chimeric receptor protein. The residues and positions which differ in Extracellular Domain 3 and Domain 4 of H13 and ERR are listed below in Table 1.

Table 1

Possible Substitutions in H13 Extracellular Domain 3 and

Domain 4 for E-MuLV Binding

Domain 3 Domain 4

Original Substituting Original Substitutim

H13 ERR H13 ERR

Residue Residue Residue Residue

V 214 I 214 N 320 I 313

E 222 K 222 N 321 D 314

E 223 N 223 D 326 G 319

G 225 S 225 V 331 Q 324

L 233 N 227 G 335 E 328

E 239 N 232

G 240 V 233

P 242 Y 235

V 244 E 237

Another means for modifying the virus binding specificity of H13 is by deletion of one or more of the

"extra" amino acid residues in H13 (in extracellular domain 4) that do not correspond to residues of ERR. Preferred

deletions are of between one and six residues from H13

positions 326 to 331 (SEQ ID NO:1), most preferably, deletion of all six of these residues.

A major advantage of transfecting non-murine cells with a chimeric receptor or substituted receptor of the present invention, rather than with the ERR protein, is the major decrease in immunogenicity. Thus, for example, human cells to be infected with a murine retrovirus are made to express a chimeric receptor comprising virtually all human sequence, but having only a few necessary amino acid residues of the murine retroviral receptor sequence needed to confer infectibility by the murine retrovirus. If cells bearing a receptor to which E-MuLV can bind are to be introduced on multiple occasions into the same human subject, the fact that the chimeric receptor is largely of human origin decreases the chances of an undesirable immune response directed to the sequences derived from receptor sequence of non-human origin. If large portions of the ERR protein were used for this purpose, the human subject would respond immunologically to the foreign epitopes on the injected cells, diminishing the utility of these cells in gene therapy.

This lack of immunogenicity is important for

survival and therapeutic efficacy of the infused retrovirally infected cells. In gene therapy using bone marrow stem cells or hepatocytes, for example, it is common to manipulate the cells in vitro with cytokines and then to infect them with the vector bearing the gene of interest. Such cells are very short lived an have yielded very short lived therapeutic changes (see, for example, Wilson, J.M. et al., Proc. Natl. Acad. Sci. USA 87:8437-8441 (1990)). The addition of

immunogenic epitopes on such cells would further shorten their half-life in vivo.

The H13 protein as well as the ERR/H13 chimeric protein can be expressed on the cell surface as an integral membrane protein in a number of cell types, particularly cells of the T lymphocyte and monocyte/macrophage lineages,

consistent with in vitro tropism of known human retroviruses such as HIV-1 and HTLV-1. Thus, the chimeric receptor will permit cells of these lineages in the human, which are

normally resistant to murine retrovirus infection, to be infected with E-MuLV.

The virus infections for which the present invention is useful include HIV-1, HIV-2, human T lymphotropic viruses that induce leukemia (HTLV-1, HTLV-2, etc.) and other human retroviruses (Weiss, R.A. et al., RNA Tumor Viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1985). The present invention is intended to encompass all the retroviruses which attach to the H13 as their cellular

receptor or enter the cell via an H-13-dependent mechanism.

Genetic constructs encoding H13 functional derivatives thereof such as those described above, can be used in gene therapy. An abnormal H13 molecule which results in enhanced susceptibility to disease, may be replaced by

infusion of cells of the desired lineage (such as hemopoietic cells, for example) transfected with a modified H13 protein, under conditions where the infused cells will preferentially replace the endogenous cell population. Genetic constructs encoding a recombinant chimeric ERR/H13 molecule are particularly useful in gene therapy.

Recombinant amphotropic retroviruses have been recognized as useful vectors for transferring genes efficiently into human cells, for example to correct enzyme deficiencies (Cone, R.D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Danos, O. et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Depending on the receptor specificity of the viral envelope protein gp70, such viruses have varying host ranges; some recognize human cells. Cone et al. (supra) and Danos et al. (supra) produced packaging cell lines which produced

amphotrophic retroviruses which could infect human cells and integrate randomly into human genomic DNA. Such vectors have been used to transfer a histochemically detectable marker gene into neurons ((Price, J. et al., Proc. Natl. Acad. Sci. USA 84: 156-160 (1987)).

For safety reasons, it is important that a retroviral vector used for gene therapy be capable of

infecting only desired cells and not cause generalized

infection of cells throughout the body of the individual being treated. In the past, this has generally been accomplished by using helper-defective virus preparations, or mutants lacking the psi packaging sequence, etc. The present invention provides an improved measure of safety compared to the prior art approaches in that it permits use of an competent E-MuLV vector, having a limited murine host range. Only those cells to be infected with the vector are given the capacity by virtue of their expression of the chimeric receptor of the present invention.

While gene transfer using retroviruses is generally more efficient than transfection with naked DNA, some cells are not easily infectible by retroviruses, making it difficult to use retroviruses as vectors for introducing new genes into such cells. According to the present invention, a human cell which is not infectable by a human retrovirus or is infectable only at very low efficiency due to lack of sufficient

retroviral receptor protein on its surface is transfected with the H13 gene or a functional derivative, and the H13 protein expressed, resulting in retrovirus receptor appearing on the cell surface. Such a transfected cell can then be infected with a human retroviral vector carrying a gene of interest, in order to transfer the gene of interest permanently into the cell. This type of manipulation has been accomplished to render hamster cells, which are not susceptible to infection with MuLV, susceptible to this virus (see Example IV, below). Following transfection with and expression of the ERR gene (the H13 homolog) in hamster cells, these cells could be infected with MuLV, and could serve as targets for MuLV-mediated gene transfer. For a general discussion of

retrovirus-mediated gene transfer, see, for example (Gllboa, E., Bio-Essavs 5:252-258 (1987); Williams, D.A. et al.,

Nature 310:476-480 (1984)).

The present invention is intended to encompass any ecotropic murine retroviruses, or any other mammalian

retroviruses with similar receptor specificity, which attach to ERR and to the chimeric H13/ERR molecule of the present invention as their cellular receptor or enter the cell via an ERR-dependent mechanism.

More broadly, the invention is directed to the general concept of generating a chimeric retroviral receptor which will allow selected cells of one animal species to be infected with a retrovirus which normally does not infect cells of that species. Thus, for example, murine retroviruses can infect chimpanzee cells if they express a chimpanzee retrovirus-murine retrovirus chimeric receptor which allows binding of the murine retrovirus. By specific changes in the amino acid sequence of, for example, the H13 molecule, it would be possible to create a receptor molecule that would confer susceptibility to any of a number of viruses. Thus a different chimeric H13-based construct can be tailor made for any given human or non-human retrovirus. One of ordinary skill in the art will readily be able to apply this teaching to any of a number of retroviruses and chimeric receptors without undue experimentation.

One of ordinary skill in the art will know how to obtain DNA encoding a retroviral receptor homologous to ERR or H13 from any species or cell type without undue experimentation. First, one will screen (using methods routine in the art) a cDNA library of the species or cell type of interest, for example, a chimpanzee T cell cDNA library, using a probe based on the sequence of ERR or H13. Next, one will clone and sequence the hybridizing DNA to obtain the sequence of the "new" retroviral receptor. By visual

inspection or with the aid of a computer program (as described herein) it is possible to identify the regions in which the sequence of the new retroviral receptor protein differs from ERR or H13. In particular, one will concentrate on the extracellular domain regions 3 or 4. Based on the sequence differences observed, it is possible, using the teachings provided herein, to create a sequence having one or more amino acid substitutions such that a chimeric receptor between the new receptor and a known receptor is created. The chimeric receptor can then be expressed in a cell of choice and its function can easily be tested using conventional virus

binding assays or virus infectivity assays.

Furthermore, according to the present invention, it is possible to modify the receptor attachment site of a virus so that it will not bind to its natural receptor. For

example, changes in the sequence of the HIV-1 CD4-binding domain will render this virus non-infective for CD4-bearing cells. Corresponding changes may be introduced into H13, so that this mutant HIV will bind to it. In this way, a safe HIV preparation can be generated which binds only to select cells bearing the appropriate variant receptor, but not to the normal targets of HIV-1.

in another embodiment, the methods and constructs of the present invention can be used to produce a bone marrow stem cells which are infectible via CD4 without disrupting the normal cellular developmental and maturational process that depend on intact CD4 expression. In this embodiment, a chimeric H13/CD4 molecule is expressed in a bone marrow stem cell, which is then infected by a E-MuLV retroviral vector having a CD4-binding domain of HIV engineered into the murine gp70 molecule. Expression of the intact or a chimeric human H13 sequence in a chimpanzee cell or cell lineage may allow an HIV infection in chimpanzees to develop into and AIDS-like

syndrome, providing an improved animal model for AIDS than the simian immunodeficiency virus infects of chimpanzees.

It is also within the scope of the present invention to express more than one intact or chimeric retroviral

receptor molecule on the surface of the same cell. Thus, by virtue of a first retroviral receptor, e.g. ERR, a human cell can be infected with one virus strain in vitro in a transient fashion, and can be manipulated by the judicious use of cytokine growth or differentiation factors. Such cells can be introduced into a recipient. At the desired time, a second virus which binds to a second genetically engineered receptor can be introduced into the individual to infect stably alter only those introduced cells bearing the second retroviral receptor.

The preferred animal subject of the present invention is a mammal. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.

Also included are soluble forms of H13 or of a chimeric receptor, as well as functional derivatives thereof having similar bioactivity for all the uses described herein. Also intended are all active forms of H13 derived from the H13 transcript, and all muteins with H13 activity. Methods for production of soluble forms of receptors which are normally transmembrane proteins are well known in the art (see, for example, Smith, D.H. et al., Science 238:1704-1707 (1987);

Fisher, R.A. et al., Nature 331:76-78 (1988); Hussey, R.E. et al., Nature 331:78-81 (1988); Deen, K.C. et al., Nature

331:82-84 (1988); Traunecker, A. et al ., Nature 331:84-86 (1988); Gershoni, J.M. et al., Proc. Natl. Acad. Sci. USA

£5:4087-4089 (1988), which references are hereby incorporated by reference). Such methods are generally based on truncation of the DNA encoding the receptor protein to exclude the transmembrane portion, leaving intact the extracellular domain (or domains) capable of interacting with specific ligands, such as an intact retrovirus or a retroviral protein or glycoprotein.

For the purposes of the present invention, it is important that the soluble H13, or a functional derivative of H13, comprise the elements of the binding site of the H13 that permits binding to a retrovirus. An H13 molecule has many amino acid residues, only a few of which are critically involved in virus recognition and binding.

As discussed herein, the H13 proteins or peptides of the present invention may be further modified for purposes of drug design, such as, for example, to reduce immunogenicity, to promote solubility or enhance delivery, or to prevent clearance or degradation.

In a further embodiment, the invention provides

"functional derivatives" of the H13 protein. By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the H13 protein. A functional derivative retains at least a portion of the function of the H13 protein which permits its utility in accordance with the present invention.

A "fragment" of the H13 protein is any subset of the molecule, that is, a shorter peptide.

A "variant" of the H13 refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well- known in the art.

Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see

European Patent Publication No. EP 75,444).

At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by

Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variants typically exhibit the same

qualitative biological activity as the nonvariant peptide. In particular, the H13 molecule having critical amino acid residues derived from ERR can be produced using site-directed mutagenesis.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al.,

Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is then annealed with the single-stranded protein-sequence¬containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence in the second strand bears the desired mutation.

This heteroduplex vector is then used to transform appropriate cells and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. The mutated protein region may be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that may be employed for transformation of an appropriate host.

An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to the N-terminus of the peptide molecule to facilitate the secretion of mature peptide molecule from recombinant hosts.

Another group of variants are those in which at least one amino acid residue in the protein molecule, and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular

Properties. W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions which may be made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of

different species, such as those presented in Table 1-2 of Schulz et al. (supra) and Figure 3-9 of Creighton (supra).

Base on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar

residues: ala, ser, thr (pro, gly);

2. Polar, negatively charged residues and their amides: asp, asn, glu, gin;

3. Polar, positively charged residues:

his, arg, lys;

4. Large aliphatic, nopolar residues:

met, leu, ile, val (cys); and

5. Large aromatic residues: phe, tyr, trp. The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation which is important in protein folding. Note the Schulz et al. would merge Groups l and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has some kinship with Ser, Thr, etc. Substantial changes in

functional or immunological properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups, which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the

substitution, for example, as a sheet or helical

conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

Examples of such substitutions are (a) substitution of gly and/or pro by another amino acid or deletion or insertion of gly or pro; (b) substitution of a hydrophilic residue, e.g., ser or thr, for (or by) a hydrophobic residue, e.g., leu, ile, phe, val or ala; (c) substituion of a cys residue for (or by) any other residue; (d) substitution of a residue having an electropositive side chain, e.g., lys, arg or his, for (or by) a residue having an electronegative charge, e.g., glu or asp; or (e) substitution of a residue having a bulky side chain, e.g., phe, for (or by) a residue not having such a side chain, e.g., gly.

Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays, either immunoassays or bioassays. For example, a variant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by

immunoaffinity chromatography using a specific antibody on a column (to absorb the variant by binding to at least one epitope).

The activity of the cell lysate containing H13 or a chimeric H13 protein, or of a purified preparation of H13, a variant thereof, of of chimeric H13, can be screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the protein molecule, such as binding to a given antibody, is measured by a competitive type immunoassay (see below). Biological activity is screened in an appropriate bioassay, such as virus infectivity, as described herein.

Modifications of such peptide properties as redox or thermal stability, hydrophobicity, susceptibility to

proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.

An "analog" of the H13 protein refers to a nonnatural molecule substantially similar to either the entire molecule or a fragment thereof.

A "chemical derivative" of the H13 protein contains additional chemical moieties not normally a part of the protein. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo- beta-(5-imidozoyl)propionic acid, chloroacetyl

phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is

preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for

derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal

phosphate; pyridoxal; chloroborohydride;

trinitrobenzenesulfonic acid; O-methylisourea; 2,4

pentanedione; and transaminase-catalyzed reaction with

glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin.

Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK_a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form o-acetyl tyrosyl species and 3-nitro derivatives,

respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)

carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl

residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio)propioimidate yield

photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;

4,229,537; and 4,330,440 are employed for protein

immobilization.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecule Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack

Publishing Co., Easton, PA (1980).

Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al., Molecular Biology of the Gene. Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y.

(1986); Lewin, B.M., Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering. 2d edition. University of California Press, publisher, Berkeley, CA (1981); and Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). These references are hereby incorporated by reference. The recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more

preferably, by application of recombinant DNA techniques.

By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.

By "cDNA" is meant complementary or copy DNA

produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

By "cDNA library" is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire expressible genome of an

organism. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in

Sambrook et al., Molecular Cloning: A Laboratory Manual, supra. Generally, RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are mammalian cell lines.

Oligonucleotides representing a portion of the H13 sequence are useful for screening for the presence of

homologous genes and for the cloning of such genes.

Techniques for synthesizing such oligonucleotides are

disclosed by, for example, Wu, R., et al., Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978)).

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid

(Watson, J.D. et al., supra). Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the amino acid. The probability that a particular oligonucleotide will, in fact, constitute the actual XXX-encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R., et al., J. Molec. Biol. 183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of

oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding the H13 sequences is identified.

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of

encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the protein.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the H13 fragment is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable"

sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the H13 gene (Sambrook et al., supra).

A suitable oligonucleotide, or set of

oligonucleotides, which is capable of encoding a fragment of the H13 gene (or which is complementary to such an

oligonucleotide, or set of oligonucleotides) is identified (using the above-described procedure), synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the H13 gene. Single stranded oligonucleotide molecules complementary to the "most probable" H13 peptide coding sequences can be synthesized using

procedures which are well known to those of ordinary skill in the art (Belagaje, R., et al., J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T., et al., In: Molecular Mechanisms in the Control of Gene Expression. Nierlich, D.P., et al., Eds., Acad. Press, NY (1976); Wu, R., et al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana, R.G., Science

201:614-625 (1979)). Additionally, DNA synthesis may be achieved through the use of automated synthesizers.

Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra). and by Haymes, B.D., et al. (In:

Nucleic Acid Hybridization. A Practical Approach. IRL Press, Washington, DC (1985)), which references are herein

incorporated by reference. Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, L.C., et al., Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985)), fibronectin (Suzuki, S., et al., Eur. Mol. Biol. Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter, P., et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, D., et al., Nature 301:214-221 (1983)) and human term placental alkaline phosphatase

complementary DNA (Kam, W., et al., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

In an alternative way of cloning the H13 gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing H13) into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-H13 antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as H13 proteins or peptides, or fragments thereof. In this embodiment, DNA, or more preferably cDNA, is extracted and purified from a cell which is capable of expressing H13 protein. The purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.

By "vector" is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A vector will contain one or more unique restriction sites, and may be capable of autonomous

replication in a defined host or vehicle organism such that the cloned sequence is reproducible.

An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences. Importantly, since eukaryotic DNA may contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferable to employ cDNA from a cell which is capable of expressing H13 in order to produce a prokaryotic genomic expression vector library. Procedures for preparing cDNA and for producing a genomic library are disclosed by Sambrook et al. (supra).

By "substantially pure" is meant any protein or peptide of the present invention, or any gene encoding any such protein or peptide, which is essentially free of other proteins or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.

By "functional derivative" of a polynucleotide molecule is meant a polynucleotide molecule encoding a

"fragment" "variant" or "analogue" of the H13 protein. Such a functional derivative may be "substantially similar" in nucleotide sequence to the H13-encoding sequence and thus encode a protein possessing similar activity to the H13 protein. Alternatively, a "functional derivative" of a polynucleotide can be a chemical derivative which retains its functions, such as the capability to express the protein, or the ability to hybridize with a complementary polynucleotide molecule. Such a chemical derivative is useful as a molecular probe to detect H13 sequences through nucleic acid

hybridization assays.

A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological

activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.

A DNA sequence encoding the HI3 protein or a

chimeric ERR/H13 protein of the present invention, or a functional derivative thereof, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation,

restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline

phσsphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such

manipulations are disclosed by Sambrook, J. et al., supra, and are well known in the art.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains

nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. Such regions will normally include those 5'- non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.

If desired, the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the protein, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be

substituted.

Two sequences of a nucleic acid molecule are said to be "operably linked" when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence. Thus, two sequences, such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if

transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another. As used herein, a "promoter" is a region of a DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence. A "promoter sequence" is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase. This functional

promoter will direct the transcription of a nucleic acid molecule which is operably linked to that strand of the double-stranded molecule which contains the "promoter

sequence."

Certain RNA polymerases exhibit a high specificity for such promoters. The RNA polymerases of the bacteriophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity. The promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to utilize (i.e. transcribe) only one strand of the two strands of a duplex DNA template. The selection of which strand is transcribed is determined by the orientation of the promoter sequence. This selection

determines the direction of transcription since RNA is only polymerized enzymatically by the addition of a nucleotide 5'phosphate to a 3' hydroxyl terminus. The sequences of such polymerase recognition sequences are disclosed by Watson, J.D. et al., supra). The promoter sequences of the present

invention may be either prokaryotic, eukaryotic or viral.

Suitable promoters are repressible, or, more preferably, constitutive. Strong promoters are preferred.

The present invention encompasses the expression of the H13 protein (or a functional derivative thereof) or a chimeric H13 protein in either prokaryotic or eukaryotic cells, although preferred expression is in eukaryotic cells, expression is preferred, most preferably in human cells. To express the chimeric protein of the present invention in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the H13 encoding sequence to a functional prokaryotic promoter, examples of which are well-known in the art. Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene-encoding sequence (see, for example. Gold, L. et al. (Ann. Rev. Microbiol.

35:365-404 (1981)).

To express the H13 protein (or a functional

derivative thereof) or a chimeric H13 protein in a prokaryotic cell (such as, for example, E. coli. B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the H13 encoding sequence to a functional prokaryotic promoter.

Examples of constitutive promoters include the int promoter of bacteriophage lambda, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc. Examples of inducible prokaryotic promoters include the major right and left

promoters of bacteriophage 1 (P_L and P_R), the trp, recA,

lacZ, lad, and gal promoters of E. coli, the α-amylase

(Ulmanen, I., et al., J. Bacteriol. 162:176-182 (1985)) and the s-28-specific promoters of B. subtilis (Gllman, M.Z., et al., Gene 32:11-20 (1984)), the promoters of the

bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987));

Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene- encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L., et al. (Ann. Rev.

Microbiol. 35:365-404 (1981)).

Eukaryotic hosts include yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture.

Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO, or cells of lymphoid origin, such as the hybridoma SP2/O-Ag14 or the murine myeloma P3-X63Ag8, and their derivatives. Preferred mammalian cells are cells which are intended to replace the function of the genetically deficient cells in vivo. Bone marrow stem cells are preferred for gene therapy of disorders of the hemopoietic or immune system.

For a mammalian cell host, many possible vector systems are available for the expression of H13. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus. Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be

modulated, of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.

For yeast host cells, any of a series of yeast gene expression systems can be utilized which incorporate promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in glucose-rich medium. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

Production of H13 or chimeric H13 molecules in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express H13 by methods known to those of skill. Thus, in one embodiment, sequences encoding H13 may be operably linked to the regulatory regions of the viral polyhedrin protein (Jasny, Science 238: 1653

(1987)). Infected with the recombinant baculovirus, cultured insect cells, or the live insects themselves, can produce the H13 protein in amounts as great as 20 to 50% of total protein production. When live insects are to be used, caterpillars are presently preferred hosts for large scale H13 production according to the invention.

As discussed above, expression of the H13 protein in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus

(McKnight, S., Cell 31:355-365 (1982)); the SV40 early

promoter (Benoist, C., et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955

(1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the H13 or chimeric protein does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as H13 encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the H13 encoding sequence).

The H13 or chimeric receptor coding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the H13 protein may occur through the transient expression of the introduced sequence. Alternatively,

permanent expression may occur through the integration of the introduced sequence into the host chromosome. In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like. The selectable marker gene can either be

directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol.

Cell. Biol. 3:280 (1983).

In another embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose.

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Expression. Academic Press, NY, pp. 563-608 (1980)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives, such plasmids are well known in the art (Botstein, D., et al.,

Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al., J. Clin. Hematol. Oncol. 10:3948 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Expression. Academic Press, NY, pp. 563-608 (1980)).

Preferred vectors for transient expression of the

H13 or chimeric receptor protein of the present invention in CHO cells is the pSG5 or pCDM8 expression vector.

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection,

conjugation, protoplast fusion, calcium phosphateprecipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment (Johnston et al., Science 240(4858): 1538 (1988)), etc.

After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of H13 or the chimeric H13 protein and its expression on the cell surface.

If so desired, the expressed H13 or chimeric protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. For example, the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose,

phosphocellulose, polyribocytidylic acid-agarose,

hydroxyapatite or by electrophoresis or immunoprecipitation. Alternatively, the chimeric proteins may be isolated by the use of specific antibodies, such as an anti-H13 antibody that still reacts with the protein containing ERR-derived amino acid substitutions. Such antibodies may be obtained by well¬known methods. Furthermore, manipulation of the genetic constructs of the present invention allow the grafting of a particular virus-binding domain onto the transmembrane and

intracytoplasmic portions of the H13 protein, or grafting the retrovirus binding domain of H13 onto the transmembrane and intracytoplasmic portions of another molecule, resulting in yet another type of chimeric molecule.

The present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as a mouse) the germ cells and somatic cells of which contain genomic DNA according to the present invention which codes for the H13 protein or a functional derivative thereof capable as serving as a human retrovirus receptor. The H13 DNA is introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably the one-cell, or fertilized ooσyte, stage, and generally not later than about the 8-cell stage. The term "transgene," as used herein, means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of protein in the transgenic animal.

There are several means by which such a gene can be introduced into the genome of the animal embryo so as to be chromosomally incorporated and expressed. One method is to transfect the embryo with the gene as it occurs naturally, and select transgenic animals in which the gene has integrated into the chromosome at a locus which results in expression. Other methods for ensuring expression involve modifying the gene or its control sequences prior to introduction into the embryo. One such method is to transfect the embryo with a vector (see above) containing an already modified gene. Other methods are to use a gene the transcription of which is under the control of a inducible or constitutively acting promoter, whether synthetic or of eukaryotic or viral origin, or to use a gene activated by one or more base pair substitutions, deletions, or additions (see above).

Introduction of the desired gene sequence at the fertilized oocyte stage ensures that the transgene is present in all of the germ cells and somatic cells of the transgenic animal and has the potential to be expressed in all such cells. The presence of the transgene in the germ cells of the transgenic "founder" animal in turn means that all its progeny will carry the transgene in all of their germ cells and somatic cells. Introduction of the transgene at a later embryonic stage in a founder animal may result in limited presence of the transgene in some somatic cell lineages of the founder; however, all the progeny of this founder animal that inherit the transgene conventionally, from the founder's germ cells, will carry the transgene in all of their germ cells and somatic cells.

Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the H13 DNA of the present invention, such as animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic mammal, are also intended to be within the scope of the present invention.

The techniques described in Leder, U.S. Patent

4,736,866 (hereby incorporated by reference) for producing transgenic non-human mammals may be used for the production of the transgenic non-human mammal of the present invention. The various techniques described in Palmiter, R. et al., Ann. Rev. Genet. 20:465-99 (1986), the entire contents of which are hereby incorporated by reference, may also be used.

The animals carrying the H13 gene can be used to test compounds or other treatment modalities which may

prevent, suppress or cure a human retrovirus infection or a disease resulting from such infection for those retroviruses which infect the cells using the H13 molecule as a receptor. These tests can be extremely sensitive because of the ability to adjust the virus dose given to the transgenic animals of this invention. Such animals will also serve as a model for testing of diagnostic methods for the same human retrovirus diseases. Such diseases include, but are not limited to AIDS, HTLV-induced leukemia, and the like. Transgenic animals according to the present invention can also be used as a source of cells for cell culture. The transgenic animal model of the present invention has numerous economic advantages over the "SCID mouse" model (McCune, J.M et al., Science 241:1632-1639 (1988)) wherein it is necessary to repopulate each individual mouse with the appropriate cells of the human immune system at great cost.

This invention is also directed to an antibody specific for an epitope of H13 protein. In additional

embodiments, the antibody of the present invention is used to prevent or treat retrovirus infection, to detect the presence of, or measure the quantity or concentration of, H13 protein in a cell, or in a cell or tissue extract, or a biological fluid.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural

characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a

molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals

immunized with an antigen. Monoclonal antibodies are a substantially

homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo.

Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected

intraperitoneally into pristane-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (see, for example, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Neuberger et al., Nature 314:268-270 (1985); Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science 240:1041- 1043 (1988); Better, M.D.

International Patent Publication WO 9107494, which references are hereby incorporated by reference).

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic

determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id

antibody). The anti-Id antibody may also be used as an

"immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may bear structural similarity to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to

identify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the H13 protein of the present invention may be used to induce anti-Id

antibodies in suitable animals, such as Balb/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional Balb/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an H13 protein epitope.

The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an epitope of the H13 protein.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')₂, which are capable of binding

antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')₂ and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of H13 protein according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments).

The antibodies, or fragments of antibodies, of the present invention may be used to quantitatively or qualita tively detect the presence of cells which express the H13 protein (or a chimeric receptor having an H13-derived epitope) on their surface or intracellularly. This can be

accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection.

The antibodies of the present invention may be employed histologically, as in immunofluorescence or

immunoelectron microscopy, for in situ detection of H13 protein. In situ detection may be accomplished by removing a histological (cell or tissue) specimen from a subject and providing the a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying on the biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the H13 protein but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as

staining procedures) can be modified in order to achieve such in situ detection.

Additionally, the antibody of the present invention can be used to detect the presence of soluble H13 molecules in a biological sample. Used in this manner, the antibody can serve as a means to monitor the presence and quantity of H13 proteins or derivatives used therapeutically in a subject to prevent or treat human retrovirus infection.

Such immunoassays for H13 protein typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leucocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying H13 protein, and detecting the antibody by any of a number of techniques well-known in the art.

The biological sample may be treated with a solid phase support or carrier (which terms are used interchangeably herein) such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled H13¬specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.

By "solid phase support" or "carrier" is intended any support capable of binding antigen or antibodies. Wellknown supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and

magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.

Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-H13 antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by

employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.

One of the ways in which the H13-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by

spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol

dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline

phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-6phosphate dehydrogenase, gluσoamylase and

acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect H13 protein through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Technigues and Biochemistry in Molecular Biology, by Work, T.S., et al., North Holland Publishing Company, New York (1978) with

particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" byT T. Chard, incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a gamma counter or a liquid scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,

allophycocyanin, o- phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as

diethylenetriaminepentaacetic acid (DTPA) or

ethylenediaminetetraacetic acid (EDTA). The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminesσent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of

luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

The antibody molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to

"extract" the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the socalled "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The

determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and

"forward" assays.

According to the present invention, it is possible to diagnose circulating antibodies in a subject which are specific for the H13 protein. This is accomplished by means of an immunoassay, as described above, using the protein of the invention or a functional derivative thereof.

Based on similar principles, since a retrovirus binds to its cellular receptor with detectable affinity, it is possible to detect the presence of a human retrovirus capable of binding to H13 in a biological sample, using the H13 protein or a functional derivative thereof as a ligand. In such an assay, the protein or functional derivative may be bound to an insoluble support or carrier, as in an

immunoassay. The biological sample, e.g. serum, suspected of having a retrovirus is then contacted with the H13-containing support and the virus allowed to bind to its receptor material. The presence of the bound virus is then revealed in any of a number of ways well known in the art, for example, by addition of a detectably-labelled antibody specific for the virus. The same assay can be used to detect the presence in a biological sample of a viral component such as a viral

protein or glycoprotein which has affinity for the H13

protein. Alternatively, the virus or viral protein may be labelled and binding measured in a competitive assay using an antibody specific for the virus-binding portion of the H13 molecule.

As used herein, the term "prevention" of infection involves administration of the H13 protein, peptide

derivative, or antibody (see above) prior to the clinical onset of the disease. Thus, for example, successful

administration of a composition prior to initial contact with a retrovirus results in "prevention" of the disease.

Administration may be after initial contact with the virus, but prior to actual development of the disease.

"Treatment" involves administration of the protective composition after the clinical onset of the

disease. For example, successful administration of a H13 protein or peptide or anti-H13 antibody according to the invention after development of a retrovirus infection in order to delay or suppress further virus spread comprises

"treatment" of the disease.

The H13 protein, peptides or antibody of the present invention may be administered by any means that achieve their intended purpose, for example, to treat local infection or to treat systemic infection in a subject who has, or is

susceptible to, such infection. For example, an

immunosuppressed individual is particularly susceptible to retroviral infection and disease.

For example, administration may be by various parenteral routes such as subcutaneous, intravenous,

intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.

An additional mode of using the compositions of the present invention is by topical application. This route of administration is particularly important in treating some types of retrovirus infections. The proteins, peptides and pharmaceutical compositions of the present invention may be incorporated into topically applied vehicles such as salves or ointments, which have both a soothing effect on the skin as well as a means for administering the active ingredient directly to the affected area.

The carrier for the active ingredient may be either in sprayable or nonsprayable form. Non-sprayable forms can be semi-solid or solid forms comprising a carrier conducive to topical application and having a dynamic viscosity preferably greater than that of water. Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like. If desired, these may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like. Preferred vehicles for non-sprayable topical

preparations include ointment bases, e.g., polyethylene glycol-1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.

Also suitable for systemic or topical application, in particular to the mucus membranes and lungs, are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material. The aerosol preparations can contain solvents, buffers, surfactants, perfumes, and. or antioxidants in addition to the proteins or peptides of the present invention. For aerosol administration, the active principles in accordance with the present invention may be packaged in a squeeze bottle, or in a pressurized container with an appropriate system of valves and actuators. Preferably, metered valves are used with the valve chamber being recharged between actuation or dose, all as is well known in the art. For topical applications, it is preferred to

administer an effective amount of a compound according to the present invention to an infected area, e.g., skin surfaces, mucous membranes, etc. This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, whether the use is prophylactic or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed. A preferred topical preparation is an ointment wherein about 0.01 to about 50 mg of active ingredient is used per cc of ointment base, the latter being preferably PEG-1000.

A typical regimen for preventing, suppressing, or treating retrovirus infection comprises administration of an effective amount of the H13 protein or functional derivative thereof, administered over a period of one or several days, up to and including between one week and about six months.

It is understood that the dosage administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.

The total dose required for each treatment may be administered by multiple doses or in a single dose. The protein, functional derivative thereof or antibody may be administered alone or in conjunction with other therapeutics directed to the viral infection, or directed to other symptoms of the viral disease.

Effective amounts of the H13 protein, functional derivative thereof, or antibody thereto, are from about 0.01 μg to about 100 mg/kg body weight, and preferably from about 10 μg to about 50 mg/kg body weight.

In one embodiment, the peptides of the present invention are provided to expectant mothers suspected of having a retrovirus infection, by either systemic or intrauterine administration. This treatment is designed to protect the fetus from spread of HIV, for example.

Alternatively, the compositions of the invention can be used intravaginally, especially during the birth process, to protect the newborn from infectious retrovirus which may be present in the birth canal.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.

Pharmaceutical compositions comprising the

proteins, peptides or antibodies of the inventioninclude all compositions wherein the protein, peptide or antibody is contained in an amount effective to achieve its intended purpose. In addition, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers

comprising excipients and auxiliaries which facilitate

processing of the active compounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions include suitable

solutions for administration by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e., the H13 protein or antibody) together with the excipient. Pharmaceutical compositions for oral administration include tablets and capsules. Compositions which can be administered rectally include suppositories.

The present invention provides methods for evaluating the presence and the level of normal or mutant H13 protein or mRNA in a subject. Absence, or more typically, low expression of the H13 gene or presence of a mutant H13 in an individual may serve as an important predictor of resistance to retrovirus infection and thus to the development of AIDS or certain types of leukemia or other retrovirus-mediated

diseases. Alternatively, over-expression of H13, may serve as an important predictor of enhanced susceptibility to retrovirus infection.

In addition, ERR or H13 mRNA expression is increased in virally-induced tumor cell lines, indicating that the level of mRNA or receptor protein expression may serve as a useful indicator of a viral infection not otherwise detectable.

Therefore, by providing a means to measure the quantity of H13 mRNA (see below) or protein (using an immunoassay as described above), the present invention provides a means for detecting a human retrovirus-infected or retrovirus-transformed cell in a subject.

Oligonucleotide probes encoding various portions of the H13 DNA sequence are used to test cells from a subject for the presence H13 DNA or mRNA. A preferred probe would be one directed to the nucleic acid sequence encoding at least 12 and preferably at least 15 nucleotides of the H13 sequence.

Qualitative or quantitative assays can be performed using such probes. For example. Northern analysis (see below) is used to measure expression of an H13 mRNA in a cell or tissue

preparation.

Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recombinant DNA

methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such

methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Sambrook et al. (supra). etc.

Recently, an in vitro enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "polymerase chain reaction" or "PCR"

(Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol.

51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., US 4,683,202; Erlich, H., US 4,582,788; and Saiki, R. et al., US 4,683,194).

The polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.

The precise nature of the two oligonucleotide probes of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses

directionality, which is conferred through the 5 '-3' linkage of the phosphate groups of the molecule. Sequences of DNA or RNA are linked together through the formation of a

phosphodiester bond between the terminal 5' phosphate group of one sequence and the terminal 3' hydroxyl group of a second sequence. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a 5' nucleotide

triphosphate to the 3' hydroxyl end of a nucleic acid

molecule. Thus, the action of a polymerase extends the 3 ' end of a nucleic acid molecule. These inherent properties are exploited in the selection of the oligonucleotide probes of the PCR. The oligonucleotide sequences of the probes of the PCR method are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired.

More specifically, the oligonucleotide sequences of the "first" probe is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3' to the desired sequence, whereas the oligonucleotide sequence of the "second" probe is selected such that it contains an

oligonucleotide sequence identical to one present 5' to the desired region. Both probes possess 3' hydroxy groups, and therefore can serve as primers for nucleic acid synthesis. In the PCR, the reaction conditions are cycled between those conducive to hybridization and nucleic acid polymerization, and those which result in the denaturation of duplex molecules. In the first step of the reaction, the nucleic acids of the sample are transiently heated, and then cooled, in order to denature any double-stranded molecules which may be present. The "first" and "second" probes are then added to the sample at a concentration which greatly exceeds that of the desired nucleic acid molecule. When the sample is incubated under conditions conducive to

hybridization and polymerization, the "first" probe will hybridize to the nucleic acid molecule of the sample at a position 3' to the sequence to be amplified. If the nucleic acid molecule of the sample was initially double-stranded, the "second" probe will hybridize to the complementary strand of the nucleic acid molecule at a position 3' to the sequence which is the complement of the sequence whose amplification is desired. Upon addition of a polymerase, the 3' ends of the "first" and (if the nucleic acid molecule was double-stranded) "second" probes will be extended. The extension of the

"first" probe will result in the synthesis of an

oligonucleotide having the exact sequence of the desired nucleic acid. Extension of the "second" probe will result in the synthesis of an oligonucleotide having the exact sequence of the complement of the desired nucleic acid.

The PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension product of the "first" probe, of necessity, contains a sequence which is complementary to a sequence of the

"second" probe, and thus can serve as a template for the production of an extension product of the "second" probe.

Similarly, the extension product of the "second" probe, of necessity, contains a sequence which is complementary to a sequence of the "first" probe, and thus can serve as a

template for the production of an extension product of the "first" probe. Thus, by permitting cycles of polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the PCR are provided by Mullis, K.B. (Cold Spring Harbor Svmp. Quant. Biol. 51:263-273 (1986)); Saiki, R.K., et al.

fBio/Technolocrv 3:1008-1012 (1985)); and Mullis, K.B., et al. fMeth. Enzymol. 155:335-350 (1987)). Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting of the present invention, unless specified. EXAMPLE I

General Materials and Methods

Cell Lines

The following cell lines were used in the studies described below: CCL120 (ATCC# CCL120), a human B

lymphoblastoid cell line; CCL119 (CEM, ATCC# CCL119), a human T lymphoblastoid cell line; SupT1, a human non-Hodgkin's T lymphoma cell line; H9, a single cell clone derived from

HUT78,a human cutaneous T cell lymphoma cell line; M0LT4

(ATCC# CRL1582), a human acute lymphoblastic leukemia cell line; HOS (ATCC# CRL1543), a human osteosarcoma cell line;

HeLa (ATCC# CCL2), a human epithelioid carcinoma cell line; CHO-K1 (ATCC #61), a Chinese hamster ovary cell line; B10T6R, a radiation-induced thymoma of B10.T(6R) mice; and RL12, a radiation-induced thymoma of C57BL/6Ka mice.

Screening

Human CEM and HUT 78 T-cell cDNA library (lambda gtll) was obtained from Clontech Laboratories Inc. (Palo Alto, California). The human lymphocyte cosmid library (pWE15) was obtained from Stratagene (LaJolla, CA). The libraries were screened by the method of Maniatis et al. (Maniatis, T. et al. Cell 15:887-701 (1978)). The BamHl-EcoRI fragment, containing the entire open reading frame of ERR cDNA (pJET) was provided by Drs. Albritton and Cunningham (Harvard Medical School,

Boston, MA). This DNA was labelled with ³²P by nick

translation to a specific activity of about 2 × 10⁶ cpm/μg and used as a hybridization probe. Southern Blot Analysis

High relative mass DNA was prepared from cells as described by Blin, N. et al. (Nucl. Acids Res. 3:2303-2308 (1976)) and modified by Pampeno and Meruelo (Pampeno, C. L. et al. J. Virol. 58:296-306 (1986)). Restriction endonuclease digestion, agarose gel electrophoresis, transfer to

nitrocellulose (Schleicher & Schuell, Inc., Keene, New

Hampshire), hybridization and washing was as described

(Pampeno, C. L. et al. supra; Brown, G. D. et al.

Immunogenetics 27:239-251 (1988)).

Northern Blot Analysis

Total cellular RNA was isolated from cells by the acid guanidinium thiocyanate-phenol-chloroform method

(Chomczynski, P. et al. Anal. Biochem. 162:156-159 (1987)). The DNA was electrophoresed in 1% formaldehyde agarose gels and transferred to Nytran filters (Schleicher & Schuell, Inc., Keene, New Hampshire). The hybridization and washing was performed according to Amari, N. M. B. et al. (Mol. Cell.

Biol. 7:4159-4168 (1987)).

DNA Seguence Analysis

cDNA clones from positive phages were recloned into the EcoRI site of plasmid vector pBluescript (Stratagene).

Unidirectional deletions of the plasmids were constructed by using exonuclease III and S1 nuclease, and sequenced by the dideoxy chain termination methods (Sanger, F. S. et al. Proc. Natl. Acad. Sci. USA 74:5463- 5467 (1977)) with Sequenase reagents (U.S. Biochemical Corp., Cleveland, Ohio).

Restriction maps of positive cosmid inserts were determined using T3 or T7 promoter-specific oligonucleotides to probe partially digested cosmid DNA as described elsewhere (Evans, G.A. et al., Meth. Enzymol. 152:604-610 (1987)). EcoRI-EcoRI or EcoRI-HindIII fragments in the cosmids were subcloned into pBluescript or pSport 1 (GIBCO BRL, Gaithersburg, MD). The exons and exon-intron junctions were sequenced using synthetic oligonucleotides as primers. Sequences were compiled and analyzed using the Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl. Acids Res.

12:387-395 (1984)). EXAMPLE II

DNA and Predicted Protein Sequence of H13 The complete nucleotide sequence of H13 (SEQ ID NO:7) including non-coding sequences at the 5' and 3' end of the coding sequence are shown in Figure 1. This sequence includes the partial sequence originally obtained from clone 7-2 (SEQ ID NO:1); nucleotides 1-6 and 1099-1102 of SEQ ID NO:1 were originally incorrectly determined. Figure l also shows the complete amino acid sequence predicted from the nucleotide sequence (SEQ ID NO: 8). This sequence includes the originally described partial amino acid sequence (SEQ ID NO:2) with the exception of the N-terminal Pro-Gly and the C- terminal Pro, which were originally incorrectly predicted from the nucleotide sequence.

The nucleotide sequence comparison between H13, ERR and TEA is shown in Figure 2 and the amino acid sequence comparison is shown in Figure 3.

The homology between the compared sequences is very high, for example 87.6% homology between H13 and ERR DNA, and 52.3% homology between H13 and TEA amino acids.

EXAMPLE III

Presence and Expression of the H13 Gene in Human Cells

By Southern analysis of DNA taken from cells of various species, it was shown that DNA capable of hybridizing with a murine ERR cDNA probe (Figure 4) and with the H13 cDNA (Figure 5) was present in cells of 5 human cell lines, including CCL120, CCL119, SupT1, H-9 and MOLT-4, and also in hamster cells (CHO-K1) and murine cells (normal Balb/c mouse thymocytes). H13 gene expression was examined using Northern analysis, using the H13 cDNA probe. The probe detected a transcript of approximately 9kb in RNA from HeLa, SupT1, HOS and CCL119 cells (Figure 6). This RNA could also be detected using a murine ERR cDNA probe (Figure 7). EXAMPLE IV

Transfection of Murine Retroviral Receptor

cDNA into Hamster Cells

Murine retroviral receptor (ERR) cDNA was cotransfected into hamster CHO cells, which can not be

infected by murine ecotropic retroviruses, with the selectable marker plasmid DNAP, pSV₂Neo, using calcium phosphate (Wigler, M. et al., Cell 14: 725-731 (1978)). The transfectant

expressing the receptor gene was, then, infected by murine radiation leukemia virus (RadLV). Two weeks later after the infection the reverse transcriptase (RT) activity of the supernatant was measured (Stephenson, J.R. et al., Virology48: 749-756 (1972)), and Northern Blot analysis was performed using a viral probe after preparing its RNA. As shown in Figure 8, the RT activity detected in untransfected CHO cells which do not express the receptor gene was indistinguishable from the activity of tissue culture medium (background). This indicates that the cells were not infected by MuLV.

Following transfection with the ERR cDNA, the RT activity of the transfected cell supernatant was much higher than background (Figure 8).

The MuLV viral probe detected transcripts in RNA prepared from the transfectant, but not in RNA prepared from untransfected CHO cells. The results indicate that the cells transfected with the ERR cDNA can acquire the susceptibility to ecotropic murine leukemia virus.

EXAMPLE V

Preparation and Use of Antibodies to H-13 It is very difficult to make an H-13-containing fusion protein having the whole predicted protein (SEQ ID NO:2) since the predicted protein is highly hydrophobic, as shown in Figure 9. In order to predict antigenic epitopes present in the protein, therefore, the computer analysis was carried out using the program of PEPTIDESTRUCTURE (Jameson et al., CABIOS 4: 181-186 (1988)). Figure 10 shows the

antigenicity profile of the H-13 protein sequence. The DNA sequence encoding a highly antigenic portion (SEQ ID NO: 2, amino acid residues 309-367) was prepared by cutting with the restriction enzymes AccI and EcoRI yielding a 180 bp AccI-EcoRI fragment. This fragment of H13 cDNA was ligated to the cloning sites of pGEX-2T plasmid vector

(Pharmacia LKB Biotechnology), which can express antigens as fusion proteins with glutathione-S-transferase (GST), in the orientation that permit[s] the expression of the open reading frames (Smith, D.B. et al., Gene 67: 31-40 (1988)).

The fusion protein was induced by addition of isopropyl-beta-thiogalactopyranoside (IPTG) to cultures, and was purified using glutathione Sepharose 4B chromatography (Pharmacia LKB Biotechnology) (see Figure 11). The purified fusion protein injected intramuscularly and subcutaneously into rabbits with Freund's complete adjuvant to obtain

antisera.

The antisera are shown to bind specifically to the H-13 protein and epitopic fragments thereof.

Membrane proteins from human cells are prepared according to standard techniques and are separated by

polyacrylamide gel electrophoresis, an blotted onto

nitrocellulose for Western Blot analysis. The H-13 specific antibodies are shown to bind to proteins on these blots.

EXAMPLE VT

Genetic Mapping of H13

Chromosomal location of the H13 gene was determined using Chromosome Blots (Bios Corp., New Haven, Connecticut) containing DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytogenet. Cell Genet. 51:1025 (1989)). By comparison of which human chromosomes remained in the human-hamster hybrid cell and the expression of H13 cDNA, the H13 gene was mapped to human chromosome 13 (see Figure 12). Human genes (or diseases caused by mutations therein ) linked to chromosome 13 include: retinoblastoma,

osteosarcoma, Wilson's disease, Letterer-Siwe disease, DubinJohnson syndrome, clotting factor Vii and X, collagen IV α1 and α2 chains, X-ray sensitivity, lymphocyte cytosolic protein-1, carotid body tumor-1, propionyl CoA carboxylase (α subunit), etc.

EXAMPLE VII

Chimeric H13/ERR DNA and Protein Molecules Several chimeric molecules between the mouse ERR sequence and the human H13 sequence were produced, and have been designated Chimeral - ChimeralV). Specifically, four regions in H13 cDNA were substituted based on the use of common restriction sites as shown in Figure 13.

These DNA sequences were transiently transfected into Chinese hamster ovary (CHO) cell lines using pSG5 or pCDM8 expression vectors.

Two days later, these transfectants were tested for their ability to support E-MuLV infection. Cells were

infected with a recombinant Moloney E-MuLV designated 2BAG (Price, J. et al., Proc. Natl. Acad. Sci. USA 84:156-160

(1987)). This recombinant virus also contained β-galactosidase and neomycin phosphotransferase (neo^R) genes which provide a selectable marker and a detectable product. The cells were then grown under selective conditions in the presence of the antibiotic G418 at a concentration of 0.6 mg/ml to select neo^R-expressing transfectants. After two weeks, numbers of G418-resistant colonies were counted.

These results indicate that portion of the ERR gene essential for E-MuLV infection is located within NcoI-BstXI restriction sites, and included extracellular Domain 3.

Extracellular Domain 3 (as shown in the upper line of Figure 13) is the region of the receptor protein which is most diverse between the human and mouse sequences, as shown in Figure 14. The sequences in Figure 14 (derived from the sequences shown in Figure 1-3) were aligned using Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl. Acids Res. 12:387-395 (1984)).

Next, oligonucleotide-directed mutagenesis was employed to produce chimeric molecules containing individual amino acid substitutions within extracellular domain 3. These were transfected as above and the transfectant cells are tested for susceptibility to infection by E-MuLV as shown above.

The results of the above studies show that the human H13 molecule acquires ability to bind to E-MuLV by

substituting the native amino acid sequence with between 1 and 4 amino acids from corresponding positions in the murine ERR protein.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue

experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of

limitation. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: MEHYELO, DANIEL

YOSHIMOTO, TAKAYUKI

(ii) TITLE OF INVENTION: Human Retrovirus Receptor and ENA Coding

Therefor

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADCRESSEE: Browdy and Neimark

(B) STREET: 419 Seventh Street, N.W.

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20004

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.24

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Livnat, Shmuel

(B) REGISTRATION NUMBER: 33,949

(c) REFE RENCE/DOCKET NUMBER: MERUELO=1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202 628-5197

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1102 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1102 (xi) SEQUENCE INSCRIPTION: SEQ ID NO:1:

CCG GGC GCC ACC TTC GAC GAG CTG ATA GGC AGA CCC ATC GGG GAG TTC

Pro Gly Ala Thr Phe Asp Glu leu Ile Gly Arg Pro Ile Gly Glu Phe 48 1 5 10 15

TCA GGG ACA CAC ATG ACT CTG AAC GCC CCC GGC GTG CTG GCT GAA AAC 96 Ser Arg Thr His Met Thr Leu Asn Ala Pro Gly Val LEU Ala Glu Asn

20 25 30

CCC GAC ATA TTC GCA GTG ATC ATA ATT CTC ATC TTG ACA GGA CTT TTA 144 Pro Asp Ile Phe Ala Val Ile Ile Ile leu Ile Leu Thr Gly Leu Leu

35 40 45

ACT CTT GGT GTG AAA GAG TCG GCC ATG GTC AAC AAA ATA TTC ACT TGT 192 Thr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile Phe Thr Cys

50 55 60

ATT AAC GTC CTG GTC CTG GGC TTC ATA ATG GTG TCA GGA TTT GTG AAA 240 Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly Phe Val Lys

65 70 75 80

GGA TCS GTT AAA AAC TGG CAG CTC ACG GAG GAG GAT TTT GGG AAC ACA 288 Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr

85 90 95

TCA GGC CGT CTC TGT TTG AAC AAT GAC ACA AAA GAA GGG AAG CCC GGT 336 Ser Gly Arg Leu Cys Leu Asn Asn Asp Thr Lys Glu Gly Lys Pro Gly

100 105 110

GTT GGT GGA TTC ATG CCC TTC GGG TTC TCT GGT GTC CTG TOG GGG GCA 384 Val Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala

115 120 125

GOS ACT TGC TTC TAT GCC TTC GTG GGC TTT GAC TGC ATC GCC ACC ACA 432 Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr Thr

130 135 140

GGT GAA GAG GTG AAG AAC OCA CAG AAG GOC ATC CCC GTG GGG ATC GTG 480 Gly Glu Glu Val Lys Asn Pro Gln Lys Ala Ile Pro Val Gly Ile Val

145 150 155 160

GCG TCC CTC TTG ATC TGC TTC ATC GCC TAC TTT GGG GTG TOG GCT GCC 528 Ala Ser Leu leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala

165 170 175

CTC AOG CTC ATG ATG CCC TAC TTC TGC CTG GAC AAT AAC AGC CCC CTG 576 Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Asn Asn Ser Pro leu

180 185 190

CCC GAC GCC TTT AAG CAC GTG GGC TGG GAA GGT GCC AAG TAC GCA GTC 624 Pro Asp Ala Phe Lys His Val Gly Trp Glu Gly Ala Lys Tyr Ala Val

195 200 205 GCC GTG GGC TCC CTC TGC GCT CTT TCC GCC AGT CTT CTA GGT TCC ATG 672 Ala Val Gly Ser Leu Cys Ala leu Ser Ala Ser Leu Leu Gly Ser Met

210 215 220

TTT CCC ATG CCT OGG GTT ATC TAT GCC ATG GCT GAG GAT GGA CTG CTA 720 Phe Pro Met Pro Arg Val Ile Tyr Ala Mat Ala Glu Asp Gly Leu Leu

225 230 235 240

TTT AAA TTC TEA GCC AAC GTC AAT GAT AGG ACC AAA ACA CCA ATA ATC 768 Phe Lys Phe Leu Ala Asn Val Asn Asp Arg Thr Lys Thr Pro Ile Ile

245 250 255

GCC ACA TTA GCC TCG GGT GCC GTT GCT GCT GTG ATG GOC TTC CTC TTT 816 Ala Thr Lsu Ala Ser Gly Ala Val Ala Ala Val Met Ala Phe Leu Phe

260 265 270

CAC CTG AAG CAC TTG GTG GAC CTC ATG TCC ATT GGC ACT CTC CTG GCT 864 Asp Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala

275 280 285

TAC TCG TTG GTG GCT GCC TGT GTG TTG GTC TTA CGG TAC CAG CCA GAG 912 Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr Gin Pro Glu

290 295 300

CAG CCT AAC CTG GTA TAC CAG ATG GCC AGT ACT TCC GAC GAG TTA GAT 960 Gln Pro Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp Glu Leu Asp

305 310 315 320

CCA GCA GAC CAA AAT GAA TTG GCA AGC ACC AAT GAT TCC CAG CTG GGG 1008 Pro Ala Asp Gln Asn Glu Leu Ala Ser Thr Asn Asp Ser Gin Leu Gly

325 330 335

TTT TTA OA CAG GCa CAG ATG TTG TCT TIG AAA AOC ATA CTC TCA CCC 1056 Phe Leu Pro Glu Ala Glu Met Phe Ser Leu Lys Thr Ile Leu Ser Pro

340 345 350

AAA AAC ATG GAG CCT TOC AAA ATG TCT GGG CTA ATT GTG AAC COS G 1102 lys Asn Met Glu Pro Ser lys Ile Ser Gly Leu Ile Val Asn Pro

355 360 365

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 367 amino acids

(B) TYPE: amino acid

(D) TOFOLDGY: linear

(ii) M0I_ECULE TYPE: protein

(Xi) SEQUENCE DESC RIPTION: SEQ ID NO: 2:

Pro Gly Ala Thr Phe Asp Glu Leu Ile Gly Arg Pro Ile Gly Glu Phe

1 5 10 15

Ser Arg Thr His Met Thr Leu Asn Ala Pro Gly Val Lsu Ala Glu Asn

20 25 30 Pro Asp Ile Phe Ala Val Ile Ile Ile Leu Ile Leu Thr Gly Leu Leu 35 40 45

Thr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile Phe Thr Cys 50 55 60

Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly Phe Val Lys 65 70 75 80

Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr

85 90 95

Ser Gly Arg Leu Cys Leu Asn Asn Asp Thr Lys Glu Gly Lys Pro Gly

100 105 110

Val Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala

115 120 125

Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr Thr 130 135 140

Gly Glu Glu Val Lys Asn Pro Gln Lys Ala Ile Pro Val Gly Ile Val 145 150 155 160

Ala Ser leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala

165 170 175 Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Asn Asn Ser Pro Leu

180 185 190

Pro Asp Ala Phe Lys His Val Gly Trp Glu Gly Ala Lys Tyr Ala Val

195 200 205

Ala Val Gly Ser Leu Cys Ala Leu Ser Ala Ser Leu Leu Gly Ser Met 210 215 220

Phe E>ro Met Pro Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu Leu 225 230 235 240

Phe Lys Phe Leu Ala Asn Val Asn Asp Arg Thr Lys Thr Pro Ile Ile

245 250 255

Ala Thr Leu Ala Ser Gly Ala Val Ala Ala Val Met Ala Phe Leu Phe

260 265 270

Asp Leu Lys Asp Leu Val Asp Leu Met Ser lie Gly Thr Leu Leu Ala

275 280 285

Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr Gln Pro Glu 290 295 300

Gln Pro Asn leu Val Tyr Gln Met Ala Ser Thr Ser Asp Glu Leu Asp 305 310 315 320

Pro Ala Asp Gln Asn Glu Leu Ala Ser Thr Asn Asp Ser Gln Leu Gly

325 330 335 Phe Leu Pro Glu Ala Glu Met Phe Ser Leu Lys Thr Ile Leu Ser Pro

340 345 350

Lys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val Asn Pro

355 360 365

4) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2425 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYFE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 199. .2064

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GATTCCGCCC GCGTGCGCCA TOCCCT CAGC TAGCAGGTGT GAGAGGCTTT CTACCGGCGG 60

TCTCCACACA GCTCAACATC TTGCCGCCTC CTCCGAGCCT CAAGCTACCG TGGACTCTGC 120

TGTGGOGTCT TGGOCCCCAG GTGOGGATCC TOCOCAGTGA GAA GTCCCAC G AGTCTTACA 180

GCAGATTCGC TCAGCACA ATG GGC TGC AAA AAC CTG CTC GGT CTG GGC CAG 231

Met Gly Cys Lys Asn Leu Leu Gly Leu Gly Gln

1 5 10

CAG ATG CTG CGC CGG AAG GTG GTG GAC TGC AGC CGG GAG GAG 279

Gln Met Lsu Arg Arg lys Val Val Asp Cys Ser Arg Glu Glu Ser Arg

15 20 25

CTG TCC CGC TGC CTC AAC ACC TAT CAC CTG CTA GCT CTT GGG GTG GGC 327 Leu Ser Arg Cys Lsu Asn Thr Tyr Asp Lsu Val Ala Leu Gly Val Gly

30 35 40

AGC ACC TTG GGC GCT GCT CTC TAT GTC CIA GCC GCT GCC GTG GCC GGT 375 Ser Thr Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Ala Val Ala Arg

45 50 55

GAA AAT GCT GGC CCT GCC ATC GTC ATC TCC TTC TTG ATT GCT GCT CTC 423 Glu Asn Ala Gly Pro Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu

60 65 70 75

GCC TCC GTG CTG GCC GGC CTG TG C TAC GGC GAG TTT GGT GCC CGT GTC 471 Ala Ser Val Leu Ala Gly Lsu Cys Tyr Gly Glu Phe Gly Ala Arg Val

80 85 90 CCC AAG ACG GGC TCA GCC TAC CTC TAC AGC TAC GTG AOG GTC GGG GAG 519 Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu

95 100 105

CTT TGG GCC TTC ATC ACT GGC TGG AAC CTG ATT CTC TCC TAC ATC ATC 567 Leu Trp Ala Phe Ile Dir Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile

110 115 120

GGT ACT TCA AGC GTC GCA AGA GCC TCG ACT GOS ACT TTT GAC GAG CTG 615 Gly Thr Ser Ser Val Ala Arg Ala Trp Ser Ala Thr Phe Asp Glu Leu

125 130 135

ATA GGC AAG CCC ATC GGA GAG TTC TCA CCT CAG CAC ATC GOC CTG AAT 663 Ile Gly Lys Pro Ile Gly Glu Phe Ser Arg Gln His Met Ala Leu Asn 140 145 150 155

GCT CCT GGG GTC CTS GCC CAA ACC COG GAC ATA TTT GCT GTG ATT ATA 711 Ala Pro Gly Val Leu Ala Gln Thr Pro Asp Ile Phe Ala Val Ile Ile

160 165 170

ATT ATC ATC TTA ACA GGA CTG TTA ACT CTT GGC GTC AAG GAG TCA GCC 759 Ile Ile Ile Leu Thr Gly Leu Leu Thr leu Gly Val Lys Glu Ser Ala

175 180 185

ATC GTC AAC AAA ATT TTC ACC TGT ATC AAT CTC CTG CTC TTC TCC TTC 807 Met Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val Leu Cys Phe

190 195 200

ATC GTC GTG TCC GGG TTC GTC AAA GGC TCC ATT AAA AAC TCG CAG CTC 855 Ile Val Val Ser Gly Phe Val Lys Gly Ser Ile Lys Asn Trp Gln Leu

205 210 215

AOG GAG AAA AAT TTC TCC TCT AAC AAC AAC GAC ACA AAC GTG AAA TAC 903 Thr Glu Lys Asn Phe Ser Cys Asn Asn Asn Asp Thr Asn Val Lys Tyr 220 225 230 235

GCT GAG GGA GGG TTT ATC OCC TTT GGA TTC TCT GCT CTC CTC TCA GGG 951 Gly Glu Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly

240 245 250

GCA GOG ACC TCC TTT TAT GCC TTC GTC GGC TTT GAC TGC ATC GCC ACC 999 Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr

255 260 265

ACA GGG GAA GAA GTC AAG AAC CCC CAG AAG GCC ATT CCT GTG GGC ATC 1047 Thr Gly Glu Glu Val Lys Asn Pro Gln Lys Ala Ile Pro Val Gly Ile

270 275 280

GTG GOG TCC CTC CTC ATT TGC TTC ATA GOG TAC TTT GGC GTC TCC GCC 1095 Val Ala Ser Leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala

285 290 295

GCT CTC AOG CTC ATG ATC CCT TAC TTC TCC CTG GAC ATC GAC AGC COG 1143 Ala Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser Pro 300 305 310 315 CTG CCT GCT GCC TTC AAG CAC CAG GGC TGG GAA GAA GCT AAG TAC GCA 1191 Leu Pro Gly Ala Phe lys His Gin Gly Trp Glu Glu Ala Lys Tyr Ala

320 325 330

GTG GCC ATT GGC TCT CTC TGC GCA CTT TCC ACC AGT CTC CTA GGC TCC 1239 Val Ala Ile Gly Ser Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser

335 340 345 ATG TTT CCC ATG CCC CGA GTT ATC TAT GCC ATG GCT GAA GAT GGA CTA 1287 Met Phe Pro Met Pro Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu

350 355 360

CTG TTT AAA TTT TTG GCC AAA ATC AAC AAT AGG ACC AAA ACA CCC GTA 1335 Leu Phe Lys Fhe Leu Ala lys Ile Asn Asn Arg Thr lys Thr Pro Val

365 370 375

ATG GCC ACT GTG ACC TCA GGC GCC ATT GCT GCT GTG ATG GCC TTC CTC 1383 Ile Ala Thr Val Thr Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu 380 385 390 395

TTT GAA CTG AAG GAC CTG GTG GAC CTC ATG TCC ATT GGC ACT 1431 Phe Glu Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu

400 405 410

GCT TAC TCT TTG CTG GCT GCC TGT GTT TTG GTC TTA CGG TAC CAG CCA 1479 Ala Tyr Ser leu Val Ala Ala Cys Val leu Val Leu Arg Tyr Gin Pro

415 420 425

GAA CAA CCT AAT CTG GTA TAC CAG ATG GOC AGA AOC ACC GAG GAG CTA 1527 Glu Gln Pro Asn Leu Val Tyr Gln Met Ala Arg Thr Thr Glu Glu Leu

430 435 440

GAT OGA GTA GAT CAG AAT GAG CTG GTG ACT GCC ACT GAA TCA 1575 Asp Arg Val Asp Gln Asn Glu Leu Val Ser Ala Ser Glu Ser Gln Thr

445 450 455

GGC TTT TTA CCG GTA GCC GAG AAG TTT TCT CTG AAA TCC ATC CTC TCA 1623 Gly Phe Leu Pro Val Ala Glu Lys Fhe Ser Leu Lys Ser Ile Leu Ser 460 465 470 475

CCC AAG AAC GTG GAG CCC TCCAAATTG TCA GGG CTA ATT GTG AAC ATT 1671 Pro Lys Asn Val Glu Pro Ser Lys Phe Ser Gly Leu lLe Val Asn ILe

480 485 490

TCA GCC GGC CTC CTA GCC GCT CTT ATC ATC ACC GTG TGC ATT GTG GCC 1719 Ser Ala Gly Leu Leu Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala

495 500 505

GTG CTTGGAAGA CAG GCC CTG GCCGAA GGG ACA CTG TGG GCA GTC TTT 1767 Val Leu Gly Arg Glu Ala Leu Ala Glu Gly Thr Leu Trp Ala Val Phe

510 515 520

GTA ATG ACA C4GGTGAGTG CTG CTC TGC ATG CTG GTG ACA GGC ATC ATC 1815 Val Met Thr Gly Ser Val Leu Leu Cys Met Leu Val Thr Gly Ile Ile

525 530 535 T GG AGA CAG CCT GAG AGC AAG ACC AAG CTC TCA TTT AAG GTA CCC TTT 1863 Trp Arg Gin Pro Glu Ser lys Thr lys Leu Ser Phe lys Val Pro Phe 540 545 550 555

GTC CCC CTA CTT CCT GTC TTG AGC ATC TTC GTG AAC ATC TAT CTC ATG 1911 Val Pro Val Lsu Pro Val Leu Ser Ile Phe Val Asn Ile Tyr Leu Mst

560 565 570

ATG CAG CTG GAC CAG GGC AOG TGG GTC OGG TTT GCA GTG TG G ATG CTG 1959 Met Gln Leu Asp Gln Gly Thr Trp Val Arg Phe Ala Val Trp Met Leu

575 580 585 ATA GCT TTC ACC ATC TAT TTC GCT TAT GGG ATC TGG CAC ACT GAG GAA 2007 lie Gly Phe Thr Ile Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu

590 595 600

GOS TCC CTG GCT GCT GGC CAG GCA AAG ACT CCT GAC AGC AAC TTG GAC 2055 Ala Ser Leu Ala Ala Gly Gin Ala Lys Thr Pro Asp Ser Asn Leu Asp

605 610 615

CAG TG C AAA TG AOGTGCAG CCCCACCCAC CAGGGTGACA GOGCTTGAOG 2104

Gln Cys Lys

620

GGTG CCOGTA GAAGCCTGGG ACCCTCACAA TCTCTCCACT CATGCCTCAG CATCAGCTCA 2164

CSCCCCCAAT GTCACCAAAG CTGGTTTG CT GCCAGCTOCT CAGATCCTG G TCATITCTGG 2224

ACACTCCCTT GGTTTACTCA TCTCXXTCTG AACAAAGAAA GCAGCCCITC TCCTTGCCGG 2284

COGGOOGGGC GCTTCGCTGC TGCGGCCCCCA GCAGAAGGGA GGCCOCCTTG TCCTCTCACT 2344

TGGGAAGCAG GCCTCOCTCC CTCCCTGGGA C CACCCTGGC ATCGCCCATG TGCACACTCC 2404

ACATGGCTAG TGAGCCTCTC C 2425

(5) INFOEMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 622 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Gly Cys Lys Asn Leu Leu Gly Leu Gly Gln Gln Met Leu Arg Arg 1 5 10 15

Lys Val Val Asp Cys Ser Arg Glu Glu Ser Arg Leu Ser Arg Cys Leu

20 25 30 Asn Thr Tyr Asp Leu Val Ala Leu Gly Val Gly Ser Thr Leu Gly Ala 35 40 45

Gly Val Tyr Val Leu Ala Gly Ala Val Ala Arg Glu Asn Ala Gly Pro 50 55 60

Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Leu Ala 65 70 75 80

Gly Leu Cys Tyr Gly Glu Fhe Gly Ala Arg Val E>ro Lys Thr Gly Ser

85 90 95

Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile

100 105 110

Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val

115 120 125

Ala Arg Ala Trp Ser Ala Thr Fhe Asp Glu Leu Ile Gly Lys Pro Ile 130 135 140

Gly Glu Phe Ser Arg Gln His Met Ala Leu Asn Ala Pro Gly Val Leu 145 150 155 160

Ala Gln Thr Pro Asp Ile Phe Ala Val Ile Ile Ile Ile Ile Leu Thr

165 170 175

Gly Leu Leu Thr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile

180 185 190

Fhe Thr Cys Ile Asn Val Leu Val leu Cys Phe lie Val Val Ser Gly

195 200 205

Fhe Val Lys Gly Ser Ile Lys Asn Trp Gln Leu Thr Glu Lys Asn Phe 210 215 220

Ser Cys Asn Asn Asn Asp Thr Asn Val Lys Tyr Gly Glu Gly Gly Phe 225 230 235 240

Met Pro Phe Gly Fhe Ser Gly Val Leu Ser Gly Ala Ala Thr Cys Phe

245 250 255

Tyr Ala Fhe Val Gly Phe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val

260 265 270

Lys Asn Pro Gln Lys Ala Ile E>ro Val Gly Ile Val Ala Ser Leu Leu

275 280 285

Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala Leu Thr Leu Met 290 295 300

Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser Pro Leu Pro Gly Ala Phe 305 310 315 320

Lys His Gln Gly Trp Glu Glu Ala Lys Tyr Ala Val Ala Ile Gly Ser

325 330 335 Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser Met Phe Pro Met Pro 340 345 350

Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu Leu Phe Lys Fhe Leu

355 360 365

Ala Lys Ile Asn Asn Arg Thr Lys Thr Pro Val Ile Ala Thr Val Thr 370 375 380

Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu Phe Glu Leu Lys Asp 385 390 395 400

Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val

405 410 415

Ala Ala Cys Val Leu Val Leu Arg Tyr Gln Pro Glu Gln Pro Asn Leu

420 425 430

Val Tyr Gln Met Ala Arg Thr Thr Glu Glu Leu Asp Arg Val Asp Gln

435 440 445

Asn Glu Leu Val Ser Ala Ser Glu Ser Gln Thr Gly Fhe Leu Pro Val 450 455 460

Ala Glu Lys Phe Ser Leu Lys Ser Ile Leu Ser Pro Lys Asn Val Glu 465 470 475 480

Pro Ser Lys Phe Ser Gly Leu Ile Val Asn Ile Ser Ala Gly Leu Leu

485 490 495

Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala Val Leu Gly Arg Glu

500 505 510

Ala Leu Ala Glu GlyThr Leu Trp Ala Val Phe Val Met Thr Gly Ser

515 520 525

Val Leu Leu Cys Met Leu Val Thr Gly lle Ile Trp Arg Gln Pro Glu 530 535 540

Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe Val Pro Val Leu Pro 545 550 555 560

Val Leu Ser Ile Phe Val Asn Ile Tyr Leu Met Met Gln Leu Asp Gln

565 570 575

Gly Thr Trp Val Arg Phe Ala Val Trp Met Leu Ile Gly Phe Thr Ile

580 585 590

Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu Ala Ser Leu Ala Ala

595 600 605

Gly Gln Ala Lys Thr Pro Asp Ser Asn Leu Asp Gln Cys Lys

610 615 620 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2397 base pairs

(B) TYFE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:410..1768

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GGGICTCTTT CCTCATCGCT GCCCTGGCCT CGGTEATCGC CGGCCCC TTGC TATGCTGAAT 60

TTGGGGCCCG ACTACCCAAG ACTGGATGTG CCTATCTATA CACTTAOGTC ACGGTCGGAG 120 AGCTGTGGGC CTTCATCACT GGCTGGAATC TCATCCTGTC ATATGTCATA GGTACGTCCA 180 GTGTCGCAAG AGCATGGACT GGCACCTTTG ACGAACTTCT TAATAAACAG ATTGGCCAGT 240

TTTTCAAAAC GTACTTCAAA ATGAATTACA CTGGTCTGGC AGAGEATCCA GACTTCTTTG 300

CCGTGTGCCT TGTATTACTC CTGGCAGGTC TTTTATCTTT TGGAGTAAAA GAGTCTGCTT 360

GGGTGAATAA ATTTTTACAG CTATTAATAT CCTGGTOCTT CTCTTTGTC ATG GTG 415

Met Val

1

GCT GGG TTT GTG AAA GGA AAT GTG GCT AAC TGG AAG ATC ACT GAA GAG 463 Ala Gly Fhe Val Lys Gly Asn Val Ala Asn Trp lys Ile Ser Glu Glu

5 10 15

TTT CTG AAAMT ATA TCA GCA ACT GCT ACAGAA CX-A CCT AAC 511

Fhe Leu Lys Asn Ile Ser Ala Ser Ala Arg Glu Pro Pro Ser Glu Asn

20 25 30

GGA ACA AGC ATCTAC GGG GCT GGC GGC TTT ATG CCC TAT GGC TTT ACA 559 Gly Thr Ser Ile Tyr Gly Ala Gly Gly Fhe Met Pro Tyr Gly Phe Thr

35 40 45 50

GGG ACG TTG GCT GCT GCT GCAACG TGC TTT TAT GCC TTT GTG GGC TTT 607

Gly Thr Leu Ala Gly Ala Ala Thr Cys Fhe Tyr Ala Fhe Val Gly Phe

55 60 65

GAC TGC ATT GCA ACA ACC GCT GAA GAG GTT CGG AAT CCA CAA AAG GGG 655 Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Arg Asn Pro Gin Lys Ala

70 75 80 ATC CCC ATC GGA ATA GTS ACG TCC TEA CIT GTC TGC TTT ATG GCT TAC 703 Ile Pro Ile Gly Ile Val Thr Ser Leu Leu Val Cys Phe Met Ala Tyr

85 90 95

TTT GGG GTT TCT GCA GCT TEA AOG CTT ATC ATC CCT TAC TAC CTC CTG 751 Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Tyr Leu Leu

100 105 110

GAT GAG AAA ACT CCA CTC CCA CTC GOG TTT GAG TAT GTC AGA TCG GGC 799 Asp Glu Lys Ser Pro Leu Pro Val Ala Fhe Glu Tyr Val Arg Trp Gly

115 120 125 130

CCC GCC AAA TAC GTT GTC GCA GCA GGC TCC CTC TGC GCC TEA TCA ACA 847 Pro Ala Lys Tyr Val Val Ala Ala Gly Ser Leu Cys Ala Leu Ser Thr

135 140 145

ACT CTT CTT GGA TCC ATT TTC CCA ATC CCT CCT CTA ATC TAT GCT ATC 895 Ser Leu Leu Gly Ser Ile Fhe Fro Met Pro Arg Val Ile Tyr Ala Met

150 155 160

GCG GAG GAT GGG TTG CTT TTC AAA TCT CTA GCT CAA ATC AAT TCC AAA 943 Ala Glu Asp Gly Leu Leu Phe Lys Cys Leu Ala Gln Ile Asn Ser Lys

165 170 175

AOG AAG ACA CCA GTA ATT GCT ACT TTG TCA TOG GCT GCA GTG GCA GCT 991 Thr Lys Thr Pro Val Ile Ala Thr Leu Ser Ser Gly Ala Val Ala Ala

180 185 190

GTC ATC GCC TTT CTT TTT GAC CTG AAG GCC CTC GTC GAC ATC ATC TCT 039 Val Met Ala Fhe Leu Fhe Asp Leu Lys Ala Leu Val Asp Met Met Ser

195 200 205 210

AIT GGC ACC CTC ATC GCC TAC TCT CTG GTC GCA GCC TCT GTC CTT ATT 087 Ile Gly T-hr Leu Met Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Ile

215 220 225

CTC AGG TAC CAA CCT GGC TTG TCT TAC GAG CAG CCC AAA TAC ACC CCT 1135 Leu Arg Tyr Gln Pro Gly Leu Cys Tyr Glu Gln Pro Lys Tyr Thr Pro

230 235 240

GAG AAA GAA ACT CTG GAA TCA TGT ACC AAT GOG ACT TTG AAG AGC GAG 1183 Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys Ser Glu

245 250 255

TCC CAG CTC ACC ATC CTG CAA GGA CAG GCT TTC AGC CTA CGA ACC CTC 1231 Ser Gln Val Thr Met Leu Gln Gly Gln Gly Phe Ser Leu Arg Thr Leu

260 265 270

TTC AGC CCC TCT GCC CTC CCC ACA CGA CAG TOG ECT TCC CTT GTG AGC 1279 Phe Ser Pro Ser Ala Leu Pro Thr Arg Gln Ser Ala Ser Leu Val Ser

275 280 285 290

TTT CTC CTG GGA TTC CTG GCT TTC CTC ATC CTC GGC TTG ACT ATT CTA 1327 Phe Leu Val Gly Fhe Leu Ala Phe Leu Ile Leu Gly Leu Ser Ile Leu

295 300 305 ACC ACG TAT GGC GTC CAG GCC ATT GCC AGA CTG GAA GCC TGG AGC CTG 1375 Thr Thr Tyr Gly Val Gin Ala lle Ala Arg Leu Glu Ala Trp Ser Leu

310 315 320

GCT CTT CTC GCC CTG TTC CTT GTC CTC TGC GCT GCC GTC ATT CTG ACC 1423 Ala Leu Leu Ala Lsu Phe Leu Val Leu Cys Ala Ala Val Ile Leu Thr

325 330 335

ATT TGG AGG CAG CCA CAG AAT CAG CAA AAA CTA GCC TTC ATG GTC COG 1471 Ile Trp Arg Gln Pro Gln Asn Gln Gln lys Val Ala Fhe Met Val Pro

340 345 350

TTC TEA CCG TTT CTG CCG GCC TTC AGC ATC CTG GTC AAC ATT TAC TTG 1519 Phe Leu Pro Phe Leu Pro Ala Phe Ser Ile Lsu Val Asn Ile Tyr Leu

355 360 365 370

ATG GTG CAG TEA AGT GOG GAC ACT TGG ATC AGA TTC AGC ATC TGG ATG 1567 Met Val Gln Leu Ser Ala Asp Thr Trp Ile Arg Fhe Ser Ile Trp Met

375 380 385

GCG CTT GGC TTT CTG ATG TAT TTG GCC TAT GGC AIT AGA CAC AGC TTG 1615 Ala Leu Gly Phe Leu Ile Tyr Fhe Ala Tyr Gly Ile Arg His Ser Leu

390 395 400

GAG GCT AAC CCC AGG GAC GAA GAA GAC GAT GAG GAT GCC TTT TCA GAA 1663 Glu Gly Asn Pro Arg Asp Glu Glu Asp Asp Glu Asp Ala Fhe Ser Glu

405 410 415

AAC ATC AAT GTA GCA ACA GAA GAA AAG TCC GTC ATG CAA GCA AAT GAC 1711 Asn Ile Asn Val Ala Thr Glu Glu lys Ser Val Met Gln Ala Asn Asp

420 425 430

CAT CAC CAA AGA AAC CTC AGC TEA CCT TTC ATA CTT CAT GAA AAG ACA 1759 His His Gln Arg Asn Leu Ser Leu Pro Fhe Ile Leu His Glu Lys Thr

435 440 445 450

ACT GAA TCT TGATGCT GGC C CTOGCTCTT ACCACGCATA CCTTAACAAT 1808

Ser Glu Cys

GACTACACTG TGGCOGGATG CCACCATCCT GCTGGGCTCT CGTGGGTC TG CTGTGGACAT 1868

GGCTTGCCTA ACTTCTACTT CXTCCTCCAG ACAGCTTCTC TTCAGATGCT GGATTCTG TG 1928

TCTGAGGAGA CTGCCTGAGA GCACTCCTCA GCEATATGTA TCOCCAAAAC ACTATGTCOG 1988

TGTGCCTACA TGTATGTCTG CGATGTGACT GTTCAATGTT CTCCCTEATT ACTCTGTGAC 2048 ATΆΆTTCCAG CATGCTAATT GGTGGCATAT ACTGCACACA CTAGTAAACA CTATATTGCT 2108

GAATAGAGAT CTATTCTGTA TATGTCCTAG GTGGCTGGGG AAATAGTGGT GGTTTCTTTA 2168 TTAGCTATAT GACCATCAGT TTGGACATAC TGAAATGCCA TCCCCTCTCA GGATGTTTAA 2228 CAGTGGTCAT GGGTGGGGAA GGGATAAGGA ATGGGCATTG TCTATAAATT CTAATG CATA 2288 TΆTCCTΓCTC CTACTTGCTA AGACAGCΠT CTTAAACGGC CAGGCAGAGT GTTTCTTTCC 2348 TCTGTATG AC AAGATGAAGA GGTAGTCTGT GGCTGGAGAT GGCCAATCC 2397

(7) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 453 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Val Ala Gly Phe Val Lys Gly Asn Val Ala Asn Trp Lys Ile Ser 1 5 10 15

Glu Glu Phe Leu Lys Asn Ile Ser Ala Ser Ala Arg Glu Pro Pro Ser

20 25 30

Glu Asn Gly Thr Ser Ile Tyr Gly Ala Gly Gly Phe Met Pro Tyr Gly

35 40 45

Phe Thr Gly Thr Leu Ala Gly Ala Ala Thr Cys Phe Tyr Ala Phe Val 50 55 60

Gly Phe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Arg Asn Pro Gln 65 70 75 80

Lys Ala Ile Pro Ile Gly Ile Val Thr Ser Leu Leu Val Cys Phe Met

85 90 95

Ala Tyr Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Tyr

100 105 110

Leu Leu Asp Glu Lys Ser Pro Leu Pro Val Ala Phe Glu Tyr Val Arg

115 120 125

Trp Gly Pro Ala Lys Tyr Val Val Ala Ala Gly Ser Leu Cys Ala Leu 130 135 140

Ser Thr Ser Leu Leu Gly Ser Ile Phe Pro Met Pro Arg Val Ile Tyr 145 150 155 160

Ala Met Ala Glu Asp Gly Leu Leu Phe Lys Cys Leu Ala Gln Ile Asn

165 170 175

Ser Lys Thr Lys Thr Pro Val Ile Ala Thr Leu Ser Ser Gly Ala Val

180 185 190

Ala Ala Val Met Ala Phe Leu Phe Asp Leu Lys Ala leu Val Asp Met

195 200 205 Met Ser Ile Gly Thr Leu Met Ala Tyr Ser Leu Val Ala Ala Cys Val 210 215 220

Leu Ile Leu Arg Tyr Gln Pro Gly Leu Cys Tyr Glu Gln Pro Lys Tyr 225 230 235 240

Thr Pro Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys

245 250 255

Ser Glu Ser Gln Val Thr Met Leu Gln Gly Gln Gly Phe Ser Leu Arg

260 265 270

Thr Leu Phe Ser Pro Ser Ala Leu Pro Thr Arg Gln Ser Ala Ser Leu

275 280 285

Val Ser Fhe Leu Val Gly Phe Leu Ala Phe Leu Ile Leu Gly Leu Ser 290 295 300

Ile Leu Thr Thr Tyr Gly Val Gln Ala Ile Ala Arg Leu Glu Ala Trp 305 310 315 320

Ser Leu Ala Leu Leu Ala Leu Phe Leu Val Leu Cys Ala Ala Val Ile

325 330 335

Leu Thr Ile Trp Arg Gln Pro Gln Asn Gln Gln Lys Val Ala Phe Met

340 345 350

Val Pro Phe Leu Pro Phe Leu Pro Ala Phe Ser Ile Leu Val Asn Ile

355 360 365

Tyr Leu Met Val Gln Leu Ser Ala Asp Thr Trp Ile Arg Phe Ser Ile 370 375 380

Trp Met Ala Leu Gly Fhe Leu Ile Tyr Phe Ala Tyr Gly Ile Arg His 385 390 395 400

Ser Leu Glu Gly Asn Pro Arg Asp Glu Glu Asp Asp Glu Asp Ala Phe

405 410 415

Ser Glu Asn Ile Asn Val Ala Thr Glu Glu Lys Ser Val Met Gln Ala

420 425 430

Asn Asp His His Gln Arg Asn Leu Ser Leu Pro Phe Ile Leu His Glu

435 440 445

Lys Thr Ser Glu Cys

450

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2157 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cENA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 148..2034

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

C GATCCTGCC GGAGCCCCGC C GCCGCOGGC TTGGATTCTG AAACCTTCCT TCTATCCCTC 60 CTGAGACATC TTTGCTGCAA GATCGAGGCT GTCCTCTGCT GAGAAGGTGG TGAGGCTTCC 120

OGTCATATTC CAGCTCTGAA CAGCAAC ATG GGG TGC AAA GTC CTG CTC AAC ATT 174

Met Gly Cys Val Leu Leu Asn Ile Ile

5

GGG CAG CAG ATC CTC OGG OGG AAG GTC GTG GAC TCT AGC GGG GAG GAG 222 Gly Gln Gln Met Leu Arg Arg Lys Val Val Asp Cys Ser Arg Glu Glu

10 15 20 25

AOG OGG CTC TCT GGC TGC CTC AAC ACT TTT GAT CTG GTC GOC CTC GGG 270 Thr Arg Leu Ser Arg Cys Leu Asn Thr Fhe Asp Leu Val Ala Leu Gly

30 35 40

GIG GGC AGC ACA CTC GCT GCT GCT GTC TAC GTC CTG GCT GGA GCT GTC 318 Val Gly Ser Thr Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Ala Val

45 50 55

GCC CCT GAG AAT GCA GGC CCT GCC AIT GTC ATC TCC TTC CTC ATC GCT 366 Ala Arg Glu Asn Ala Gly Pro Ala Ile Val Ile Ser Phe Leu Ile Ala

60 65 70

GCG CTG GCC TCA GTG CTG GCT GGC CTG TCC TAT GGC GAG TTT GCT GCT 414 Ala Leu Ala Ser Val Leu Ala Gly Leu Cys Tyr Gly Glu Phe Gly Ala

75 80 85

CGG GTC CCC AAG ACS GGC TCA GCT TAC CTC TAC AGC TAT GTC ACC GTT 462 Arg Val Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val

90 95 100 105

GGA GAG CTC TCG GCC TTC ATC ACC GGC TCG AAC TEA ATC CTC TCC TAC 507 Gly Glu Leu Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr

110 115 120

ATC ATC GCT ACT TCA AGC CTA GOS AGG GCC TCG AGC GCC ACC TTC GAC 558 He Ile Gly Thr Ser Ser Val Ala Arg Ala Trp Ser Ala Thr Phe Asp

125 130 135

GAG CTG ATA GGC AGA CCC ATC GGG GAG TEC TCA OGG ACA CAC ATC ACT 606 Glu Leu Ile Gly Arg Pro Ile Gly Glu Phe Ser Arg Thr His Met Thr

140 145 150 CTG AAC GCC CCC GGC GTG CTG GCT GAA AAC CCC GAC ATA TTC GCA GTG 654

Leu Asn Ala Pro Gly Val leu Ala Glu Asn Pro Asp Ile Phe Ala Val

155 160 165

ATC ATA ATT CTC ATG TTG ACA GGA CTT TEA ACT CTT GCT GTG AAA GAG 702 Ile Ile Ile Leu Ile Leu Thr Gly Leu Leu Thr Lsu Gly Val Lys Glu

170 175 180 185

TCG GCC ATG GTG AAC AAA ATA TTC ACT TGT ATT AAC CTC CTG GTC CTG 750 Ser Ala Met Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val Leu

190 195 200

GGC TTC ATA ATG GTG TCA GGA TTT GTG AAA GGA TCG GTT AAA AAC TG G 798 Gly Fhe Ile Met Val Ser Gly Fhe Val Lys Gly Ser Val Lys Asn Trp

205 210 215

CAG CTG ACG CAG GAG GAT TTT GGG AAC ACA TCA GGC CCT CT C TGT TTG 846

Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr Ser Gly Arg Leu Cys Leu

220 225 230

AAC AAT GAC ACA AAA GAA GGG AAG CCC GCT CTT GCT GGA TTG ATG CCC 894 Asn Asn Asp Thr Lys Glu Gly Lys Pro Gly Val Gly Gly Fhe Met Pro

235 240 245

TTC GGG TTC TCT GCT GTC CTG TCG GGG GCA GOG ACT TGC TTC TAT GCC 942

P he Gly Phe Ser Gly Val Leu Ser Gly Ala Ala Thr Cys Phe Tyr Ala

250 255 260 265

TTG GTG GGC TTT GΑC TGC ATG GCC ACC ACA GCT GAA GAG GTG AAG AAC 990 Phe Val Gly Fhe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val lys Asn

270 275 280

CCA CAG AAG GCC ATG CCC GTG GGG ATG GTG G03 TCC CTC TTG ATG TGC 1038

Pro Gin Lys Ala Ile Pro Val Gly Ile Val Ala Ser Leu Leu Ile Cys

285 290 295

TTC ATC GCC TAC TTT GGG GTG TCG GCT GCC CTC AOG CTC ATG ATG CCC 1086 Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro

300 305 310

TAC TTC TGC CTG GAC AAT AAC AGC CCC CTG CCC GAC GCC TTT AAG CAC 1134 Tyr Phe Cys Leu Asp Asn Asn Ser Pro Leu Pro Asp Ala Phe Lys His

315 320 325

GTG GGC TGG GAA GCT GCC AAG TAC GCA GTG GCC GTG GGC TCC CTC TG C 1182 Val Gly Trp Glu Gly Ala Lys Tyr Ma Val Ala Val Gly Ser Leu Cys

330 335 340 345

GCT CTT TCC GCC AGT CTT CTA GCT TCC ATG TTT CCC ATG CCT OGG GTT 1230 Ala Lsu Ser Ala Ser Leu Leu Gly Ser Met Phe Fro Met Pro Arg Val

350 355 360

ATC TAT GCC ATG GCT GAG GAT GGA CTG CTA TTT AAA TTC TEA GCC AAC 1278 Ile Tyr Ala Mst Ala Glu Asp Gly Leu Leu Phe Lys Phe Leu Ala Asn

365 370 375 GTC AAT GAT AGG ACC AAA ACA CCA ATA ATG GCC ACA TEA GCC TOS GGT 1326 Val Asn Asp Arg Thr lys Thr Pro Ile Ile Ala Thr Leu Ala Ser Gly

380 385 390

GCC GTT GCT GCT GTG ATG GCC TTC CTC TTT GAC CTG AAG GAC TTG GTC 1374 Ala Val Ala Ala Val Met Ala Fhe Leu Fhe Asp Lsu Lys Asp Lsu Val

395 400 405

GAC CTC ATG TCC ATT GGC ACT CTC CTG GCT TAC TOG TTG GTG GCT GCC 1422 Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val Ala Ala

410 415 420 425

TCT GTG TTG GTC TEA OGG TAC CAG CCA GAG CAG CCT AAC CTG GTA TAC 1470 Cys Val Leu Val Lsu Arg Tyr Gin Pro Glu Gln Pro Asn Lsu Val Tyr

430 435 440

CAG ATG GCC ACT ACT TCC GAC GAG TEA GAT CCA GCA GAC CAA AAT GAA 1518 Gln Mst Ala Ser Thr Ser Asp Glu Leu Asp Pro Ala Asp Gin Asn Glu

445 450 455

TTG GCA AGC ACC AAT GAT TCC CAG CTG GGG TTT TEA CCA GAG GCA GAG 1566 Leu Ala Ser Thr Asn Asp Ser Gin Leu Gly Phe Leu Pro Glu Ala Glu

460 465 470

ATG TEC TCT TTG AAA ACC ATA CTC TGA OCC AAA AAC ATG GAG CCT TCC 1614 Met Fhe Ser Leu Lys Thr Ile Leu Ser Pro Lys Asn Met Glu Pro Ser

475 480 485

AAA ATG TCT GGG CTA ATT GIG AAC ATT TCA ACC AGC CTT ATA GCT CTT 1662 Lys lie Ser Gly leu Ile Val Asn Ile Ser Thr Ser Leu Ile Ala Val

490 495 500 505

CTC ATG ATG ACC TTC TG C ATT GTG ACC GTG CTT GGA AGG GAG GCT CTC 1710 Leu Ile Ile Thr Phe Cys Ile Val Thr Val Leu Gly Arg Glu Ala Lsu

510 515 520

ACC AAA GGG GOG CTG TG G GCA GTC TTT CTG CTC GCA GGG TCT GCC CTC 1758 Thr Lys Gly Ala leu Trp Ala Val Phe Lsu Lsu Ala Gly Ser Ala Leu

525 530 535

CTC TCT GCC GTG GTG AGG GGC GTC ATG TGG AGG CAG CCC GAG AGC AAG 1806 Leu Cys Ala Val Val Thr Gly Val Ile Trp Arg Gln Pro Glu Ser Lys

540 545 550

ACC AAG CTC TCA TTT AAG GTT CCC TTC CTG CCA GTG CTC CCC ATG CTG 1854 Thr Lys Lsu Ser Phe Lys Val Pro Phe Lsu Pro Val Leu Pro Ile Lsu

555 560 565

AGC ATC TTC CTG AAC GTC TAT CTC ATG ATG CAG CTG GAC CAG GGC ACC 1902 Ser Ile Phe Val Asn Val Tyr Leu Met Met Gln Lsu Asp Gln Gly Thr

570 575 580 585

TGG CTC OGG TTT GCT GTG TGG ATG CTG ATA GGC TTC ATC ATC TAC TTT 1950 Trp Val Arg Fhe Ala Val Trp Met Leu Ile Gly Fhe Ile Ile Tyr Fhe

590 595 600 GGC TAT GGC CTC TCG CAC AGC GAG GAG GOS TCC CTC GAT GCC GAC CAA 1998 Gly Tyr Gly Leu Trp His Ser Glu Glu Ala Ser Leu Asp Ala Asp Gln

605 610 615

GCA AGG ACT CCT GAC GGC AAC TTC GAC CAG TGC AAG TGAOGCACAG 2044 Ala Arg Thr Pro Asp Gly Asn Leu Asp Gln Cys Lys

620 625

CCCC CCGOC C GGAGGTGGC AGCAGCOCOG AGC4GA0GC0C OCAGAGGAGC GGGAGGCAOC 2104 CCACCCTCCC CACCAGTGCA ACAGAAACCA CCTGOCTCCA CAOOCTCACT GCA 2157

(2) INFORMATION FΌR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENCTH: 629 amino acids

(B) TYFE: amino acid

(D) TOPOLOGY: linear

(ii) MOIECUIE TYFE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Gly Cys Val Leu Leu Asn Ile Ile Gly Gln Gln Met Leu Arg Arg

5 10 15

Lys Val Val Asp Cys Ser Arg Glu Glu Thr Arg Leu Ser Arg Cys Leu

20 25 30

Asn Thr Fhe Asp Leu Val Ala Leu Gly Val Gly Ser Thr Leu Gly Ala

35 40 45

Gly Val Tyr Val Leu Ala Gly Ala Val Ala Arg Glu Asn Ala Gly Pro

50 55 60

Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Leu Ala

65 70 75 80

Gly Leu Cys Tyr Gly Glu Fhe Gly Ala Arg Val Pro Lys Thr Gly Ser

85 90 95

Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile

100 105 110

Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val

115 120 125

Ala Arg Ala Trp Ser Ala Thr Fhe Asp Glu Leu Ile Gly Arg Pro Ile

130 135 140

Gly Glu Phe Ser Arg Thr His Met Thr Leu Asn Ala Pro Gly Val Leu

145 150 155 160 Ala Glu Asn Pro Asp Ile Phe Ala Val Ile Ile Ile Leu Ile Leu Thr 165 170 175

Gly Leu Leu Thr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile

180 185 190

Fhe Thr Cys Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly

195 200 205

Phe Val Lys Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe 210 215 220

Gly Asn Thr Ser Gly Arg Leu Cys Leu Asn Asn Asp Thr Lys Glu Gly 225 230 235 240

Lys Pro Gly Val Gly Gly Fhe Met Pro Phe Gly Phe Ser Gly Val Leu

245 250 255

Ser Gly Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile

260 265 270

Ala Thr Thr Gly Glu Glu Val Lys Asn Pro Gln Lys Ala Ile Pro Val

275 280 285

Gly Ile Val Ala Ser Leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val 290 295 300

Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Asn Asn 305 310 315 320

Ser Pro Leu Pro Asp Ala Phe Lys His Val Gly Trp Glu Gly Ala Lys

325 330 335

Tyr Ala Val Ala Val Gly Ser leu Cys Ala Leu Ser Ala Ser Leu Leu

340 345 350

Gly Ser Met Phe Pro Met Pro Arg Val Ile Tyr Ala Met Ala Glu Asp

355 360 365

Gly Leu Leu Phe Lys Phe Leu Ala Asn Val Asn Asp Arg Thr Lys Thr 370 375 380

Pro Ile Ile Ala Thr Leu Ala Ser Gly Ala Val Ala Ala Val Met Ala 385 390 395 400 Phe Leu Phe Asp Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr

405 410 415 Leu Leu Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr

420 425 430

Gln Pro Glu Gln Pro Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp

435 440 445

Glu Leu Asp Pro Ala Asp Gln Asn Glu Leu Ala Ser Thr Asn Asp Ser 450 455 460 Gln Leu Gly Phe Leu Pro Glu Ala Glu Met Phe Ser Leu Lys Thr Ile 465 470 475 480 Leu Ser Pro Lys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val

485 490 495

Asn Ile Ser Thr Ser Leu Ile Ala Val Leu Ile Ile Thr Phe Cys Ile

500 505 510

Val Thr Val Leu Gly Arg Glu Ala Leu Thr Lys Gly Ala Leu Trp Ala

515 520 525

Val Phe Leu Leu Ala Gly Ser Ala Leu Leu Cys Ala Val Val Thr Gly 530 535 540

Val Ile Trp Arg Gln Pro Glu Ser Lys Thr Lys Leu Ser Phe Lys Val 545 550 555 560

Pro Fhe Leu Pro Val Leu Pro Ile Leu Ser Ile Phe Val Asn Val Tyr

565 570 575

Leu Met Met Gln Leu Asp Gln Gly Thr Trp Val Arg Phe Ala Val Trp

580 585 590

Met Leu Ile Gly Phe Ile Ile Tyr Fhe Gly Tyr Gly Leu Trp His Ser

595 600 605

Glu Glu Ala Ser Leu Asp Ala Asp Gln Ala Arg Thr Pro Asp Gly Asn 610 615 620

Leu Asp Gln Cys Lys

625

Claims

WHAT IS CLAIMED IS:

1. A DNA molecule consisting essentially of a nucleotide sequence which encodes the H13 protein or encodes a functional derivative thereof.

2. A DNA molecule according to claim 1 comprising the nucleotide sequence SEQ ID NO: 7

3. A DNA according to claim 1 which is an expression vector.

4. A host transformed or transfected with a vector according to claim 3.

5. A host according to claim 4 which is a mammalian cell.

6. A protein molecule H13 substantially free from impurities of human origin with which it is natively

associated, said protein comprising the amino acid sequence SEQ ID NO: 8, or a functional derivative thereof.

7. A method for inhibiting the infectivity of a retrovirus comprising contacting said retrovirus with an effective amount of the protein molecule or derivative

according to claim 6 and allowing said molecule to prevent said virus from attaching to a cell thereby inhibiting said infectivity.

8. A method according to claim 7 wherein said virus is human immunodeficiency virus.

9. The method of claim 8 wherein said contacting is in vivo.

10. A pharmaceutical composition useful for

preventing or treating a retrovirus infection, comprising a protein molecule or functional derivative according to claim 6 and a pharmaceutically acceptable carrier.

11. A method for preventing or treating a retrovirus infection in a subject comprising administering an effective amount of a composition according to claim 10.

12. An antibody specific for a protein molecule or derivative according to claim 6.

13. An antibody according to claim 12 which is monoclonal.

14. A pharmaceutical composition useful for

preventing, suppressing or treating a retrovirus infection, comprising an antibody according to claim 12 and a

pharmaceutically acceptable carrier.

15. A method for preventing or treating a retrovirus infection in a subject comprising providing to that subject an effective amount of a composition according to claim 14.

16. A method for producing the H13 protein useful for preventing or treating a retrovirus infection in a

subject comprising the steps of:

(a) providing a DNA molecule according to claim 1 in expressible form;

(b) expressing said DNA molecule in host cell in culture, thereby producing the H13 protein; and (c) obtaining said H13 protein from said culture.

17. A method according to claim 16 further comprising:

(d) purifying said H13 protein.

18. A method for detecting the presence in a sample of a human retrovirus, or a retroviral protein or peptide derived therefrom, wherein said retrovirus, retroviral protein or retroviral peptide is capable of binding to the H13

protein, comprising:

(a) incubating said sample which is suspected of containing said retrovirus, protein or peptide in the presence of an H13 protein or functional derivative according to claim 6;

(b) permitting said H13 protein or functional

derivative to bind to said retrovirus,

retroviral protein or retroviral peptide; and (c) detecting said retrovirus, retroviral protein or retroviral peptide which is bound to said H13 protein or functional derivative,

thereby detecting the presence of said retrovirus, retroviral protein or retroviral peptide in said sample.

19. A method for detecting the presence in a sample of an antibody specific for an epitope of the H13 protein, comprising:

(a) incubating said sample which is suspected of containing said antibody in the presence of an H13 protein or a functional derivative

according to claim 7;

(b) permitting said antibody to bind to the H13

protein or functional derivative; and

(c) detecting said antibody which is bound to said H13 protein or functional derivative,

thereby detecting the presence of said antibody in said sample.

20. A transgenic non-human mammal essentially all of whose germ cells and somatic cells contain a DNA sequence encoding H13 (SEQ ID NO: 8) or encoding a functional derivative thereof.

21. A transgenic mammal accordance to claim 20 in which said DNA molecule has been introduced into said mammal or an ancestor of said mammal at an embryonic stage.

22. A DNA molecule encoding a chimeric retroviral receptor protein, comprising

(a) a first nucleotide seguence which encodes

retroviral receptor protein I of a first animal species; and

(b) substituted therein, a sufficient number of

nucleotides from a second nucleotide sequence encoding retroviral receptor protein II of a second animal species,

wherein said substituting nucleotides confer on said chimeric retroviral receptor protein the ability to bind a retrovirus which binds to receptor protein II but not to receptor protein I, allowing said chimeric protein to function as a retroviral receptor for said retrovirus.

23. A DNA molecule according to claim 22 wherein said first nucleotide sequence comprises the coding portion of human H13 DNA (SEQ ID NO:7).

24. A DNA molecule according to claim 2 encoding a chimeric H13/ERR chimeric protein wherein said second

nucleotide sequence comprises the coding portion of murine ERR DNA (SEQ ID NO: 3), and said substituting ERR nucleotides are those encoding an amino acid residue selected from the group consisting of Ile2l4, Lys222, Asn223, Ser225, Asn227, Asn232, Val233, Tyr235, Glu237, Ile313, Asp314, Gly319, Gln324, Glu328 and any combination of the above.

25. A DNA molecule according to claim 22 which is an expression vector.

26. A host transformed or transfected with a vector according to claim 25.

27. A host according to claim 26 which is a

mammalian cell.

28. A chimeric retroviral receptor protein molecule encoded by a DNA molecule according to claim 22.

29. A chimeric retroviral receptor protein molecule encoded by a DNA molecule according to claim 23.

30. A chimeric retroviral receptor protein molecule encoded by a DNA molecule according to claim 24.

31. A method for rendering a cell of species I susceptible to infection by, and retrovirus-mediated gene transfer by, a retroviral vector normally incapable of

infecting a cell of species I, comprising the steps of:

(a) transforming a cell of species I with an

expressible DNA molecule according to claim 22; (b) expressing the chimeric retroviral receptor

protein on the surface of said cell in culture, thereby rendering said cell susceptible to infection by said retroviral vector.

32. A method for rendering a human cell susceptible to infection by, and retrovirus-mediated gene transfer by, a retroviral vector normally incapable of infecting a human cell, comprising the steps of:

(a) transforming a human cell with an expressible DNA molecule according to claim 23;

(b) expressing the chimeric retroviral receptor

protein on the surface of said human cell in culture,

thereby rendering said human cell susceptible to infection by, and retrovirus-mediated gene transfer by, said retroviral vector.

33. A method for rendering a human cell susceptible to infection by, and retrovirus-mediated gene transfer by, an ecotropic murine leukemia virus vector which is incapable of infecting a human cell, comprising the steps of:

(a) transforming a human cell with an expressible

DNA molecule according to claim 24;

(b) expressing the chimeric H13/ERR retroviral

receptor protein on the surface of said human cell in culture,

thereby rendering said human cell susceptible to infection by said ecotropic murine leukemia viral vector.

34. A method for transferring a gene to a cell of species I for use in gene therapy, comprising:

(a) culturing a cell intended to receive said

transferred gene;

(b) transforming said cell with a DNA molecule

according to claim 22, thereby providing said cell with a chimeric retroviral receptor protein;

(c) infecting said cell with a retroviral vector normally incapable of infecting a cell of species I, said retroviral virus being capable of infecting said cell expressing said chimeric receptor, said retroviral vector further carrying the gene to be transferred; and

(d) allowing the gene carried by said retroviral vector to be expressed in said cell,

thereby transferring said gene.

35. A method for transferring a gene to a human cell for use in gene therapy, comprising:

(a) culturing a human cell intended to receive said transferred gene;

(b) transforming said cell with a DNA molecule

according to claim 23, thereby providing said cell with a chimeric retroviral receptor protein; (c) infecting said cell with a retroviral vector normally incapable of infecting a human cell, said retroviral vector being capable of infecting said human cell expressing said chimeric receptor, said retroviral vector further carrying the gene to be transferred,

(d) allowing the gene carried by said retroviral vector to be expressed in said human cell.

36. A method for transferring a gene to a human cell for use in gene therapy, comprising:

(a) culturing a human cell intended to receive said transferred gene;

(b) transforming said cell with a DNA molecule

according to claim 24, thereby providing said cell with an H13/ERR chimeric retroviral receptor protein;

(c) infecting said cell with a ecotropic murine

retroviral vector normally incapable of

infecting a human cell, said retroviral vector being capable of infecting said human cell expressing said H13/ERR chimeric receptor, said retroviral vector further carrying the gene to be transferred,

(d) allowing the gene carried by said retroviral vector to be expressed in said human cell.