CA2097705A1

CA2097705A1 - Human retrovirus receptor and dna coding therefor

Info

Publication number: CA2097705A1
Application number: CA002097705A
Authority: CA
Inventors: Daniel Meruelo; Takayuki Yoshimoto
Original assignee: Individual
Current assignee: New York University NYU
Priority date: 1990-12-14
Filing date: 1991-12-13
Publication date: 1992-06-15
Also published as: WO1992010506A1; EP0562013A4; EP0562013A1; JPH06503476A; AU9152091A

Abstract

A human protein molecule termed H13 has strong sequence homology to murine retrovirus receptor proteins and encodes a human retrovirus receptor. DNA encoding the H13 protein, cells transformed and transfected with this DNA and antibodies specific for H13 are disclosed. The H13 protein or its functional derivative can be used for preventing or treating retrovirus infection by administration to a subject of the H13 protein or a functional derivative thereof, or an anti-H13 antibody. Transgenic animals, useful as animal models for diagnosis or therapy of human retrovirus infections, are made by transfecting embryonic cells with the H13-encoding DNA. A chimeric retrovirus receptor protein comprises the H13 sequence, having substituted therein, amino acid residues encoding a murine retroviral receptor. Expression of the chimeric receptor in human cells allows infection or retrovirus-mediated gene transfer with murine retroviruses, which provides an extra measure of safety for in vivo gene therapy. DNA encoding the chimeric retrovirus receptor protein, cells transformed with this DNA, and methods for rendering a cell susceptible to infection by a retrovirus normally incapable of infecting that cell are disclosed.

Description

W O 92/10506 2 ~ 9 7 7 0 5 PC~r/US91/09382 HUMAN RETROVIRUS RECEPTOR AND DNA CODING THEREFOR

B~CRGROUND 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 10 prevent or treat retrovirus infection. The invention also concerns substitutio~s 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 15 vectors.

Description of the Backaround 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 20 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.
312:763-767 (1984)); Klatzmann, D., Nature 312:767 (1984);
25 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-l (Greve, J.M. et al., Cell 56:839 (1989);
30 Staunton, D.E. et al., Cell 56:849 (1989)). Rabies virus binds to the acetylcholine receptor (Lentz, T.L., Science 215:

wo 92/105n6 ~ 7 V 5 PCT/U591/09382 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., 5 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 10 cell surface or within an acidified endosome after receptor-mediated endocytosis (White et al., Ouant. Rev. Bio~hvs. 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 15 cell surface molecules other than CD4 ~ay 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 Z0 have been transfected with the CD4 gene and express this molecule on their surface are o~ten 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-25 dividing quiescent cells; for infection by HIV in vitro, thesecells 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.

30 Murine Retrovirus Rece~tors 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, 35 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)).

WO92/10~06 2 0 9 ~ I 0 5 PCT/US9l/09382 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., 5 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 10 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
15 (Rein, A. et al., Virolooy 136:144-152 (1984)).
Hybrid cells which were created by fusion of primary mouse lymphocytes with nonpermissive Chinese hamster lung cel~s retained susceptibility to E-MuLV infection and bound gp70 to the membrane (Gazdar, A.F., Cel.l 11:949-956 (1977)).
20 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-l locus on mouse chromosome 5 (Oie et al., Nature 274: 60-62 (1978); Ruddle et al., J. Exp.
~ 148:451-465 (1978)). Purification of the protein encoded 25 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 30 hypothesis that a single gene encodes the receptor protein.
~urthermore, 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 35 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

2 ~ 9 r~ rl 0 5 PCT/US91/09382 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 _etroviral Eeceptor (ERR) was identified (Albritton, L.W. et al., Cell 57:659-666 (1989)). This study demonstrated that susceptibility to E-~uLV infection was ac~uired by the expression of a single mouse gene in human EJ
10 cells. Furthermore, this gene appears to define Rec-1, the genetic locus on mouse chromosome 5 associated with ecotropic virus infectivity tOie 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 15 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 20 infection by functioning as a true receptor that binds specifically to E-MuLV gp70 in analogous fashion to HIV gpl20 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 25 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.
30 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 35 al., Cell 49:659-668 (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. 57:1048-105~
(1986)). The potential role for a protein in virus fusion WO92/10506 2 ~ 9 7 7 9 5 PCT/US91/09382 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 5 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 lO comparison of the predicted amino acid sequences of several of these proteins ~o 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, suDra).

15 ~ Cell Earlv 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
20 clones which identify new functions, MacLeod, C.L. et al.
(Mol. Cell. Biol. 10:3663-3674 (l990)) 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 25 model system is known as the SL12 T lymphoma (Hays et al., Int. J. Cancer 38:597-601 (1986)); MacLeod et al., Cancer Res.
44:1784-1790 (1984)); ~ 74:875-882 (1985); Proc. Natl.
Acad. Sci. USA 83:6989-6993 (1986); Cell Growth & Differ.
1:271-279 (1990)). The two cell clones derived from a single 30 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 35 syngeneic animals (MacLeod et al., 1985, 1986, supra). In contrast, the cells of sister clone SLl2.4 express mRNAs for all the components of the T cell receptor (TCR)-CD3 comple~

WO92/10506 2 0 9 7 7 ~ ~ PCT/US91/09382 except TCR-~ and resemble thymocytes at an intermediate stage of development (MacLeod et al., 1986, supra; Wilkinson et al , EMBO J. 7:101-109 (1988)). SL12.4 cells are much less tumorigenic than SL12.3 cells (MacLeod et al., 1985, su~ra).
A combination of subtraction hybridization-enriched probes (Hedrick et al., Nature 308:149-153 (1984); MacLeod et al., J. BioL. Chem. 1:271-279 tlg9o); Timberlake, Dev. Biol.
78:497-S03 (19~0)) and classical differential screening Sambrook et al., Mo~ecular Clonina: A Laboratorv Manual, 2nd 10 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 15 limited number of tissues. The gene expressed by this clone was designated TEA (T cell early activation, SEQ ID NO:5).
1~ 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 20 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, su~ra).
30 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 35 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 WO92/10506 2 ~ 9 7 7 0~ PCT/US91/09382 transmembrane-spanning protein which is induced durin~ 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 5 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 10 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 15 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-l gene). This finding suggests that the ~ gene product might function as a murine 20 retroviral receptor. In contrast to the Rec-l 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 25 distribution could be responsible for the tissue specificity of retroviruses which are restricted to cells of the lymphoid lineage tQuint 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 30 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 35 al., 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 WO92/10506 ~ o~ PCT/US91/09382 receptor. Related to these findings is the fact that a human cDNA which confers susceptibility to infe~tion by gibbon ape leukemia virus has a sequence similar to a phosphate transporter protein in fungi (Vile et al., supra). It is not 5 known whether Tea also encodes a permease.
~ IV is an example of a virus exhibiting receptor-mediated tissue restriction, apparently based on its use of the CD4 protein as its primary receptor. However, cell-specific receptors are unlikely to be the sole determinant of 10 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.
15 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., Viroloay 136:144-152 (1984)).
Despite the high degree of similarity between TEA
20 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 25 new family members.
~ enetically 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 30 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 35 cells. One relatively efficient means for achieving transfer of genes is by retrovirus-mediated gene transfer (Gilboa, E., Bio-Essavs 5:252-258 (1987): Williams, D.A. et al., Nature 310:476-480 (1984); Weiss, R.A. et al., RNA Tumor Viruses, 2~977~
g 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 5 (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)). 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 10 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 15 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 20 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 AInS. See Yoshimoto, T. et al., Viroloay 25 185:10-17 (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 30 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 WO92t10506 PCT/US91/09382 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 5 amino acid sequence of the full-length Hl3 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 Hl3 protein has 629 amino acids lO 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 15 which encodes the Hl3 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 Hl3, the seguence 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-l, comprising contacting the virus with an effective amount of the Hl3 protein molecule or functional derivative thereof and allowing the molecule to prevent the 30 virus from attaching to the cell thereby inhibitinq infection.
The present invention includes a method for preventing, suppressing, or treating a retrovirus infection, such as HIV-l, in a subject comprising providing to that subject an effective amount of the Hl3 molecule or functional 35 derivative.
The invention is further directed to an antibody specific for the Hl3 molecule or an epitope thereof, including polyclonal, monoclonal, and chimeric antibody. An additional W092/10506 2 0 9 7 7 0 ~ PCT/US91/09382 embodiment involves a method for preventing, suppressing, or treating a retrovirus infection, such as HIV-l 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 Hl3 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 i~cludes 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 Hl3 protein molecule or a functional derivative thereof or an antibody specific for the Hl3 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 Hl3 protein, alone or in combination with the ~D4 gene.
This transgenic animal serves as a ~odel for human retrovirus 30 infection and allows testing of anti-viral therapies.
The methods of the present invention which identify normal or mutant Hl3 genes or measure the presence or amount of Hl3 protein associated with a cell or tissue can serve as methods for identifying susceptibility to human retrovirus 35 infection, as in AIDS or certain forms of leukemia.
The present invention further provides a DNA
molecule encoding a chimeric retroviral receptor protein, comprising:

WO92~10506 2 0 ~ PCT/US91/09382 (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 10 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 15 nucleotide sequence comprises the coding portion of murine ERR
DNA (SEQ ID ~0:3), and the substituting ERR nucleotides are those encoding an amino acid residue selected from the group consisting of Ile214, Lys222, Asn223, SerZ25, Asn227, Asn232, Val233, Tyr235, Glu237, Ile313, Asp314, Gly319, Gln324, Glu328 20 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 WO92/10506 2 0 9 7 7 0 ~ PCT/US91/09382 normally incapable of infeoting 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, 5 (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, lO most preferably an ecotropic murine leukemia virus, and the chimeric receptor is preferably a chimeric Hl3/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: .
15 (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 ell, 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 ~13 and ERR cDNA sequence (SEQ ID No:7 and 10 3, respectively). The sequences were analyzed using the Genetics computer group sequence analysis software package (Devereux, J. çt al., Nuçl. Acids Res. 12:387-395 (1984)).
Figure 3 shows the alignment of H13, ERR and TEA
deduced amino acid sequences. Vertical lines indicate 15 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 310-20 337).
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, BIOT6R) origin, probed with the KpnI-KpnI fragment 25 (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:l).
DNA hybridized was EcoRI-digested DNA of human (CCL120, CCL

WO92/10506 2 ~ 3 7 7 0 ~ PCT/US91/09382 119, SupTl, H9, MOLT4), hamster (C~O-Kl) 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 5 hybridized with the H13 cDNA (SEQ ID NO:1).
Figure 7 is an autoradiogram showing the hybridization pattern of RNA of human (CEM, Hg, MOLT4, SupTl, CCL120, CCLll9), hamster (CHO Kl) and mouse (RL12) origin, probed with the KpnI-KpnI fragment (390 bp) of murine ERR
10 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 Rl cells, the transfectants expressing 15 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 25 H13 predicted protein, analyzed using the PEPTIDESTRUCTURE
program. One of the highly antiqenic peptides (amino acid residues 309-367) was prepared using an AccI-EcoRI ~ragment as shown in Figure 14.

W092/10506 2 0 9 7 7 0 ~ PCT/US91/09382 Figure ll depicts a polyacrylamide gel electropherogram showing the synthesis of a fusion protein including the Hl3 protein with glutathione-S-transferase (GST). The fusion protein was prepared by ligating the 180 bp 5 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 lO to human chromosome 13. The autoradiogram (Figure 12A) shows the hybridization pattern of EcoRI-digested DNA from human-hamster somatic cell hybrids probed with Hl3 cDNA (SEQ ID
NO:l). Lane l and 11 contain DNA from human and hamster, respectively. Lanes 2-10 contain DNA whi~h is derived from the 15 chromosomes as designated in the table in Figure 12B.
Figure 13 is a schematic diagram of the genetic structure of the Hl3 and ERR genes, and four chimeric constructs therebetween. The infectivity of E-MuLV on human cells transfected with the various constructs is also 20 indicated.
Figure 14 shows a comparison of sequences (nucleotide and amino acid) of the region of Hl3 and ERR
termed Extracellular Domain 3 (see also SEQ ID NO:7, SEQ ID
NO:8 and Figure l). This region of the receptor protein is 25 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 severa 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 5 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).

DE$5~IPT~ON OF TRE~ PREFERRED EHBODIMENTS
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 15 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 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.
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 W092/10506 ~0 9 7 7 ~ ~ PCT/US91/09382 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 5 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 10 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 15 present invention can be purified biochemically or physicochemically from a variety of cell or tissue sources.
For preparation OL naturally occurring H13 protein, tissues such as human lymphatic organs and cells such as human lymphoid cells are preferred. Alternatively, methods are well 20 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 25 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 5 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 10 possible, while still retaining sufficiently high affinity in binding to HIV to inhibit i~fection. 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 15 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 20 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 25 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.

WO92/10506 ~0 ~,~ r~ ~ ~ PCT/US91/09382 ~ espite the degree of similarity in amino acid sequence (87.6% identity) and structure (14 transmembrane spanning domains), between the human Hl3 protein of the present invention and the murine ERR, H13 fails to bind 5 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 10 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 o~ a cell to infection by E-MuLV.
The chimeric mouse-human E-MuLV receptor of the 15 present invention can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic or non-~ammalian eukaryotic cell. Preferably, however, a chimeric ~13/ERR protein molecule is produced by recombinant means and expressed in ~ammalian cells, most 20 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 ` 25 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 20~770~

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.
Hl3 extracellular domain 3 and Hl3 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 Hl3. Domain 3 comprises residues between lO positions 210 and 250 (SEQ ID N0:7). 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
N0:4~. Domain 4 of Hl3 comprises residues 31-337 (SEQ ID
NO:7). Preferred substitution is with one or more amino acid 15 residues from the corresponding domain of ERR, between amino acid residues 303 and 330 (SEQ ID NO:4).
Substitution of between l and 4 residues is preferred. Substitution of as few as one amino acid may alter the virus specificity of the chimeric receptor protein. The 20 residues and positions which differ in Extracellular Domain 3 and Domain 4 of Hl3 and ERR are listed below in Table l.

WO92/lOS06 ~ 0 9 7 7 o ~ PCT/US91tO9382 Table 1 Possible Substitutions in H13 Extracellular Domain 3 and Domain 4 for E-MuLV Binding Domain 3 Domain 4 Original SubstitutingOriginal Substituting Residue Residue Residue Residue E 223 N 223 D 32~ G 319 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) ~hat do not correspond to residues of ERR. Preferred 5 deletions are of between one and six residues from H13 positions 326 to 331 (SEQ ID NO:l), ~ost 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 10 present invention, râther than with ~he 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 15 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, thè fact that the chimeric receptor is largely of human origin decreases 20 the chances of an undesirable immune response directed to the sequences derived from receptor sequence of non-human origin.
If large portions of the ER~ protein were used for this purpose, the human subject would respond immunologically to WO92/10506 2 0 ~ 7 7 0 ~ PCT/US91/09382 the foreign epitopes on the injected cells, diminishing the utility of these cells in gene therapy.
This lacX of immunogenicity is important for survival and therapeutic efficacy of the infused retrovirally 5 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 10 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 15 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-l and HTLV-1. Thus, the chimeric receptor will 20 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-l, HIV-2, human T lymphotropic viruses 25 that induce leukemia (HTLV-l, HTLV-2, etc.) and other human retroviruses (~eiss, R.A. et al.j 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 30 receptor or enter the cell via an H-13-dependent mechanism.
Genetic constructs encoding H13 functional deriva-tives 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 35 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.

W O 92/10~06 PC~r/US91/09382 ~V9'`170~

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 5 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 tl988)).
Depending on the receptor specificity of the viral envelope protein gp70, such viruses have varying host ranges; some 10 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 qene 15 into neurons ttPrice, J. et al., Proc. Natl. Acad. Sci. USA
84:156-160 tl987)).
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 20 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 Dsi packaging sequence, etc. The present invention provides an improved measure of safety compared to the prior ; 25 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 35 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 2~9770~

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 5 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 10 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 (Gilboa, E., Bio-Essays 5:252-258 tl987); 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 si~ilar 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 20 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 25 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 30 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 35 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 2097~5 W092t10506 PCT/US91/09382 Hl3 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, 5 using a probe based on the sequence of ERR or Hl3. 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 lO sequence of the new retroviral receptor protein differs from ERR or Hl3. 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 15 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-l CD4-binding domain will render this virus non-infective for CD4-bearing 25 cells. Corresponding changes may be introduced into Hl3, 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-l.
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 35 chimeric Hl3/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.

2~77 0~

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 i~proved animal model for AIDS than the 5 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 10 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 15 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 20 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 25 having similar bioactivity for all the uses described herein.
Also intended are all active forms of H13 deri~ed 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 30 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
35 85:4087-4089 (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 W092/lOS06 ~ ~ 7 ~ O ~ PCT/US91/09382 (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 5 important that the soluble Hl3, or a functional derivative of Hl3, comprise the elements of the binding site of the Hl3 that permits binding to a retrovirus. An Hl3 molecule has many amino acid residues, only a Pew of which are critically involved in virus recognition and binding.
As discussed herein, the Hl3 proteins or peptides of the present invention may be further modified for purposes of drug design, such as, for example, to reduce immunogenicity, to promo~e solubility or enhance delivery, or to prevent clearance or degradation.
In a further embodiment, the invention provides "functional derivatives" of the Hl3 protein. By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the Hl3 protein. A functional derivative retains at least a portion of the function of the 20 Hl3 protein which permits its utility in accordance with the present invention.
A "fragment" of the Hl3 protein is any subset of the molecule, that is, a shorter peptide.
A "variant" of the Hl3 refers to a molecule sub-25 stantially similar to either the entire peptide or a fragmentthereof. 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 30 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 35 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 WO92/10506 2 0 9 7 7 0 ~ PCT/US91/09382 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 5 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.
10 In particular, the Hl3 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 15 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). ~his primer is 20 then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Xlenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence in the second strand bears the desired mutation.
25 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 30 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 35 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 W092/]0506 2 ~ 9 r~ ~f o 5 PCT/US91/09382 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 5 Creighton, T.E., Proteins: Structure and Molecular Pro~erties, 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 lO of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (su~ra) and Figure 3-9 of Creighton (su~ra).
Base on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five 15 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, gln;

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 30 formation which is important in protein folding. Note the Schulz et al. would merge Groups 1 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 35 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 W092/lOS06 2 ~ 9 7 7 0 ~ PCT/US91/09382 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.
5 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) 10 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, 15 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 20 the exact effect of the substituti~n, 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 25 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 30 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 35 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).

6 ~ ~ 7 7 ~ PCT/US91/09382 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 5 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 Hl3 protein refers to a non-natural molecule substantially similar to either the entire 10 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 15 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 20 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 25 phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
Histidyl residues are derivatized by reaction with 30 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 6Ø
Lysinyl and amino terminal residues are reacted with 35 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 WO92/10506 2 0 9 7 7 ~ ~ PCTtUS91/09382 imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with 5 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 re~uires that the reaction 10 be performed in alXaline conditions because of the high PXa f 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 15 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, 20 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) 25 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 30 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 35 matrix or to other macromolecular carriers. Commonly used cross-linking agents include, e.g., l,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuCCinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional WO92/10506 2 0 ~ r~ r~ ~ 5 PCT/VS91/09382 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 5 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;
10 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 15 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 eli.minate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, 25 in Reminqton'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 ~3ioloay o~ ~he Gene, Volumes I and II, The 30 Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al., Molecular Cell Bioloay, 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 35 Manipulation: An Introduction to Genetic Enqineerinq, 2d edition, University of California Press, publisher, Berkeley, CA (1981); and Sambrook, J. et al., Molecular Clonina. A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring 2a9770~

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 5 preferably, by application of reco~binant 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 10 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 ~olecules 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 25 Sambrook et al., Molecular Clonin~: 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 qenes.
Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, R., et al., Pro~. Nucl. Acid.
35 Res. Molec. Biol. 21:101-141 (1978)).
Because the genetic code is degenerate, more than o~e codon may be used to encode a particular amino acid (Watson, J.D. et al., supra). Using the genetic code, one or WO92/10506 2 0 3 ~ 7 0 ~ PCT/US91/09382 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 S 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"
lO of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable"
nucleotide sequence capable of encoding the Hl3 sequences is identified.
Although occasionally an amino acid sequence may be 15 encoded by only a single oligsnucleotide, 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 20 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 25 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, 30 containing the theoretical "most probable" sequence capable of encoding the Hl3 fragment is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable"
sequance, or set of sequences. An oligonucleotide containing 35 such a complementary sequence can be employed as a probe to identify and isolate the Hl3 genP (Sambrook et al., supra).
A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of WO92~10506 PCT/US91/09382 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, 5 more preferably, a cDNA preparation derived from cells which are capable of expressing the H13 gene. Single stranded oligonucleotide molecules comple~entary to the "most probable"
H13 peptide coding sequences can be synthesized using procedures which are well known to those of ordinary skill in 10 the art (Belagaje, R., et al., J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T., et al., n: Molecular Mechanisms in the Control of Gene Expression, Nierlich, D.P., et al., Eds., Acad. Press, NY (1976); Wu, R., et al., Proa. Nucl. Acid Res.
Molec. Biol. 21:101-141 tl978); Khorana, R.G., Science 15 203:614-625 (1979)). Additionally, DNA synthesis may be achieved through the use of automated synthesizers.
Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (suDra), and by ~aymes, B.D., et al. (In:
Nucleic Acid Hybridization. A Practical Approach, IRL Press, 20 Washington, DC (1985)), which references are herein incorporat~d 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 (198S)), fibronectin 25 (Suzuki, S., et al., Eur. Mol. Biol. oraan. 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 30 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) 35 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 WO92~10506 PCT/US91/09382 2 0977~5 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, 5 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 15 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 20 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.
25 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 D~A may contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic 30 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 WO92/1050~ 2 ~ ~ 7 7 0 ~ PCT/US91/09382 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 5 "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 ~13 protein. Alternatively, a "functional derivative" of a 10 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 15 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 ~iological 20 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 H13 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, 30 restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Sambrook, J. et al., supra, and 35 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 WO92/10506 ~ ~ 7 7 ~ ~ PCT/US91/09382 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 5 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 10 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 15 sequence, CAAT sequence, and the like.
If desired, the non-coding region 3' to the gene sequence coding for the protein may ~e obtained by ~he above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as 20 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 prcvided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, 25 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 30 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 35 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.

WO92/10506 2 0 9 7 ~ O ~ PCT/US91/09382 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 5 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 10 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 15 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 20 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 ~1., supra). The promoter sequences of the present 25 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 30 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, 35 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 W092/10506 ~ ~ 9 ~`~ rl ~ ~ PCT/USg1/09382 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 5 derivative thereof) or a chimeric H13 protein in a prokaryotic cell (such as, for example, ~. 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 10 bacteriophage lambda, the kl~ promoter of the ~-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 (PL and PR), the trp, recA, 15 lacZ, lacI, and aal promoters of E. coli, the ~-amylase (Ulmanen, I., et al., ~. Bac~e~ol. 162:176-182 ~1985)) and the s-28-specific promoters o~ B. subtilis (Gilman, M.Z., et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bac~llus (Gryczan, T.J., In: The Molecular 20 Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Stre~tomvces 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, 25 S. (Ann. Rev. Genet. 18:415-442 (1984)).
Proper expression in a prokaryotic cell also reguires 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.
30 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 35 at correct sites. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VER0 or CH0, or cells of lymphoid origin, such as the hybridoma SP2/0-Agl4 or the murine myeloma P3-X63Ag8, and their derivatives.

W~92/]0~06 2~77a5 PCT/US91/09382 Preferred mammalian cells are cells which are intended to replace the function of the genetically deficient cells in vivo. Bone marrow s~em 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 Hl3. A wide variety of transcriptional a~d translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory lO 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, 15 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, 20 expression can be repressed or initiated, or are subject to chemical regulation, e.q., 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 25 coding for glycolytic enzymes produced in large guantities 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 Hl3 or chimeric Hl3 molecules in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express Hl3 by methods known to those of skill. Thus, in one embodiment, sequences encoding Hl3 may be operably linked to the regulatory regions 35 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 Hl3 protein in amounts as great as 20 to 50~ of total protein ~9770~

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 5 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. A~
10 Gen. 1:273_288 (1982)); the TX promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature rLondon) 290:304-310 (1981)); the yeast aal4 gene promoter (Johnston, S.A., et al., Proc._Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, 15 P.A., et al., Proc. Natl. ~cad. Sci. rUSA) 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 20 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 25 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 30 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 35 expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

W0~2/10506 PCT/US91/09382 2a~ ~05 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 5 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 10 directly linked to the DNA gene sequences to be expressed, or introduced lnto 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 15 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 20 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., iami Wntr. Sym~.
25 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., 30 In: Cell Biolooy: 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., 35 Miami Wntr. Sym~. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cvcle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-20 WO92/10506 ~ V9 7 7 ~ ~ PCT/US91/09382 (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 Ex~ression, 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 se~uence containing the construct(s) has been prepared for expression, the vector or 10 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 phosphate-precipitation, and application with polycations such as 15 diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment (Johnston ~_~1., Science ?40(4858~: 1538 (1988)), etc.
After the introduction of the vector, recipient 20 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 25 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 30 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 35 still reacts with the protein containing ERR-derived amino acid substitutions. Such antibodies may be obtained by well-known methods.

2~.~7705 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 5 retrovirus binding domain of ~13 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, lO 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 ~NA is introduced into the animal to be made transgenic, or an 15 ancestor of the animal, at an embryonic stage, preferably the one-cell, or fertilized oocyte, 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 20 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 25 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 30 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, 35 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 WO92/10506 ~ 7 ~ 5 PCT/US91/09382 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 5 somatic cells. Introduction of the transgene at a later embryonic stage in a founder animal may result in limited presence of the tr~nsgene 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 lO 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 Hl3 DNA of the present invention, such as animals produced when fewer than 15 all of the cells of the morula are transfected in the process of producing the transgenic ~ammal, are also intended to be within the scope of the pre6ent invention.
The techniques described in Leder, U.S. Patent 4,736,866 (hereby incorporated by reference) for producing 20 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 Hl3 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 Hl3 molecule as a receptor.
30 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, 35 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.

W092/10506 2 0 9 7 ~ ~ ~ PCT/US91/09382 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 5 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 10 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, 15 and anti-idiotypic (anti-Id) antibodies.
An antibody is said to be "capable of binding" a molecule if it is capable o~ specifically reacting with the molecule to thereby bind ~he molecule to the antibody. The term "epitope" is ~eant to refer to that portion of any 20 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 25 characteristics as well as specifiG 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 30 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.

W092/lOS06 ~ 0 9 7 r) ~ 5 PCT/US91/09382 - 5~ -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 5 (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 10 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. M~bs of isotype IgM or IgG may be purified from such 15 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 20 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., Na~e ~14:268-270 (1985); Sun 25 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 30 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 35 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).

W092/10506 2 0 9 7 ~ ~ 5 PCT/US91/09382 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 5 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 10 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 15 (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 20 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 25 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. ~ed. 24:316-325 (1983)).
It will be appreciated that Fab and F(ab')2 and 30 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 35 (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-WO92/10506 2 ~ 9 7 7 0 5 PCT/US91/09382 tively detect the presence of cells which express the Hl3 protein (or a chimeric receptor having an Hl3-derived epitope) on their surface or intracellularly. This can be accomplished by immunofluorescence techniques employing a 5 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 Hl3 lO 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 15 sample. Through the use of such a procedure, it is possible to determine not only the presence of the Hl3 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 20 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 Hl3 molecules in a biological sample. Used in this manner, the antibody can 25 serve as a means to monitor the,presence and ~uantity of Hl3 proteins or derivatives used therapeutically in a subject to prevent or treat human retrovirus infection.
Such immunoassays for Hl3 protein typically comprise incubating a biological sample, such as a biological fluid, a 30 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 Hl3 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

7 ~ ~

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 5 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. Well-known supports, or carriers, include glass, polystyrene, 10 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 15 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 20 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 25 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, fil-30 tering and the like may be added to the assays as is customaryor 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, 35 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. ~nzymes W092/10506 2 0 9 ~ 7 0 S PCT/US91/09382 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 5 phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydroqenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by lO colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzy~atic reaction of a substra~e in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety 15 of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect Hl3 protein through the use of a radioimmunoassay (RIA). A
good description of RIA may be found in Labo~atory Techniques and Biochemistry in Molecular BioloqY, by Work, T.S., et al., 20 North Holland Publishing Company, New York (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard, incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a gamma counter or a 25 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 30 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 l52Eu, or others of the 35 lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

W O 92/10506 2 ~ 3 7 7 o 5 P ~ /US91/09382 The antibody also can be detectahly labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determlned by detecting the presence of luminescence that arises during the 5 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 bioluminescent compound may be used to 10 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 15 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 20 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 25 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 30 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 35 "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 2 ~ 7 0 ~

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 so-5 called "simultaneous" and "reverse" assays are used. Asimultaneous 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 10 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 15 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 se~ond incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested 20 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 25 to diagnose circulating antibodies in a subject which are specific for the H13 protein. This is accomplished by means of an immunoassayl as described above, using the protein of the invention or a functional derivative thereof.
Based on similar principles, since a retrovirus 30 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 35 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 W092/10506 2 0 ~ 7 7 ~ ~ PCT/US91/09382 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 5 biological sample of a viral component such as a viral protein or ~lycoprotein 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 lO molecule.
As used herein, the term "prevention" of infection involves administration of the Hl3 protein, peptide derivative, or antibody ~see above) prior to the clinical onset of the disease. Thus, for example, successful 15 ad~inistration 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 20 protective composition after the clinical onset of the disease. For example, successful administration of a Hl3 - protein or peptide or anti-~13 antibody according to the ; invention after development of a retrovirus infection in order to delay or suppress further virus spread comprises 25 "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 30 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, ~5 intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.

~ o 9 r~ 7 o ~i 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 5 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 lO 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 15 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 20 agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like. Preferred vehicles f~r non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol-lOOO (PEG-lOOO); conventional creams such as HEB cream;
25 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.
30 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 35 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.

WO92/10506 2 ~ 9 7 7 0 ~ PCT/US91/09382 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 5 about O.OOl mg to about l 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 O.Ol to about 50 mg of active lO ingredient is used per cc of ointment base, the latter being preferably PEG-lOO0.
A typical regimen for preventing, suppressing, or treating retrovirus infection comprises administration of an effective amount of the Hl3 protein or functional derivative 15 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 n vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if 20 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 25 of skill in the art.
~ he 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 30 directed to the viral infection, or directed to other symptoms of the viral disease.
Effective amounts of the Hl3 protein, functional derivative thereof, or antibody thereto, are from about O.Ol ~g to about lO0 mg/kg body weight, and preferably from about 35 lO ~g 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 ~ ~Y ~7~60 intrauterine administration. This treatment is designed to protect the fetus ~rom spread of HIV, for example.
Alternatively, the compositions of the invention can be used intravaginally, especially during the birth process, to 5 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 lO 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 15 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 20 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 O.Ol to 99 percent, preferably from about 25 20 to 75 percent of active component (i.e., the Hl3 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 Hl3 protein or mRNA in a subject. Absence, or more typically, low expression of the Hl3 gene or presence of a mutant Hl3 in an individual may serve as an important predictor of resistance 35 to retrovirus infection and thus to the development of AIDS or certain types of leukemia or other retrovirus-mediated diseases. Alternatively, over-expression of Hl3, may serve WO 92tlO506 2 0 9 7 7 0 5 Pcr/~ls91/09382 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 5 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 10 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 15 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 20 preparation.
Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recom`binant DNA
methodologies capable of amplifying purified nucleic acid 25 fragmen~s 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 30 Cohen et al. tU.S. Patent 4,237,224), Sambrook et al. (supra), etc.
Recently, an in vitro enzymatic method has been de-scribed which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been 35 referred to as the "polymerase chain reaction" or "PCR"
(Mullis, K. et al., Cold Sprin~ Harbor S~mp. Ouant. 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., ~09770.~

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 5 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 10 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 15 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 seguence and the terminal 3' hydroxyl group of a second 20 sequence. Poly~erase 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 p~lymerase extends the 3' end of a nucleic acid molecule. These inherent properties are 25 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 30 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 35 "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.

WO92/10506 2 a ~ 7 7 0 ~ PCT/US91/09382 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 5 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 lO 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 a~plified. If the nucleic acid molecule of the sample was initially double-stranded, 15 the "second" probe will hybridize to the compl~mentary strand of the nucleic acid molecule at a position 3' to the seguence 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) 2~ "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 25 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 30 "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 l'first" probe, and thus can serve as a 35 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 W092/10506 ~ 0~,~ 7 PCT/US91/09382 the PCR are provided by Mullis, K.B. (Cold Sprlnq Harbor SYm~.
Ouant. 8iol. 51:263-273 (1986)); Saiki, R.K., et al.
(Bio/Technoloay 3:1008-1012 (1985)); and Mullis, K.B., et al.
(Meth. Enzvmol. 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 Mate~als and Methods Cell Lines The following cell lines were used in the studies described below: CCL120 (ATCC~ CCL120), a human B
15 lymphoblastoid cell line; CCLll9 (CEM, ATCC# CCLll9), a human T lymphoblastoid cell line; SupTl, 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; MOLT4 (ATCC# CRL1582), a human acute lymphoblastic leukemia cell 20 line; HOS (ATCC# CRL1543), a huma~ osteosarcoma cell line;
HeLa (ATCC# CCL2), a human epithelioid carcinoma cell line;
CHO-Kl (ATCC #61~, a Chinese hamster ovary cell line; BlOT6R, a radiation-induced thymoma of BlO.T(6R) mice; and RL12, a radiation-induced thymoma of C57BL/6Ka mice.
25 Screeninq 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 30 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 35 translation to a specific activity of about 2 x 106 cpm/~g and used as a hybridization probe.

WO92/10~06 2 0 9 7 7 0 ~ PCT/US91/09382 Southern Blot Analvsis 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 5 al. J. Virol. 58:296-306 (1986)). Restriction endonuclease digestion, agarose gel electrophoresis, transfer to nitrocellulose (Schleicher & Schuell, lnc., Reene, New Hampshire), hybridization and washing was as described (Pampeno, C. L. et al. supra; Brown, G. D. et al.
10 Immunogenetics 27:239-251 (1988)).
Northern Blot Analysis Total cellular RNA was isolated from cells by the acid guanidinium thiocyanate-phenol-ChlorofQrm method (Chomczynski, P. et al. Anal. Biochem. 162:156-159 (1987)).
15 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)).
20 DNA Sequence 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 Sl nuclease, and sequenced by the 25 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 30 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 35 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)).

W092/lOS06 'JO 9 7 7 o 5 PCT/US91/09382 EXAMPLE II
DNA and Predicted Protein Seouence of H13 The complete nucleotide sequence of H13 (SEQ ID
NO:7) including non-coding sequences at the 5' and 3' end of 5 the coding sequence are shown in Figure 1. This sequence includes the partial sequence originally obtained from clone 7-2 (SEQ ID NO:l); nucleotides 1-6 and 1099-1102 of SEQ ID
NO:1 were originally incorrectly determined. Figure 1 also shows the complete amino acid sequence predicted from the 10 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 20 52.3~ homology between H13 and TEA amino acids.

EXa~PlE I~
Presence and Expression of_~he ~13 Gene in Human Cells By Southern analysis of DNA taken from cells of various species, it was shown that DNA capable of hybridizing 5 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, SupTl, H-9 and MOLT-4, and also in hamster cells (CHO-Kl) and murine cells (normal Balb/c mouse thymocytes). H13 gene expression was examined using Northern 10 analysis, using the H13 cDNA probe. The probe detected a transcript of approximately 9kb in RNA from HeLa, SupTl, HOS
and CCL119 cells (Figure 6). This RNA could also be detected using a murine ERR cDNA probe (Figure 7).

~XA~PLE IV
Transfection of Mu~ine Retroviral Rece~tor cDNA into Hamster Cells Murine retroviral receptor (ERR) cDNA was 5 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 10 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., Virologv 48: 749-756 (1972)), and Northern Blot analysis was performed using a viral probe after preparing its RNA. As shown in 15 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
20 activity of the transfected cell supernatant was much higher than background (Figure 8).
The ~uLV 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 25 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 30 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 35 al., CABIOS 4: 181-186 (1988)). Figure 10 shows the antigenicity profile of the H-13 protein sequence.

W092/10506 2 ~ 9 7 7 ~ S PCT/US91/09382 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 5 ligated to the cloning sites of pGEX-2T plasmid vector (Pharmacia LXB 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 glu~athione Sepharose 4B chromatography (Pharmacia LKB Biotechnology) (see Figure 11). The purified fusion protein injected intramuscularly and subcutaneously 15 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 20 according to standard technigues 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.

EX~LE Vl Genetic ~ap~in~ 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., Cytoqenet. Cell Genet. 51:1025 30 (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, 35 osteosarcoma, Wilson's disease, Letterer-Siwe disease, Dubin-Johnson syndrome, clotting factor Vii and X, collagen IV ~1 and ~2 chains, X-ray sensitivity, lymphocyte cytosolic WO92/10506 2 0 9 ~ 7 0 5 PCT/US91/09382 protein-l, carotid body tumor-l, propionyl CoA carboxylase (~
subunit), etc.

EXAMPL~ 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 ChimeraI - ChimeraIV). 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 tCHO) 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 15 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 ~-galactosidase and neomycin phosphotransferase (neoR) genes ~; which provide a selectable marker and a detectable product.
20 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 n~_R-expressing transfectants. After two weeks, numbers of G418-resistant colonies were counted.
These results indicate that portion of the ERR gene 25 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 30 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 35 employed to produce chimeric molecules containing individual amino acid substitutions within extracellular domain 3. These were transfected as a~ove and the transfectant cells are W O 92/~0506 PC~r/US91/09382 20~7705 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 5 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 10 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 15 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 20 the present di-~closure as co~e 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 25 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 30 should and are intended to be comprehended within the meaning and range of equivalents of the disclosed em~odiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

WO 92/10506 2 0 ~ 7 1 0 ~i PCr/US91/09382 S~NOE IISlING

(1) OENER~ lNFORM~llCN:

Yo6~rol q~YU~
(ii) ll~rlE OF ~ON: }~nan Retn~vi~ Receptor ar~ ~ Coding I5~erefor (iii) N~ OF S~OE:S: 8 (iv) ~REæS~NDENOE ADE~S:
(A) A~i~x B~ and Ne~nark (B) Sl~;~r: 419 Seventh Street, N.W.
(C) CI'rY: Wa~i~
(D) Sl~E: DC
(E) ~RY: ~SA
(F) ZIP: 20004 (v) a~WER RE~ F~:
(A) MEDIt~l TY~: ~o~y disk (B) C~II~: IEM PC npatible (D) SOFIW~ PatentIn Release #1.24 (vi) a~EæNr A~rC~rION n~:
(B) ~ U~E:
(C) CLAssIFlt~oN
(viii) A~/AOE2~r IN~ON:
(A) NPME: Li~mat, 5hnuel (B) REX;ISTRAI~ON Nt~: 33,949 (C) ~;F~OE/DC~l NU~: M~J~l (ix) ~?~CP~ON ~IION:
(A) 1 = E: 202 628-5197 (2) INF~RM~O~ FCR SE~ ID NO:l:
(i) SEX~ENOE CHPRACIERI~
(A) ~æI~I: 1102 base pairs (B) TYPE: nucleic acid (C) S~: single (D) I~POIDGY: linear (ii) r~I~:CULE TYPE: c~NA

( ix) ~lURE:
(A) NAME/ÆY: C~;
(B) I~ON: 1..1102 W O 92/10506 PCT/US9l/09382 ~ ~ 9 ~ ~U~

(Xl) SEQUENOE DESCK~ ON: SEQ ID ND:1:

CCG GGC GCC ACC ITC GAC GAG CTG ATA GGC AGA CCC ATC GGG GAG TTC
Pr~ Gly Ala qhr Phe Asp Glu Leu Ile Gly Arg Pr~ Ile Gly Glu Phe 48 TCA CGG ACA CAC ATG ACT CTG AAC GCC CCC GGC GTG ~1~ GCT GAA AAC 96 Ser Arg Ihr His Met qhr Leu Asn Ala Pro Gly Val Leu Ala Glu As~

CCC GAC AlA TrC GCA GIG ATC ATA AIT CTC ArC Tl~ ACA GGA CIT IIA 144 Prc Asp Ile Phe Ala Val Ile Ile Ile Leu Ile T~- Ihr Gly Leu Leu ACT CIT GGT GTG AAA GAG T~ GCC ATG GTC AAC M A ATA TTC ACT TGT 192 q~r Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile Phe Ihr Cys AIT AAC GTC CrG GTC CTG GGC ITC ATA AIG GTG TCA GGA m GTG A~A 240 Ile Asn Val L~u Val Leu Gly Phe Ile Met Val Ser Gly Phe Val Lys GGA TCG GIT AAA AAC ~GG CAG CTC ACG G~G GAG G~T m GGG AAC AC~ 288 Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr TC~ GGC CGT CTC TGT TTG AAC AAT G~C ACA AAA GAA GGG AAG CCC GGT 336 Ser Gly Arg Leu Cys Leu Asn Asn Asp IThr Lys Glu Gly Lys PrD Gly GIT GGT GGA TT~ ATG OOC TTC GGG TrC TCT GGT GTC CTG TCG GGG GCA 384 Val Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala Ala Thr Cys Phe Tyr Ala Fhe Val Gly Phe Asp Cy~ Ile Ala Thr Thr GGT GAA GAG GTG AAG AAC CCA CAG A~G GCC A~C CCC OEG GGG ATC GT~ 480 Gly Glu Glu Val Lys Asn Pr~ Gln Lys Ala Ile Pr~ Val Gly Ile Val GCG TCC crc TTG ATC TGC TTC ATC GCC TAC TTT GGG GTG TCG GCT GCC 528 Ala Ser Leu Leu Ile CY5 Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala CTC ACG CTC A~G ATG ~ TAC TTC TGC CTG GAC AAT AAC AGC CCC CTG 576 Leu Thr Leu Met Met ~ ~r ~e Cys Leu Asp Asn Asn Ser Pr~ Leu CCC GAC GCC TTT A~G CAC GTG GGC T~G GAA GGT GCC AAG TAC GCA GTG 624 Pr~ Asp Ala Phe Lys His Val Gly Trp Glu Gly Ala Lys Tyr Ala Val GCC GIG GGC TCC CTC rGC GCT ~T TCC GCC AGT CTr Cl~ GGT TCC ATG 672 Ala Val Gly Ser Leu Cys Ala Leu Ser Ala Ser Leu Leu Gly Ser Met m ccc AIG CCT CGG GTT ATC lAr GCC ATG GCT GAG GAr GGA CTG CI~ 720 Phe Pr~ Met Pr~ Arg Val Ile Tyr Ala Met Ala Glu Asp Gly T~ll Leu TTT AAA TTC q ~ GCC A~C GTC MT G~T AGG ACC MA ACA CCA A~ AIC 768 Phe Lys Phe T~- Ala Asn Val Asn Asp Arg Thr Lys Thr Pro Ile Ile GCC ACA TTA GCC ICG GCr GCC GlT GCT GCT GTG Al~; GCC ITC 1~ m 816 Ala Thr Leu Ala Ser Gly Ala V 1 Ala Ala Val Met Ala Phe LPI1 Phe GAC ~ AAG G~C TTG ~rG G~C CTC ATG T~C Al'r GGC ACT CTC CTG GCT 864 Asp Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Val T~l Arg Tyr Gln PrD Glu CæG CCT AAC CIG GTA ~hC CAG ATG GCC AGT ACT TCC G~C G~G TTA G~T 960 Gln PrD Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp Glu Leu Asp CC~ GCA GAC CA~ A~T GAA TTG GC~ AGC ACC AA~ GAT TCC CAG CTG GGG 1008 Pr~ Ala Asp Gln Asn Glu T~l Ala Ser Thr Asn Asp Ser Gln T~l Gly m TI~ CC~ GAG GC~ G~G A~G TTC TCT TTG AAA ACC ATA C~C TC~ CCC 1056 Phe T~U Pro Glu Ala Glu Met Fhe Ser L2U Lys m r Ile Leu Ser Pro 3~0 345 350 A~A AAC AlG G~G CCT TCC A~A AIC TCT GGG CTA AIT GTG AAC COG G 1102 Lys Asn Met Glu Pr~ Ser Lys Ile Ser Gly Leu Ile Val Asn PrD

(2) INFORM~IION FOR SEQ ID N0:2:
(i) SEÇUENCE CH~RA~l~KlSTICS:
(A) LENGIH: 367 aminD acids (B) TYPE: amlnD acid tD) TOPOLDGY: linear (ii) MOLECULE TYFE: protein (xi) SEÇpENOE DESCRIPIION: SEQ ID NO:2:
Pro Gly Ala Thr Phe Asp Glu Leu Ile Gly Arg Pro Ile Gly Glu Phe Ser Arg Thr His Met Thr Leu Asn Ala Pro Gly Val Leu Ala Glu Asn W O 92/10506 ~ U ~ PC~r/US91/09382 Pr~ A~p Ile Phe Ala Val Ile Ile Ile Leu Ile Leu qhr Gly T~l Leu Ihr Leu Gly Val Lys Glu S~r Ala Mk~t Val PY~n Lys Ile Phe Thr Cys Ile A~n Val Leu Val Leu Gly Fhe Ile ~t Val Ser Gly Phe Val Lys ly S~r Val Lys Asn Trp Gln Leu Thr Glu Glu ~Y~p Phe Gly Asn Ihr ~Yr Gly A~ng Leu Cys Leu A~;n Asn P~p I~Lr Lys Glu Gly Lys Pro Gly ~l Gly Gly Phe Met PrD Phe Gly Phe S~r Gly Val Leu Ser Gly Ala Ala q~lr Cys Fhe Tyr Ala Phe Val Gly Phe AY~P Cys Ile Ala Thr Thr Gly Glu Glu Val Lys AY3n Pr~ Gln Lys Ala Ile Pr~ Val Gly Ile Val la Sb~r Leu Leu Ile Cys Fhe Ile Ala q~nr Fhe Gly Val Ser Ala Ala eu q~Lr Ifau Met Mbt Pso q~r Phe Cys Leu A~p A~;n Asn Ser Pro Leu ro A~p Aaa Phe Lys His Val Gly q~p Glu Gly Ala Lys Tyr Ala Val Ala Val Gly Ser leu ~s Ala Leu Ser Ala Ser Leu Leu Gly Ser Met Phe Pro ~ t Pro Arg Val Ile q~r Ala ~ t Ala Glu ~ p Gly Leu T~

he Lys Fhe T~ Ala Asn V 1 ~n A ~ Arg Thr Lys Thr Pro Ile Ile la Thr Leu Ala Ser Gly Ala Val Ala Ala Val Met Ala Phe Leu Fhe ~p Leu Lys A~p Leu Val A~p Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val Aaa Ala Cys Val Leu Val Leu A ~ ~r Gln Pro Glu Gln Pro A~n Leu Val Tyr Gln Met Ala Ser qllr Ser ~p Glu Leu Asp Pro Ala Asp Gln Asn Glu Leu ~aa Ser mr Asn Asp Ser Gln Leu Gly W O 92/10506 2 ~ ~ 7 7 0 ~ PCT/US91/09382 Phe Leu Pro Glu Ala Glu Met Fhe Ser Leu Lys Thr Ile Leu Ser Pr~

~ys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val Asn PrD

4) DNFOR~aIlCN FOR SEQ ID ND:3:

(A) IENGTH: 2425 base pairs (B) TYPE: nucleic acid (C) SlRANDELNESS: s mgle (D) TDPOLDGY: lLnear (ii) MDLECUIE IYPE: cDNA
(ix) P~:
(A) N~ME/XEY: CDS
(B) IDChIIoM: 199..2064 (xi) SE~UENCE DeSCRIPq5QN: SEQ ID N0:3:

G~ITCCECCC GCCIOCGCC~ qCCCClC~GC IaoCaGGTGT G~Ga~GCTTr CT~CCCECGG 60 I~TCC~C~C~ GCIC~ACArC IIGCCGCCIC CTCCGAGCCr G~AGCI~CCG IGCPCICIGC 120 qGTG.~GT:r IG30000CAG GrGOE G~IOC I~C~aLIGA G~AGrCCCAC G~GTCTT~C~ 180 GCALo~l~CC TCAGC~CA AIG GGC TGC AA~ AAC CIG CTC CTG GGC C~G 231 Met Gly Cys Lys Asn Leu T~l Gly Leu Gly Gln CAG ATG CTG CGC CGG A~G GIG GTG GAC TGC AGC CGG GAG G~G AGC CGG 279 Gln Met T~7 Arg Arg Lys Val Val Asp Cys Ser Arg Glu Glu Ser Arg CTG TCC CGC TGC crc AAC ACC TAT GAC CTG GTA GCT CTT GGG G~G GGC 327 T~- Ser Arg Cys Leu Asn Thr Tyr Asp Leu Val Ala Leu Gly Val Gly AGC ACC ~1~ GGC GCT GGT GTC TAT GTC CTA GCC GGT GCC GTG GCC CGl~ 375 Ser Thr Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Ala Val Ala Arg GAA AAT GCT GGC CCT GC~ ATC GTC ATC TC~ TIC TrG ATT GCT GCT CT~ 423 Glu Asn Ala Gly Pr~ Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu GCC TCC GTG CrG GCC G C CTG TGC TAC GGC G~G TTT GGT GCC CGT GTC 471 Ala Ser Val Leu Ala Gly Leu Cys Tyr Gly Glu Phe Gly Ala Arg Val go ~0~'~7~

CCC AAG ACG GGC TCA GCC TAC CTC TAC A~C TAC GTG ACG GIG GGG GAG 519 Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu CTr TGG GCC TTC AIC ACT GGC TGG AAC CTG ATT CTC TC~ TAC ATC ATC 567 T~- Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile 110 115 ~.20 GGT ACT TC'A AGC GIG GCA AGA GCC TGG AGT GCG ACT m GAC GAG CIG 615 Gly qhr Ser Ser Val Ala Ar3 Ala Trp Ser Ala Thr Phe Asp Glu Leu A~A GGC AAG CCC ATC GGA GAG TTC TCA CGT C~G C~C ATG GCC CIG A~T 663 Ile Gly Lys Pro Ile Gly Glu Fhe Ser Arg Gln His Met Ala Leu Asn GCT C T G G GTG CTG GC~C C~AA AOC CCG GAC ATA m GCT ~1~ ATT ATA 711 Ala Pro Gly Val Leu Ala Gln Thr Pr~ Asp Ile Phe Ala Val Ile Ile ATT A~C ATC TTA ACA GGA CTG TTA ACT CTT G~C GTG AAG GAG TCA GC~ 759 Ile Ile Ile ~ Thr Gly Leu Leu Thr Leu Gly Val Lys Glu Ser Ala 1~5 180 185 AT~ GTC AAC AAA AIT TTC ACC TCT ATC AAT ~1~ CTG G~C ITG TGC ITC 807 Mbt Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val T~l Cys Phe ATC G~G ~1~ TCC GGG TrC GTG AAA GGC TCC A~T M A AAC I~G CAG CTC 855 Tle Val Val Ser Gly Phe Val Lys Gly Ser Ile Lys Asn Tr,o Gln Leu ADG GAG AAA AAT ITC TCC T&T AAC AAC AAC G~C AC~ AAC GTG AAA I~C 903 Ihr Glu Lys Asn Phe Ser Cys Asn Asn Asn Asp Thr Asn V~l Lys Tyr G~T G~G G~ GGG TTT A~G CCC m GGA TTC TCT GGT GTC CTG TCA GGG 951 Gly Glu Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val T~ Ser Gly GCA GCG ACC TGC m T~T GCC TTC GTG GGC m GAC TGC ATC GCC ACC 999 Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr ACA G~G GAA GAA GTC AAG AAC CCC CAG AAG GCC ATT c~r GTG GGC ATC 1047 Thr Gly Glu Glu Val Lys Asn PrD Gln Lys Ala Ile Pr~ Val Gly Ile GTG GCG TCC CTC CTC ATT TGC TTC ATA GCG TAC m GGC GTG TCC GCC 1095 Val Ala Ser Leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Al~

GCT CTC ACG crc ATG ATG cc~r TAC TTC TGC CTG GAC A'TC GAC AGC C~G 1143Ala Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser PrD

W O 92/10506 2 0 9 7 7 0 ~ PCT/US91/09382 CTG CCT GGT GCC TTC A~G CAC CAG GGC TGG GAA GAA GCT AAG TAC GCA 1191 T~l Pr~ Gly Ala Phe Lys His Gln Gly Trp Glu Glu Ala Lys Tyr Ala GTG GCC ATT GGC TCT CTC TGC GCA CTT TCC ACC AGT CTC CrA GGC TCC 1239 Val Ala Ile Gly Ser Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser ATG m ccc ATG CCC CGA GIT ATC TA~ GCC ATG GCT GAA GAT GGA CTA 1287 Met Phe Pro Met Pr~ Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu ~1~ m AAA m TTG GCC AAA ATC AAC A~T AGG ACC AAA AC~ CCC Gl~ 1335 Leu Fhe Lys Phe Leu Ala Lys Ile Asn Asn Arg lhr Lys Thr Pro Val ATC GCC ACT GIG ACC TCA GGC GCC AIT GCT GCT GTG ATG GCC TrC CTC 1383 Ile Ala Thr Val Thr Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu m G~A CIG AAG GAC CTG GTG GAC CTC ATG TCC A~T GGC ACT crc CTG 1431 Fhe Glu Leu Lys Asp T~l Val Asp Leu Met Ser Ile Gly Thr Leu Leu GCT TAC TCT TTG GTG GCT GCC TGr GIT TTG GTC TTA CGG TAC CC~ 1479 Ala Tyr Ser T~l Val Ala Ala Cys Val Leu Val T~- Arg Iyr Gln Pr~

GAA CAA CCT AAT C~G GTA TAC A'rG GCC ACA ACC ACC GAG G~G CI~ 1527 Glu Gln Pro Asn Leu Val Tyr Gln Met Ala Arg qhr Thr Glu Glu Leu GAT oG~ GTA GAT AAT GAG C~G GTC A~T GCC AGT GAA TCA C~G ACA 1575 Asp Arg Val Asp Gln Asn Glu T~l Val Ser Ala Ser Glu Ser Gln Thr GG~ TTT TTA CCG GTA GCC GAG AAG m TCT ~1~ A~A TCC ATC crc TCA 1623 Gly Phe Leu Pro Val Ala Glu Lys Phe Ser Leu Lys Ser Ile Leu S r CCC AAG AAC GTG GAG OOC TCC AAA TTC TC~ ~GG CTA ATT GTG AAC ATT 1671 Pro Lys Asn Val Glu Pro Ser Lys Phe Ser Gly Leu Ile Val Asn Ile TC~ G C GGC crc crA GCC GCT CTT ATC ATC ACC GIG TGC ATT GTG GOC 1719 Ser Ala Gly Leu Leu Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala Gl~ CTT GGA AGA GAG GCC CTG GCC GAA GGG ACA CTG TGG GCA GTC TTT 1767 Val L~u Gly Arg Glu Ala Leu Ala Glu Gly Ihr Leu Trp Ala Val Phe GTA ATG ACA GGG TC~ GTC CTC CI~- TGC ATG CTG G~G ACA GGC AIC AIC 1815 Val Met Thr Gly Ser Val Leu Leu Cys Met Leu Val Thr Gly Ile Ile W O 92/10506 ~ ~ ~ 7 7 o ~ PCT/VS91/09382 TGG AGA C~G CCT GAG AGC AAG ACC AAG CTC TCA m AAG GTA CCC m 1863 Trp Arg Gln Pro Glu Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe GrC CCC GTA CIT CCT ~-~ TIG AGC ATC TTC GIG AAC AIC T~r CTC ATG 1911 Val Pro Val Leu Pr~ Val Leu Ser Ile Phe Val Asn Ile Tyr Leu Met ATG CAG CIG GAC C~G GGC ASG IGG GIC CGG m GC~ GTG TGG ATG CTG 1959 Met Gln T~- Asp Gln Gly Ihr Trp Val Arg Phe Ala Val Trp Met Leu AIA GGT TrC ACC ATC TAT TrC GGT TAT G~G ATC IGG CAC AGT G~G G~A 2007 Ile Gly Phe Thr Ile Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu GCG TCC CTG GCT GCT GGC C~G GC~ AAG Acr CCT GAC bGC AAC TTG GAC 2055 Ala Ser Leu Ala Ala Gly Gln Ala Lys Thr E~o Asp Ser Asn Leu Asp CAG TGC AAA ~GPoGIGCA~ c~Acc~ac CAGGGIGACA GCGGTTGACG 2104 G}n Cys Lys GGIGC~ OE ra GPACc~nGCG A~ICAChA T~TCTCCACT car~OCTC~G GATcAGcqC~ 2164 CaC~rDAr GTCACCAAAG clGGrrIacr GC~ALCTCGT GAG~5CCrGG ICarTICTGG 2224 ALPGICC~Tr GCTTIPC~CA l~lCDClCr: AACA~AGAAA GCAGCOCIrC TC~TTGCCGG 2284 IGCGGC~OCA GCaEAAGGGA GGCC~CCTTC ICCTCTCACT 2344 loGGAAGIAG GC~IC~CTOC CIOOCTGaG~ CC~CCCTGGC AICGC~IG TGCAC~CTCC 2404 ALP3CCCTAG IGPGC~TCT~ C 2425 (5) INF~Na~ION FOR SEQ ID N0:4:
ti) SE~y~NOE CH~RACTERISTIC5:
(A) LENGIH: 622 a~ acids (B) TYPE: amino acid (C) STFANDer~ESS: s mgle (D) TOPOLDGY: linear (ii) MDLECULE TYPE: protein (xi) SEÇUENOE DESCRIPqION: SEQ ID NO:4:
Met Gly Cys Lys Asn Leu Leu Gly Leu Gly Gln Gln Met Leu Arg Arg Lys Val Val Asp Cys SOE Ar~ 51U G1U SOE Ar~ Leu Ser Ar~ Cys Leu ~0~7705 Asn Thr Tyr Asp Leu Val Ala Leu Gly Val Gly Ser Thr Leu Gly Ala Gly Val Tyr Val Leu Ala Gly Ala Val Ala ~ Glu Asn Ala Gly Pro Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Leu Ala Gly Leu Cys Tyr Gly Glu Fhe Gly Ala Arg Val Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile 10~ 105 110 qhr Gly Irp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val Ala Arg Ala Trp Ser Ala Ihr Phe Asp Glu Leu Ile Gly Lys Pro Ile Gly Glu Phe Ser Arg Gln His Met Ala Leu Asn ~la Pro Gly Val Leu Ala Gln Thr Pro Asp Ile Phe Aaa Val Ile Ile Ile Ile Ile Leu Thr Gly Leu Leu Thr Leu Gly V~l Lys Glu Ser Ala Met Val Asn Lys Ile 180 lB5 190 Fhe Thr Cys Ile Asn Val leu Val Leu Cys Phe Ile Val Val Ser Gly Phe Val Lys Gly Ser Ile Lys Asn Trp Gln Leu Thr Glu Lys Asn Phe Ser Cys Asn Asn Asn Asp Thr Asn Val Lys I~r Gly Glu Gly Gly Phe Met Pro Phe Gly Phe Ser Gly Val Leu Ser Gly Ala Ala Ihr Cys Phe Tyr Ala Phe Val Gly Phe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Lys Asn Pro Gln Lys Ala Ile Pro Val Gly Ile Val Ala Ser Leu Leu Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Phe Cys Leu Asp Ile Asp Ser Pro L u Pro Gly U a Phe Lys His Gln Gly Trp Glu Glu Ala Lys Tyr Ala Val Ala Ile Gly Ser W O 92/10506 ~ V 9 7 7 0 ~ PCT/US91/09382 Leu Cys Ala Leu Ser Thr Ser Leu Leu Gly Ser Met Phe PrD Met PrD

Arg Val Ile Tyr Ala Met Ala Glu Asp Gly T~l Leu ~he Lys Phe Leu Ala Lys Ile Asn Asn Arg Ihr Lys Thr Pro Val Ile Ala Thr Val Thr Ser Gly Ala Ile Ala Ala Val Met Ala Phe Leu Phe Glu Leu Lys Asp Leu Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr Gln Pro Glu Gln PrD Asn Leu 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 C~r Glu Ser Gln Thr Gly Phe Leu PrD Val Ala Glu Lys Phe Ser Leu Lys Ser Ile Leu Ser Pr3 Lys Asn Val Glu PrD Ser Lys Phe Ser Gly Leu Ile Val Asn Ile Ser Ala Gly Leu Leu Ala Ala Leu Ile Ile Thr Val Cys Ile Val Ala Val Lsu Gly Arg Glu Ala T~l Aaa Glu Gly Ihr Leu ~ Ala Val Phe Val Met Thr Gly Ser Val Leu T~ll Cys Met Leu Val Ihr Gly Ile Ile Trp Arg Gln Pr~ Glu . Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe Val PrD Val Leu PrD

Val Leu Ser Ile Phe Val Asn Ile Tyr T~l Met Met Gln Leu ~ Gln Gly Thr Trp Val Arg Phe Ala Val Trp Met Leu Ile Gly Phe Thr Ile Tyr Phe Gly Tyr Gly Ile Trp His Ser Glu Glu Ala Ser Leu Ala Ala Gly Gln Ala Lys Thr Pro Asp Ser Asn Leu Asp Gln Cys Lys WO 92/10506 2 0 9 7 7 0 ~ PCr/US91/09382 (2) ~FORM~ON FOR S}~Q ID NO:5:

(i) SE~IOE a~ACrERIS~CS:
(A) ~I~: 2397 base pairs (B) I'Y~: nucleic acid (C) ~s: s~le (D) IOPO~GY: l~near (ix) E~lU~:
(A) tlP~: CDS
(B) IDCl~llON: 410 . .1768 (xi) SE~ENOE I~ESCRI~r[ON: SE~ :5:

GGG~ A~;Cr mn:GCCr a3~I~XC a~CITn.C I~lGCm~AT 60 llGa~: AGIA~; ACI~ ~= CAClTACGIC AX~I~G 120 AGCI~I ~ Cl ~ ~ TC l ~ ~ I~rC ~ TA GGrACGI~C~ 180 GTGTCGCAAG AGcaIGGAGr GGCAC~IITG ACGhbCTTCr TAAI~A~CAG A7lGGccaoT 240 mDc;AAAc G~ACIICYAA A5GAAlTACA CqGGTClGGC AGAoTATccA GACTTCTTTG 300 CCG~GrGCCT IGIATTACr~ ClGGCAGGrC mrYrcm IGEACTPAAA Gprcl~cTT 360 rGTGpAT~A AmrrhcaG CIATI~UTAT CCn3aTCCTT CTCTIDCTC AT& GTG 415 Met Val GCT GGG m GIb A~A GG~ AAT GTG GCT A~C T æ AAG ATC AGT GAA GAG 463 Ala Gly Phe Val Lys Gly Asn Val Ala Asn Trp Lys Ile Ser Glu Glu TIT CTC AAA AAT ATA TCA GCA AGT GCT ALA GAA CCA C'CT ~ GAG AAC 511Phe Leu Lys Asn Ile Ser Ala Ser Ala Arg Glu Pro Pro Sex Glu Asn GGA ACA AGC ATC TAC GGG GCT GGC GGC TIT ATG CCC TAT GGC m ACA 559 Gly Thr Ser Ile Tyr Gly Ala Gly Gly Phe Met Pr~ Tyr Gly Phe Thr GGG ACG TrG GCT GGT GCT GCA ACG TGC m TAT GC~ m GTG GGC m 607 Gly Thr Leu Ala Gly Ala Ala Thr Cys Phe Tyr Ala Phe Val Gly Phe GAC T&C ATT GCA ACA ACC GGT GAA GAG G~T oGG AAT C~A CAA AAG GOG 655 Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Arg Asn Pr~ Gln Lys Ala W O 92/10~06 ~ 7 0 ~ PCT/US91/09382 ATC CCC ATC GGA ATA GTG AOG TCC T~A CIT GTC TGC m AT~ GCT TAC 703 Ile Pro Ile Gly Ile Val Thr Ser Leu Leu Val Cys Phe Met Ala Tyr m GGG GIT TCT GCA GCT TTA ACG CIT ATG ATG CCT TAC TAC crc CTG 751 Phe Gly Val Ser Ala Ala Leu Thr Leu Met Met Pro Tyr Tyr Leu Leu G~T GAG AAA AGT CC~ CTC CC~ GTC GCG m GAG TAT GTC AGA TGG G~C 799 Asp Glu Lys Ser Pr~ Leu Pro Val Ala Phe Glu Tyr Val Arg Trp Gly 115 120 ~75 130 CCC GCC AAA TAC GIT GTC GCA GCA GGC TCC CTC TGC GCC Tr~ TC~ ACA 847 Pr~ Ala Lys Tyr Val Val Ala Ala Gly Ser Leu Cys Ala Leu Ser Thr AGT Cl'l' CIT GGA TC~ AIT TTC OCA ATG CCT C~T G~ AIC TAT GCr ATG 895Ser Leu Leu Gly Ser Ile Phe Pro Mbt Pr~ Arg Val Ile Tyr Ala Met GC~ GAG GAT GGG TTG CIT TTC AAA TGT CTA GCT CAA ATC AAT TCC AAA 943 Ala Glu Asp Gly T~u T~l ~e Lys Cys Leu Ala Gln Ile Asn Ser Lys ACX~ AAG AC~ ~rA ATT GCT ACT ~G ~ TC; GGT GC2~ GTG GC~ GCT 991 q~r Lys l~r ~ro Val Ile Ala Ihr Le~ Ser Ser Gly Ala Val Ala Ala GTG AIIG GOC m CTT I~CT G~C CTG AAG GOC CTC GTG G~C AIG A~rG TCT 039 V~l ~et Ala Phe L~u Phe Asp Leu ~rs Ala T~l Ual Asp Met Met Ser AIT GGC ACC CTC A~ GCC TAC ICT CTG GrG GC~ GOC IGT GTG ~11 A~ 087 Ile Gly fflr L~u Met Ala l~r Ser Leu Val Ala Ala Cys Val Leu Ile AGG TAC CAA CCT GGC qTG TGT q~C GPY; CP~G COC AAA I~C AOC CCT 1135 Leu Ar~ ~r Gln }~ ly ~u ~ys l~yr Glu Gln ~ ~s Tyr Ihr ~ro GAG AAA GAA ACT CTG GAA T~A IGT ACC AAT GCG ACT TIG AAG AGC GAE 1183 Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys Ser Glu Ser Gln Val Thr Met Leu Gln Gly Gln Gly Fhe Ser keu Arg mr Leu ~e Ser E~ro Ser Ala Lu ~ Ihr Ar~ Gln Ser Ala Ser Leu Val Ser TTT ~1~ GTG GGA TTC CrG GCT TTC CIC ATC CTG GGC TTG AGT AIT CTA 1327 E~e Leu Val Gly Phe Leu Ala Phe Leu Ile Leu Gly Leu Ser Ile Leu W O 92/10506 2 o 9 7 rl O ~ PCT/US91/09382 ACC ACG lAr GGC GTC CAG GCC Alr GCC AGA CT~ GAA GCC TGG AGC CTG 1375 Thr Thr Tyr Gly Val Gln Ala Ile Ala Arg Leu Glu Ala Trp Ser Leu GCT CTT CTC GCC CrG TTC W l GTC ~ C TGC GCT GCC GTC ~TT CTG ACC 1423 Ala Leu T~l Ala Le~ Phe Leu Val Le~ Cys Ala Ala Val Ile Leu Thr AIT TGG A~G CAG CCA CAG AAr CAG C~ AAA GIA GCC TT~ ATG GTC CCG 1471 Ile Irp Arg Gln PrD Gln Asn Gln Gln Lys Val Ala Phe Met Val Pro TTC TTA CCG m CTG OCG GCC TTC AGC AIC CTG GTC AAC ATT IAC TTG 1519 Fhe Leu Pr~ ~he Leu PrD Ala Fhe Ser Ile Leu Val Asn Ile Tyr Leu AIG GTC C~G TT~ A~T GaG GAC ACT TGG ATC AG~ TTC AGC ATC TGG ATG 1567 Mbt V~l Gln Leu Ser Ala Asp Thr Trp Ile Arg Phe Ser Ile Trp Met GCG CIT GGC TTr CTG AIC TAT TrC GCC I~T GGC AIT AGA C~C AGC TIG 1615 Ala Leu Gly ~he T~- Ile Tyr Phe Ala Tyr Gly Ile Arg His Ser Leu G~G GGT AAC CCC AEG G~C G~A GAA G~C G~T G~G G~T GCC TTT TCA GAA 1663 Glu Gly Asn PrD Arg Asp Glu Glu Asp Asp Glu Asp Ala Phe Ser Glu A~C ATC AAr GTA GCA ACA GAA GAA AAG TCC GTC A~G CAA GCA AAT GAC 1711 Asn Ile Asn Val Ala Thr Glu Glu Lys Ser Val Met Gln Ala Asn AsF

CAT CAC CA~ hGA A~C crc AGC TT~ CCT TTC ATA CTT CAT G~A AA5 ACA 1759 His His Gln Arg Asn Leu Ser Leu PrD Phe Ile T~1 HiS Glu Lys Thr A~T G~A IGT TG nGcIaGc CCICEGTCIT AC~ACGC~TA CCII~ACAAT 1808 Ser Glu Cys GAGI~CACIG T&GCCGGATG CCACCAICGT GC3GGGCTGT CGnG3GTCqa CTGT~G~CAT 1868 GGClTGCCIa ACITGTACIT CrTCCqCC2a ACAGCrTCnC TrcaGaDGaT GGATrCTGlG 1928 ICIG2GGAGA cTGccIGAGa GCACTCCTCA GCTATATGTA TCo~C~AA~C AGTATCICCG 1988 TGIGOGTACA TGTAT~lw ~ CGArCIG~T GTT~PPICIT GTCCGITATT A ~ AC 2048 AT~ATICCAG CATGGIAATT GGT~GCATAT ACTGCACACA CTAGTAAACA GTATATIGCT 2108 GA~TAGAGAT GT;TTCTGTA TATGTCCTAG GTGGCIGGGG AA~TAGIGGT GGTTTCTITA 2168 TIa3GT~IAT GACCATCAGT TIGGACATAC T&AAAIGCCA TCCO~IaIC~ GGAIGIITAA 2228 C~GTGGTcaT GGGDGGGGAA GGGaIaAEGA AIGGGCATrG TCT~IAAATT GI~ATGCATA 2288 TATCCITCTC CTACTIGCTA AGACAGCTTT cTTAAAaGGc C~GGGAGAGT GrTnCrTTcC 2348 WO 92/10506 ~ o ~ r~ t~ ~J 5 PCI/US91/09382 ~ 84 --I~l~.l~C MG~ a~i~5r (a3c~Ga~ ~ 2397 (7) INFORM~lION F~R SEQ ID N0:6:
(i) SE~ENCE CH~RACrERISrICS:
(A) ~: 453 am~no acids (B) TY~: amino acid (C) Sl~: single (D) lOPOLOGY: linear (ii) ~IE0I.E T~: pr~tein (xi) SE~ENCE DESC~:ON: SE~Q ID N0:6:
Met Val Ala Gly }~e V~l Lys Gly Asn Val Ala Asn l~p Lys Ile Ser Glu Glu E~e Leu Lys Asn Ile Ser Ala Ser Ala Ar~ Glu E~o E~ Ser Glu Asn Gly Ihr Ser Ile ~r Gly Ala Gly Gly ~e Met ~ l~rr Gly ~e q~r Gly Ihr Leu Ala Gly Ala Ala mr ~ys ~e Tyr Ala l~e Val Gly E~e Asp C~ys Ile Ala n,r ~hr Gly Glu Glu Val Ar~ Asn ~ro Gln Lys Ala Ile ~ Ile Gly Ile Val mr Ser Leu Leu Val Cys Phe Met Ala l~ e Gly Val Ser Ala Ala Leu mr Leu Met Met ~7 Tyr Tyr Leu T~ Asp Glu Lys Ser ~ro Leu Pro Val Ala Ehe Glu Tyr Val Arg Trp Gly ~ro Ala Lys Tyr Val Val Ala Ala Gly &r Leu Cys Ala Leu Ser Ihr Ser T~77 Leu Gly Ser Ile ~e ~ Met ~ro Ar~ Val Ile Tyr Ala Met Ala Glu Asp Gly Leu Leu ~e Lys Cys Leu Ala Gln Ile Asn &r Lys mr Lys Ihr ~ro Val Ile Ala mr Leu Ser Ser Gly Ala Val Ala Ala Val Met Ala ~e Leu ~e Asp Leu Lys Ala Leu Val Asp Met W O 92/10506 20g 7 70S PCT/US91/09382 Met Ser Ile Gly Thr Leu Met Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Ile T ~ Arg Tyr Gln Pro Gly Leu Cys Tyr Glu Gln Pro Lys Tyr Thr Pro Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys Ser 51u Ser Gln Val Thr Met Leu Gln Gly Gln Gly Fhe Ser Leu Arg Thr Leu Ehe Ser Pro Ser Ala Leu Pro Thr Arg Gln Ser Ala Ser Leu V~l Ser Phe Leu Val Gly Phe Leu Ala Phe Leu Ile T~l Gly Leu 5er Ile Leu Ihr Thr Tyr Gly Val Gln Ala Ile Ala Arg Leu Glu Ala Trp Ser Leu Ala Leu Leu Ala Leu ~he leu Val Leu Cys Ala Ala V~l Ile L~u Thr Ile Trp Arg Gln Pro Gln Asn Gln Gln Lys Val Ala Phe Met VA1 Pro Phe Leu Er~ E~e Leu F~o Ala he Ser Ile T~l Val A~n Ile Tyr Leu Met V~l Gln Leu Ser Ala Asp Thr Trp Ile Arg Phe Ser Ile Trp Met Ala Leu Gly E!he Leu Ile Tyr Phe Ala Tyr Gly Ile Arg His Ser Lsu Glu Gly Asn PrD Arg Asp Glu Glu Asp Asp Glu Asp Ala Ehe Ser Glu Asn Ile Asn Val Ala mr Glu Glu Lyæ Ser Val Met Gln Ala Asn Asp His His Gln Ary Asn Leu Ser Leu Pro Phe Ile Leu His Glu Lys Thr 5er Glu Cys (2) INF~RXPIION F~R SEQ ID N0:7:
(i) 5EQUEN OE CH~R~l~KlSTYCS:
(A) LENGTH: 2157 base pairs (B) TYPE: nucleic acid (C) STRaN~EINESS: single (D) T~POLDGY: linear ~) ()9 770~
~ 86 -(:~x) ~:
(A) N~ME/KEY: CDS
(B) LDC~IION: 1482034 (Xi) SE~UENOE ~ESCRIPqIQN: SEQ ID NO:7:

COalCCTGOC G~AECCrCGC CGCCGCrGaC IIGG~rlC~G AAA ~ TGIATCCCTC 60 CTG~G~C~rC Tq1GCIGChA Ga~CEaGGCT G~CCIC53Gr GaG~GGrGG I~aGGCqIC~ 120 CGTCAIarIC CPGCTCIG;; C~GCAAC AIG GGG IGC A~A GTC CTG CTC AAC AIT 174 Met G1Y CYS VA1 T~- L u Asn Ile Ile GGG CAG CAG ATG CTG CGG CGG AAG GTG GTG G~C IGT AGC CGG GAG GhG 222 Gly G1n G1n Met LeU Arg Arg Lys Val Val Asp Cys Ser Arg G1U G1U

ACG CGG CTG TCT OGC TGC CTG AAC ACT TTT GAT CIG GTG GCC crc GGG 270 Thr Arg T~ Ser Arg Cys Leu Asn Thr Phe Asp LRU Val Ala L~u Gly GIG GGC AGC AC~ CTG GGr GCT GGr GTC TAC GIC C~G Gcr GG~ GCT GTG 318 Val Gly Ser Thr T~l Gly Ala Gly Val Tyr V~ T~l Ala Gly Ala Val G C OGT G~G AAT GCA GGC OCT GCC ATT GTC AIC TCC TTC CIG A~C GCT 366 Ala Arg Glu Asn Ala Gly PrD Ala Ile Val Ile Ser Phe Leu Ile Ala GCG CrG GCC TCA GIG CTG G T GGC CrG TCC IAT GGC GAG TIT GGT GCT 414 Ala Leu Ala Ser Val Leu Ala Gly Leu C~s Tyr Gly Glu Fhe Gly Ala ~ GrC CCC AAG AOG GGC TCA Gcr TAC CTC TAC AGC TAT GTC ACC' GTT 462 Arg Val Pro Lys Thr Gly Ser Ala Tyr Leu ~yr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr ATC ATC GGr ACT TCA AGC GTA GCG AGG GCC T~G AGC GCC ACC TrC GAC 558 Ile Ile Gly Thr Ser Ser Val Ala Ary Ala Trp Ser Ala Thr Phe Asp Glu Leu Ile Gly Arg Fr~ Ile Gly Glu Fhe Ser Arg Thr His Met Thr 20~770~

CTG AAC GCC CCC GGC GTG CTG GCT GAA AAC CCC GAC ATA ITC GCA Gl~ 654 Leu Asn Ala Pro Gly Val Leu Ala Glu Asn Pro Asp Ile Phe Ala Val ATC ATA ATT CTC ATC ITG ACA GGA CTT TrA ACT CIT GGT GIG AAA GAG 702 Ile Ile Ile Leu Ile Leu Thr Gly Leu Leu Thr T~l Gly Val Lys Glu TCG GCC AT~ GTC AAC AAA ATA ITC ACT TGT ATT AAC GTC CIG GTC CTG 750 Ser Ala Met Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly Phe Val Lys Gly Ser Val Lys Asn Trp CAG CTC ACG GAG GAG GAr TTT GGG AAC A~ TC,A GGC CGT CTC TGT TrG 846 Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr Ser Gly Arg Leu Cys Leu Asn Asn Asp ~hr Lys Glu Gly Lys Pro Gly Val Gly Gly Phe Met PrD

TTC GGG TTC TCT GGT GTC CTG qCG G~G GC~ G~G Acr T~C TTC ~T GC~ 942 Phe Gly Fhe Ser Gly Val Leu Ser Gly Ala Ala Thr Cys Yhe Tyr Ala TTC GTG GGC ITT GAC TGC ATC GCC AOC ACA GGT GAA G~G GqG AAG AAC 990 Fhe Val Gly Ehe Asp Cys Ile Ala Thr Thr Gly Glu Glu Val Lys Asn CCA CAG AAG.GCC ATC CCC G~l~ GGG AIC G$G GCG TCC CTC ITG A~C TGC 1038Pro Gln Lys Ala Ile Pro Val Gly Ile Val Ala Ser Leu Leu Ile Cys ITC ATC G C TAC TIT GGG GTG TCG GrT GCC CTC ACG CTC ATG AIG CCC 1086 Phe Ile Ala Tyr Phe Gly V~l Ser Ala Ala T~- Thr Leu Met ~et Pro TAC TTC TGC CTG GAC A~T AAC AGC CCC CTG CCC GAC GrC TTT A~G CAC 1134 Tyr Phe Cys Leu Asp Asn Asn Ser Pro Leu Pro Asp Ala Phe Lys His GTG GGC TGG GAA GGT GCC AAG TAC GCA GTG GCC ~1~ GGC TCC CTC TGC 1182 Val Gly Trp Glu Gly Ala Lys Tyr Ala Val Ala Val Gly Ser Leu Cys GCT CTT TCC GCC A~T CTT CIA GGT TCC ATG m ccc ATG CCT CGG GIT 1230 Ala Leu Ser Ala Ser Leu Leu Gly Ser Met Phe Pro Met Pro Arg Val A'TC TAT GCC ATG GCT GAG GAT GGA. C~G CTA m AAA TTC TTA GCC A~C 1278 Ile Tyr Ala Mbt Ala Glu Asp Gly Leu Leu Phe Lys Phe Leu Ala Asn W O 92/10506 2 0 ~ ~ 7 ~ 5 PCT/US91/093X2 GTC AAT G~T AGG ACC AAA ACA CCA ATA ATC GCC AC~ TTA GCC TCG GGT 1326 Val Asn Asp Arg Thr Lys Thr Pr~ Ile Ile Ala Thr Leu Ala Ser Gly GCC GTT G~l GCT GTG ATG GCC TTC CTC TTT GAC CTG AhG GAC TTG GIG 1374 Ala Val Ala Ala Val ~et Ala Phe Leu Phe Asp Leu Lys Asp Leu Val G~C CTC AIG T~ ATT G~C AST CTc CIG GCT TAC TCG TIG GTG GCT GCC 1422 Asp Leu Met Ser Ile Gly Thr Leu L~u Ala Tyr Ser Leu Val Ala Ala TGT G~G TIG GTC TIA u~ C~G CCA G~G CAG CCT AAC CTG GIA T~C 1470 Cys Val Leu Val Leu Arg Tyr Gln PrD Glu Gln Pr~ Asn Leu Val Tyr C~G AT~ GCC AGT ACT TCC G~C G~G TT~ GAT CC~ GC~ GAC CAA A~T GAA 1518 Gln Met Ala Ser Thr Ser Asp Glu Leu Asp Pro Ala Asp Gln Asn Glu TTG GCA AGC ACC AAT GAT TCC C~G C~G GGG m Tl~ cc~ GAG GCA GAG 1566 T~ Ala Ser Thr Asn Asp Ser Gln Leu Gly Phe Leu Pr~ Glu Ala Glu AIG TTC TCT llG AAA A~C ATA CTC TCA CCC AAA AAC A~G G~G CCT TCC 1614 Met Phe Ser Leu Lys Thr Ile Leu Ser PrD Lys Asn Met Glu Pr~ Ser AA~ ATC TCT GGG CI~ ATT GTG AAC AIT TC~ ACC AGC CTT AT~ GCT GIT 1662 Lys Ile Ser Gly T~l Ile Val Asn Ile Ser Thr Ser Leu Ile Ala Val CTC ATC AIC ACC TTC T~C AIT GTG ACC GIG CTT GGA AGG G~G G T CTC 1710 T~l Ile Ile Thr Phe Cys Ile Val Thr Val Leu Gly Arg Glu Ala Leu A~C AAA GGG GCG CTG TGG GC~ G~C TTT Cl~ crc GC~ GGG TCT GOC CIC 1758 Thr Lys Gly Ala Leu Trp Ala Ual Fhe Leu Leu Ala Gly Ser Ala Leu CTC T~r GCC GTG G~ A~G GGC GTC AIC ~GG A~;G CAG CCC GAG A~;C AAG 1806 I~ Cys Ala Val Val qhr Gly V~l Ile qrp An~ Gln ~ro Glu Ser Lys ACC AAG CTC TCA TTT AAG G~T CCC TTC CrG CCA GrG C~C COC ATC c~rG 1854 Thr Lys T~l Ser Phe Lys Val Pr~ Phe Leu Pro Val Leu Pro Ile Leu AGC ATC TTC C~l~ AAC GTC TAT crc ArG A~ CAG C~ G~C CAG G5C ACC 1902 Ser Ile Phe Val Asn Val Tyr Leu Met Met Gln Leu Asp Gln Gly Thr Tæ GTC ~;G m G T GTG TGG ATG CIG ATA GGC TrC ATC ATC TAC m 1950 Trp Val Ary Phe Ala Val Trp Met Leu Ile Gly Phe Ile Ile Tyr Phe W O 92/10506 2 0 9 7 7 0 5 PCT/USg1/09382 GGC TAT GGC CIG TGG CAC AGC GAG GAG GCG TCC CTG GAr GCC GAC CAA 1998 Gly Tyr Gly Leu Trp His Ser Glu Glu Ala Ser Leu Asp Ala Asp Gln GCA AGG ACT CCT GAC GGC AAC TTG GAC CAG T&C AAG T&ACGCACAG 2044 Ala Arg Thr Pro Asp Gly Asn Leu Asp Gln Cys Lys COCDGCCrCC CEGPGGIGGC PGCa~CCCCG AGYGaLGCCC CC~GAGGACC GGG~GGCACC 2104 CCACCCTCCC CACC~GTGCA ACAG~AAOCA CCIaCGICC~ CACCCTCACT GCA 2157 (2) INFORMAIICN FOR SEQ ID ND:8:
(i) SE~UENOE CH~RACTERISTICS:
(A) LENGTff: 629 amuno acids - (B) TYPE: a ~ acid (D) IOFOLDGY: linear (ii) MOL ~ TYPE: prokein (xi) Sæ~UENOE DESCRIPqION: SEQ ID NO:8:

Met Gly Cys Val Leu Leu Asn Ile Ile Gly Gln Gln Met Leu Arg Arg Lys Val Val Asp Cys Ser Arg Glu Glu Thr Arg Leu Ser Arg Cys Leu Asn Thr Phe Asp Leu Val Ala Leu Gly Val Gly Ser Thr heu Gly Ala Gly Val Tyr Val T~l Ala Gly Ala Val Ala Arg Glu Asn Ala Gly Pro Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu Ala Ser Val Ieu Ala Gly Leu Cys Tyr Gly Glu Phe Gly Aaa Arg Val Pro Lys Thr Gly Ser Ala Tyr Leu Tyr Ser Tyr Val Thr Val Gly Glu Leu Trp Ala Phe Ile Thr Gly Trp Asn Leu Ile Leu Ser Tyr Ile Ile Gly Thr Ser Ser Val llS 120 125 Ala Arg Ala Trp Ser Ala Thr Phe Asp Glu Leu Ile Gly Arg Pro Ile Gly Glu Phe Ser Arg Thr His Met m r Leu Asn Ala Pro Gly Val Leu Ala Glu Asn PrD Asp Ile Phe Ala -Val Ile Ile Ile Leu Ile Leu Thr Gly Leu Leu ~hr Leu Gly Val Lys Glu Ser Ala Met Val Asn Lys Ile Phe Thr Cys Ile Asn Val Leu Val Leu Gly Phe Ile Met Val Ser Gly Fhe Val Lys Gly Ser Val Lys Asn Trp Gln Leu Thr Glu Glu Asp Phe Gly Asn Thr Ser Gly Arg T~l Cys Leu Asn Asn Asp Ihr Lys Glu Gly Lys Pr~ Gly Val Gly Gly Phe Met Pr~ Phe Gly Phe Ser Gly Val Leu Ser Gly Ala Ala ffl r Cys Phe Tyr Ala Phe Val Gly Fhe Asp Cys Ile Ala Thr Thr Gly Glu Glu V~l Lys Asn Pr~ Gln Iys Ala Ile PrD Val Gly Ile Val Ala Ser L u L~u Ile Cys Phe Ile Ala Tyr Phe Gly Val Ser Ala Ala T~l Thr T~l Met Met Pro Tyr Phe Cys Leu Asp Asn Asn Ser Pr~ Leu Pr~ Asp Ala Phe Lys His Val Gly TSp Glu Gly Ala Ly~

Tyr Ala Val Ala Val Gly Ser Ieu Cys Ala Leu Ser Ala Ser Leu T~

Gly Ser Met Phe Pro Met Pro Arg Val Ile Tyr Ala Met Ala Glu Asp Gly Leu Leu Phe Lys Phe Leu Ala Asn Val Asn Asp Arg Thr Lys Thr Pro Ile Ile Ala Thr L u Ala Ser Gly Ala Val Ala Ala Val Met Ala Phe Leu Phe Asp Leu Lys Asp T~l Val Asp Leu Met Ser Ile Gly Thr Leu Leu Ala Tyr Ser Leu Val Ala Ala Cys Val Leu Val Leu Arg Tyr Gln PrD Glu Gln Pr~ Asn Leu Val Tyr Gln Met Ala Ser Thr Ser Asp Glu Leu Asp Pro Ala Asp Gln Asn Glu Leu Ala Ser Ihr Asn Asp Ser W O 92/10506 2 0 9 7 7 ~ 5 PCT/US91/09382 Gln Leu Gly Fhe Leu Pro Glu Ala Glu Met Phe Ser Leu Lys Thr Ile T~l Ser Pro Lys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val Asn Ile Ser Thr Ser Leu Ile Ala Val Leu Ile Ile Ihr Fhe Cys Ile Val Thr V~l Leu Gly Arg Glu Ala Leu Ihr Lys Gly Ala LPU Trp Ala V 1 Phe Leu Leu Ala Gly Ser Ala T~7 Leu Cys Ala Val Val Thr Gly Val Ile Trp Arq Gln Pro Glu Ser Lys Thr Lys Leu Ser Phe Lys Val Pro Phe Leu Pro Val Leu Pro Ile T~- Ser Ile Phe Val Asn Val Tyr T~l Met M~t Gln Leu Asp Gln Gly Thr Trp Val Arg Phe Ala Val Trp xet T~- Ile Gly Phe Ile Ile ~ Phe Gly Tyr Gly Leu ~ His Ser Glu Glu Ala Ser Leu Asp Ala Asp Gln Ala Arq Thr Pro Asp Gly Asn Leu Asp Gln Cys Lys

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 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 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 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 Ile214, 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.