MXPA01000473A - Methods and compositions for modulating the interaction between the apj receptor and the hiv virus - Google Patents

Abstract

The orphan seven transmembrane domain receptor, APJ, can function as a coreceptor for cellular infection by the HIV virus. The establishment of cell lines that coexpress CD4 and APJ provide valuable tools for continuing research on HIV infection and the development of anti-HIV therapeutics.

Description

METHODS AND COMPOSITIONS TO MODULATE THE INTERACTION BETWEEN APJ RECEIVER AND HIV VIRUS BACKGROUND OF THE INVENTION The entry of HIV-1 into the cells involves the binding of the viral envelope protein (env) to CD4, followed by interaction with one of several coreceptors (reviewed in EA Berger, 1997, AIDS, 11: S3-S16; et al., 1997, J. Leukocyte Biol., 62: 20-29, Doms et al., 1997, Virology, 235: 279-190, and Moore et al., 1997, Curr. Opinion Immunol., 9: 551 -562). The binding of the env protein to the appropriate co-receptor is considered to activate conformational changes in env that mediate the function between the viral membrane and the host cell membrane. The chemokine receptors CCR5 and CXCR4 have been identified as the main co-receptors of VTH-1 in all strains of HIV-1 tested to date using either or both of these molecules as second receptors. CCR5 supports infection by strains of R5 virus (with tropism M), whereas CXCR4 supports infection by X4 virus isolate (tropism T) (Alkhatib et al., 1996, Science, 272: 1955-1958; Berger et al. , 1998, Na ture, 391: 240, Choe et al., 1996, Cell, 85: 1135-1148, Deng et al., 1996, Na ture, 381: 661-666, Doranz et al., 1996, Cell, 85: 1149-1158; Dragic et al., 1996, Na ture, 381: 667-673; and Feng et al., "1996, Science, 272: 872-877.) Vi viral env proteins R5-X4 (double tropism). ) can, together with CD4, use either CCR5 or CXCR4 for cellular entry.The differential use of CCR5 and CXCR4 by strains of HIV, together with the expression patterns of CD4 positive cells, largely explains viral tropism at the level of In addition to CCR5 and CXCR4, it has been shown that many other chemokines and orphan receptor with seven transmembrane domains function as co-receptors for one or more strains of virus in vi tro, including CCR2b, CCR3, CCR8, CX3CR1, GPR1, GPR15, STRL33, US28 and ChemR23 (Choe et al., 1996, Cell, 85: 1135-1148, Deng et al., 1997, Na ture 388: 296-300, Doranz et al., 1996, Cell , 85: 1149-1158, Farzan et al., 1997, J. Exp. Med. 186: 405-411; Liao et al., 1997, J7 Exp. Med. 195: 2015-2023; Pleskoff et al., 1997 , Science, 276: 1874-1878; Reeves et al., 1997, Virology 231: 120-134; Rucker et al., 1997, J. Virol. 71: 8999-9007). In general, these alternative co-receptors support virus infection with less efficiency than CCR5 or CXCR4. However, the use of alternative co-receptors can help certain aspects of HIV-1 tropism and pathogenesis in vivo. For example, neurological disease is a serious and relatively frequent consequence of HIV-1 infection, where microglia is the primary target of infection by viruses in the central nervous system (Bagasra et al., 1996, AIDS, 10). : 573-585; Sharer et al., 1992, J. Neuropa th.

Exp. Neurol. , 51: 3-11; Wiley et al., 1986, Proc. Na ti. Acad.

Sci. , USA, 83: 7089-7093). Microglia express both CCR3 and CCR5 and it has been suggested that the use of CCR3 by a strain of virus can be related to neurotropism (He et al., 1997, Na ture, 385: 645-649). The identification of additional coreceptors for the HIV virus can provide an important tool for investigating and controlling HIV infection.

BRIEF DESCRIPTION OF THE INVENTION In one aspect of the invention, the invention relates to a recombinant eukaryotic cell transformed with a polynucleotide encoding an APJ polypeptide or a polynucleotide encoding a CD4 polypeptide, or both, wherein the cell coexpresses the APJ and CD4 polypeptides. The invention also relates to an antibody to which it specifically binds to an extracellular APJ domain, wherein the antibody inhibits HIV infection of a target cell that coexpresses the APJ and CD4 polypeptides, or wherein the antibody inhibits the membrane fusion between a first cell that coexpresses the APJ and CD4 polypeptides, and a second cell expressing an HIV env protein. The invention also relates to a substantially purified peptide fragment of APJ, wherein the peptide inhibits HIV infection of a target cell that coexpresses APJ and CD4 polypeptides, or wherein the peptide inhibits cell fusion between a first cell that coexpresses APJ and CD4 polypeptides, and a second cell that expresses a protein env of HIV. In another aspect of the invention, the invention relates to methods for identifying compounds that modulate the interaction between an HIV virus and an APJ receptor. The invention also relates to a method for inhibiting HIV infection of a target cell expressing APJ and CD4 polypeptides, comprising contacting the target cell with an effective amount of an agent that binds or blocks APJ. The invention also relates to a method of treating a subject having or at risk of having an HIV infection or a related disorder, comprising administering a therapeutically effective amount of an antibody to the subject to APJ.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the cell-cell fusion mediated by HIV-1 or HIV-2 env proteins. All of the indicated env proteins are derived from HIV-1, with the exception of HIV-2 / ST, which is derived from HIV-2. QT6 cells expressing CD4, the indicated co-receptor and luciferase under the control of the T7 promoter are mixed with cells expressing T7 polymerase and the HIV-1 or HIV-2 env protein indicated. The degree of cell-cell fusion is determined 8 hours after mixing by measuring the luciferase activity. The results are normalized by adjusting the degree of fusion obtained when coexpressing up to 100% of CD4 either "with CCR5 (for env proteins R5) or CXCR4 (for env proteins X4) .The degree of fusion obtained with the major co-receptors of HIV-1 is generally 40 to 100 times above background levels.The error bars here and in the subsequent figures represent the standard error of the mean derived from multiple independent experiments.Figure 2 is a graph that shows cell-cell fusion mediated by SIV env proteins Cell-cell fusion was determined as in Figure 1, but using SIV instead of HIV env proteins.The results are normalized by adjusting the degree of fusion obtained when they are coexpressed CD4 and CCR5 at 100% The degree of fusion obtained with the different env proteins is generally 400 to 100 times above the background levels (defined as CD4 only) Figure 3 is a graph that shows pseudotype virus infection. HEK 293 cells expressing CD4 and the indicated co-receptor are infected with pseudotypes of luciferase virus having the indicated HIV env or SIV env protein, and the luciferase activity is determined 2-3 days after infection.

Figure 4 is a graph showing viral infection of HEK 293 cells expressing CD4 and the indicated co-receptor and also containing a plasmid encoding luciferase under the control of HIV-1 LTR. The cells are infected with live HIV-1 IIIB (HxB) HIV-1 89.6. The values are normalized by establishing the degree of infection obtained with either CCR5 (env proteins R5) or CXCR4 (env proteins X4) at 100%. Figure 5 is an image of a Southern blot showing the entry of virus into cells expressing CD4 and APJ. QT6 cells stably express CD4 and transiently express the desired co-receptor and are infected with cell-free virus treated with DNase. The viral specific LTR DNA sequence is detected two days after infection by amplification by PRC, followed by separation of the products on a 2% agarose gel and determination of the sequences using a labeled probe. Figure 6 is an image of a Northern blot showing the expression of APJ in human brain tissue. Membranes containing poly A + RNA from various regions of the human brain are obtained from Clontech and incubated with a labeled cDNA probe specific for APJ, overnight. The membranes are then exposed to a Fuji imaging plate for 4 hours. The following tissues were examined: Panel (A): (1) Amygdala; (2) caudate nucleus; (3) corpus callosum; (4) hippocampus; (5) complete brain; (6) substantia nigra; (7) subthalamic nucleus; (8) thalamus; Panel (B): (1) cerebellum; (2) cerebral cortex; (3) marrow; (4) spinal cord; (5) occipital lobe; (6) frontal lobe; (7) temporal lobe; (8) putamen. Figure 7 is an image of a transfer Northern showing the expression of APJ in human peripheral tissue. Membranes containing poly A + RNA from various human tissues are obtained from Clontech and incubated with a labeled cDNA probe specific for APJ overnight. The membranes are then exposed to a Fuji image plate for 4 hours. The following tissues were examined: (1) spleen; (2) thymus; (3) prostate; (4) testicles; (5) ovary; (6) small intestine; (7) mucosa of the colon; (8) total peripheral blood lymphocytes. Figure 8 is an image of a stained agarose gel showing the expression of APJ in primary cells and in C cell lines. The RNA of the indicated cells was used in RT-PCR reactions in one tube and 10 μl of each reaction 25 μl is run on a 2% agarose gel. The size of the predicted APJ band is 481 base pairs. Both the plasmid DNA and the RNA isolated from stable U87-APJ transfected cells are included as positive controls and water is used as a template for a negative control. Figure 9 is the nucleotide and deduced amino acid sequence for human APJ (O 'Do d et al., 1993, Gene 136: 355-360) (SEQ ID NO: 1). The positions of the seven transmembrane regions are as follows: transmembrane region 1 (TM 1) corresponds to amino acids 26-54; the transmembrane region 2 (TM2) corresponds to amino acids 66- 5 90; the transmembrane region 3 (TM3) corresponds to "" amino acids 104-125; the transmembrane region 4 (TM4) corresponds to amino acids 144-167; the transmembrane region 5 (TM5) corresponds to amino acids 199-225; the transmembrane region 6 (TM6) corresponds to amino acids 246-271; the transmembrane region 7 (TM7) corresponds to amino acids 285-312. The extracellular portions of the APJ polypeptide are located in the amino terminal segment before the transmembrane domain 1 (for example amino acids 1-25), between the transmembrane domains 2 and 3 (for example amino acids 91-153), between the transmembrane domains 4 and 5 (for example amino acids 168-198) and between transmembrane domains 6 and 7 (for example amino acids 272-284).

DETAILED DESCRIPTION OF THE INVENTION 20 In accordance with this invention, it has been discovered that the orphan receptor with seven transmembrane domains is, APJ (O 'Do d et al., 1993, Gene 136: 355-360), functions as an efficient co-receptor. for numerous strains of HIV-1 and SIV. APJ serves as a highly efficient co-receptor for some strains of X4 virus (with tropism T) and R5-X4 (with double tropism) while two R5 isolates (with tropism M) use APJ less efficiently. APJ also serves as a co-receptor for several strains of SIV. Further in accordance with this invention, the widely disseminated expression of APJ in the human central nervous system has been discovered. The efficient use of APJ by numerous strains of virus, together with the expression of APJ in the central nervous system, indicates that the use of this receptor may be important in the neuropathogenesis of HIV. Also in accordance with this invention, the expression of APJ has been discovered in a CD4 positive T cell line, C8166. In one embodiment, the present invention relates to recombinant cell lines, cells which coexpress the APJ and CD4 polypeptides, and which contain an exogenous polynucleotide that already encodes an APJ polypeptide or a CD4 polypeptide. The present invention also relates to recombinant cell lines, cells which coexpress APJ and CD4 polypeptides, and which contain an exogenous polynucleotide that encodes an APJ polypeptide and an exogenous polynucleotide that codes for a CD4 polypeptide. As used herein, a "CD4 polypeptide" means a mammalian CD4 polypeptide, preferably a human or simian CD4 polypeptide, or a biologically active fragment thereof. As used herein, an "APJ polypeptide" means a mammalian APJ polypeptide, preferably a human or simian APJ polypeptide, or a biologically active fragment thereof. As used herein, the term "biologically active" refers to polypeptides that are capable of specifically interacting with an HIV or SIV virus or with polypeptides having at least one epitope for an antibody immunoreactive with the APJ or CD4 polypeptide. The invention relates not only to the APJ and CD4 polypeptides that occur naturally, but also to mutant APJ and CD4 polypeptides. For example, changes in the amino acid sequence of APJ are contemplated in the present invention. APJ can be altered by changing the DNA encoding the protein. "Preferably, only conservative alterations in the amino acid sequence are considered, using amino acids having the same property or similar properties Illustrative amino acid substitutions include the following changes: alanine a serine, arginine to lysine, asparagine to glutamine or histidine, aspartate to glutamate, cysteine to serine, glutamine to asparagine, glutamate to aspartate, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine Lysine to arginine, glutamine or glutamate, methionine to "leucine or isoleucine; "phenylalanine" to tyrosine, leucine or methionine, serine to trionine, trionine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, valine to isoleucine or leucine, etc. The polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode for APJ or CD4 polypeptides Such polynucleotides include polynucleotides that occur naturally, synthetic and intentionally manipulated. For example, portions of the mRNA "sequence can be altered due to alternating RNA splicing patterns or the use of alternating promoters for RNA transcription." As another example, APJ and CD4 polynucleotides can be subjected to site-directed mutagenesis. The polynucleotide sequence for APJ and CD4 also include antisense sequences The invention also includes an APJ polypeptide having biological activity or a CD4 polypeptide having biological activity Suitable cell types include, but are not limited to cells of the following types : NIH 3T3 (murine), Mv 1 lu (mink), BS-C-1 (African green monkey), human embryonic kidney (HEK) 293 cells (ATCC CRL 1573) and QT6 quail cells. For example, in the Cell Line Catalog of the American Type Culture Collection (ATCC), these cells can be stably transformed or transiently transformed by a method known to those skilled in the art.

See, for example, Ausubel, et al., Introduction of DNA Into Mammalian Cells, in Curren t Protocols in Molecular Biology, sections 9.5-9.5.6 (John Wiley &Sons, Inc. 1995). In the context of the invention, the "stable" transformation means that the cells are immortal to the extent that * they have experienced at least 50 divisions. The exogenous APJ or CD4 polynucleotides can be expressed using inducible or constitutive regulatory elements for expression. Commonly used constitutive or inducible promoters, for example, are known in the art. For example, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian virus (e.g., the long terminal repeat sequence of retroviruses, the adenovirus late promoter, the 7.5K virus promoter can be used. of vacinia). Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of exogenous polynucleotides for APJ or CD4. The coding sequence for the desired protein and the operably linked promoter can be introduced into a recipient cell either as a non-replicating DNA (or RNA) molecule, which can be a linear molecule or, more preferably, with a covalent circular molecule closed. Since such molecules are incapable of autonomous replication, the expression of the desired molecule can be represented by the transient expression of the introduced sequence. Alternatively, the permanent expression may be presented through the integration of the sequence introduced into the host chromosome. In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of "autonomous replication in the host host.For this purpose a wide variety of vectors can be used.The importance factors in the selection of a plasmid or vector Particular viral include the following: the ease with which the receptor cells containing the vector can be recognized and selected from the recipient cells which do not contain the vector, the number of vector copies which are desired in a particular host; and whether it is desirable to "drive" the vector between host cells of different species Several possible vector systems are available for expression One class of vector uses DNA elements which provide extrachromosomal plasmids that replicate autonomously, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus or SV40 virus. Second class of vectors includes vaccinia virus expression vectors. A third class of vectors is based on the integration of the desired gene sequence in the host chromosome. Cells which have stably integrated the DNA introduced into their chromosomes can also be selected by introducing one or more markers (eg, an exogenous gene) which allows selection of the host cells which contain the expression vector. The label can provide, for example, prototyping to an auxotrophic host or resistance to biocide, for example antibiotic resistance or resistance to heavy metals, such as resistance to copper.The selectable marker gene can be directly linked to the DNA sequences that They may be expressed, or they may be introduced into the same cell by cotransformation.Additional elements may also be necessary for optimal mRNA synthesis.These elements may include splicing signals, as well as transcription promoters, extenders and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol. Cell, Biol., 3: 280 (1983), and others, Once the vector or DNA sequence containing the construct is have prepared for expression, a DNA construct can be introduced (transformed) into an appropriate host.Of those techniques which can be used include, for example, photoplast fusion, calcium phosphate precipitation, electroporation, microinjection, liposome delivery, viral infection or other conventional techniques. In another embodiment, the present invention relates to transgenic animals that have cells that co-express CD4 and APJ polypeptides. Such transgenic animals represent a model system for the study of HIV infection and the development of more effective anti-HIV therapies. The term "animal", as used herein, indicates all species of mammals, except humans. It also includes an individual animal at all stages of development, '- which include embryonic and fetal stages. Farm animals (pigs, goats, sheep, cows, horses, rabbits, etc.), rodents (such as mice) and domestic pets (for example cats and dogs) are included within the scope of the present invention. A "transgenic" animal is any animal that contains cells that present genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, for example by microinjection or by infection with a recombinant virus. In the present context, "transgenic" does not encompass the concept of classical cross-insemination or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule. Although it is greatly preferred that this molecule "be integrated into the chromosomes of the animal, the present invention also contemplates the use of extrachromosomally replicating DNA sequences, which can be engineered into yeast artificial chromosomes. "transgenic animal" also includes a transgenic animal of "germ cell line." A transgenic animal with a germ cell line is a transgenic animal in which genetic information has been captured and incorporated into a germ cell line, so both confers the ability to transfer the information to the offspring, if such an offspring in fact owns part or all of that information, these can also be considered transgenic animals.It is highly preferred that the transgenic animals of the present invention be introduced into embryos of a single cell or polynucleotide that codes for a pol APJ peptide or a polynucleotide encoding a CD4 polypeptide, or both, such that these polynucleotides are stably integrated into the DNA of the germline cells of the mature animal and are inherited in a normal Mendelian manner. Advances in micromanipulation technology now allow for the induction of exogenous polynucleotides in or of several fertilized mammals. For example, totipotent or pluripotent cells can be transformed by microinjection, calcium phosphate-mediated precipitation, liposome fusion, retroviral infection or other medium, transformed cells are then introduced into the embryo and the embryo then develops into a transgenic animal . In a preferred embodiment, developing embryos are infected with a retrovirus containing the desired polynucleotide and the transgenic animals produced from the infected embryo. In a more preferred method, the appropriate polynucleotides are co-injected into the pronucleus or cytoplasm of embryos, preferably at the stage of a cell and the embryos are allowed to develop into mature transgenic animals. These techniques are well known. For example, reviews of standard laboratory procedures for exogenous DNA microinjection into used ova of mammals (mouse, pig, rabbit, sheep, goat, cow) include: Hogan et al., Manipula ting The Mouse Embryo (Cold Spring Harbor Press 1986); Krimpenfort et al., Bio / Technology 9:86 (1991); Palmiter et al., Cell. 41: 343 (1985); Kraemer et al., Genetic Manipulation of The Early Mammalian Embryo (Cold Spring Harbor Laboratory Press 1985); Hammer et al., Na ture 315: 680 (1985); Purcel et al., Science 244: 1281 (1986); Wagner et al., U.S. Patent No. 5,175,385; Krimpenfort et al., U.S. Patent No. 5,175,384, the respective contents of which are incorporated herein by reference. The polynucleotide encoding APJ or CD4 can be fused in an appropriate reading frame under transcriptional and translational control to produce a genetic construct which is then amplified, for example, by preparation in a bacterial vector according to conventional methods. See, for example, the standard work: Sambrook et al., Molecular Cloning: a Labora tory Manual (Cold Spring Harbor Laboratory Press 1989); whose content is incorporated herein by reference. The amplified construct is subsequently cut from a vector and purified for use in the production of transgenic animals. The production of transgenic animals containing the gene for human CD4 has been described. See Snyder et al., Mol. Reprod. & Devel. 40: 419-428 (1995); Dunn et al., J. Gen. Virology 76: 1327-1336 (1995), the content of which is incorporated herein by reference. In another embodiment, the present invention relates to antibodies that bind APJ and that inhibit the entry of HIV into a positive CD4 target cell or that inhibit cell-cell fusion between a first cell type expressing CD4 and APJ polypeptides, and a second type of cells that expresses the env protein. As used herein, an env protein means an env protein derived from an HIV virus, either HIV-1 or HIV-2, or derived from a SIV virus. The expression of an env protein by a cell will typically result in the expression of the gpl20 portion of the env protein on the cell surface. The antibodies of the invention can also inhibit the binding of gpl20 to APJ. Such antibodies can represent research and diagnostic tools in the study of HIV infection and the development of more effective therapies against HIV. In addition, the pharmaceutical compositions comprise antibodies to APJ and may represent an effective therapy against HIV. A suitable antibody for blocking env-mediated cell-cell fusion, HIV entry into a positive CD4 cell or binding of gpl20 to APJ is specific for at least a portion of an extracellular region of the APJ polypeptide, for example, the first extracellular region (amino acids 1-25), the second extracellular region (amino acids 91-103), the third extracellular region (amino acids 168-198), or the fourth extracellular region (amino acids 272-284), as shown in Figure 9. Preferred antibodies are those which recognize an epitope comprising a portion of either a first extracellular region or a second extracellular region. Particularly preferred antibodies are those which recognize an epitope comprising the amino acid sequence Asn-Tyr-Tyr-Gly (SEQ ID NO: 3) contained within the first extracellular region. Antibodies of the invention against APJ include polyclonal antibodies, monoclonal antibodies and polyclonal and monoclonal antibody fragments. The preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992), which are incorporated herein by reference. The preparation of monoclonal antibodies in the same way is conventional. See, for example, Kohler &; Milstein, Na ture 256: 495 (1975); Coligan et al., Curren t Protocols in Immunology, sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Labora tory Manual, page 726 (Cold Spring Harbor Pub. 1988), which is incorporated herein by reference. Briefly, monoclonal antibodies can be obtained by infecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing B lymphocytes with myeloma cells to produce Hybridomas, clone the hybridomas, select the positive clones that produce antibodies to the antigen and isolate the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by various well-established techniques. Such isolation techniques include affinity chromatography with protein-A Sepharose, size exclusion chromatography, purification of antigen affinity and ion exchange chromatography. See, for example, Coligan et al., Current Protocols in Immunology, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG), in Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press, 1992). Methods of in vi tro and in vivo multiplication of antibodies are well known to those skilled in the art. The in vi tro multiplication can be carried out in an appropriate culture medium such as Dulbeco modified Eagle medium or RPMI 1640 medium, optionally replenished by mammalian serum such as fetal bovine serum or trace elements and supplements that support growth such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. In vitro production provides relatively pure antibody preparations and allows for broad scale to obtain large quantities of the desired antibodies. The large-scale hybridoma culture can be carried out by homogeneous suspension cultures in an air lift reactor, in a continuous agion reactor or in a culture of immobilized or entrapped cells. In vivo multiplication can be carried out and injected cell clones into histocompatible mammals with parental cells, for example osingeneic mice to cause the growth of antibody producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) before injection. After 1 to 3 weeks, the monoclonal antibody is recovered from the body fluid of the animal. Therapeutic applications for antibodies to APJ described herein are also part of the present invention. For example, the antibodies of the present invention can also be derived from subhuman primate antibodies. General techniques for generating therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication WO 91/11465 (1991) and Losman et al., Int. J. Cancer 46: 310 (1990), which is incorporated herein by reference. Alternatively, a therapeutic or diagnostically useful APJ antibody can be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determinant regions from the variable and heavy chains of mouse immunoglobulin into a human variable domain, then substituting the human residues in regions of major structure of mouse counterparts. The use of antibody components derived from humanized monoclonal antibodies eliminates the potential problems associated with immunogenicity of murine constant regions. General techniques for cloning variable domains of murine immunoglubulin are described, for example, by Orlandi et al., Proc. Na ti. Acad. Sci. USA 86: 3833 (1989), which is incorporated herein by reference in its entirety. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Na ture 321: 522 (1986); Riechmann et al., Na ture 332: 323 (1988); Verhoyen et al., Science 239: 1534 (1988); Cárter et al., Proc. Na ti. AcadX'Sci. USA 89: 4285 (1992); Sandhu, Cri t. Rev. biotech, 12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1933), which are incorporated herein by reference. The antibodies of the invention can also be derived from human antibody fragments isolated from a combination immunoglobulin library. See, for example, Barbas et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al., Ann. Rev. immunol. 12: 433 (1994), which are incorporated herein by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from Stratagene Cloning Systems (La Jolla, CA). In addition, the antibodies of the present invention can be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic exposure. In this technique, elements of human heavy and light chain loci are introduced into strains of mice derived from embryonic cell lines containing targeted interruptions of the endogenous heavy and light chain loci. Transgenic mice can synthesize human antibodies specific for human antigens, and mice can be used to produce hybridomas secreting human antibodies. The methods for obtaining human antibodies from transgenic mice are described by Green et al., Na ture Genet. . 7:13 (1994); Lonberg et al., Na ture 368: 856 (1994); and Taylor et al., In t. Immunol. 6: 579 (1994), which are incorporated herein by reference. The antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by digestion with pepsin or papain of the whole antibodies, by conventional methods. For example, antibody fragments can be produced by enzymatic separation of antibodies with pepsin to provide a 5S fragment designated F (ab ') 2. This fragment can be further separated using a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from the separation of disulfide bonds, to produce 3.5S Fab 'monovalent fragments and an Fc fragment directly. These methods are described, for example by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647, and references contained therein. These patents are incorporated herein by reference in their entirety. See also Nisonhoff et al., Arch. Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959); Edelman et al., Methods in Enzymology, Vol. 1, page'- 422 (Academic Press 1967); and Coligan et al., Curren t Protocols in Immunology, sections 2.8.1-2.8.10 and 2.10.1-2.10.4. Other methods for separating antibodies, such as the separation of heavy and light chains to form monovalent light-heavy chain fragments, the additional separation of fragments or other enzymatic, chemical or genetic techniques, can also be used, insofar as the fragments bind to the antigen that is recognized by the intact antibody. For example, the Fv fragments comprise an association of VH and VL chains. This association may be non-covalent, as described in Inbar et al., Proc. Na ti. Acad. Sci. USA 69: 2659 (1972). Alternatively, the variable chains can be linked by an intramolecular disulfide bond or can be crosslinked by chemicals such as glutaraldehyde. See, for example, Sandhu, Cri t. Rev. biotech, 12: 437 (1992). Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a unique polypeptide chain with a binding peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird et al., Science 242: 423-426 (1988); Ladner et al., U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11: 1271-77 (1993); and Sandhu, Crit. Rev. biotech, 12: 437 (1992). Another form of an antibody fragment is a peptide that codes for a unique region of complementarity determinant (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding CDRs of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, Vol. 2, page 106 (1991). Antibodies that bind to the chemokine receptor CXCR4, another HIV co-receptor, have been shown to block the function of HIV strains that use the CXCR4 receptor for infection (Feng, et al., Science 272: 872 (1996); Endres et al. al., Cell 87: 745 (1996)). In another embodiment, the present invention relates to "APJ variants". As used herein, a variant of APJ means a molecule that simulates at least part of the structure of APJ and that inhibits the entry of HIV into a target cell that expresses CD4 and APJ polypeptides or that inhibits cell function - cell between a first type of cell expressing CD4 and APJ polypeptides, and a second type of cells expressing the env protein. The env protein of certain HIV isolates can participate in HIV infectivity by binding to APJ on the cell surface. While not wishing to be bound by any particular theory of the invention, the inventors consider that APJ variants can interfere with HIV infectivity by competing with APJ upon binding to env. In one embodiment, the present invention relates to APJ variants that are peptides and peptide derivatives that have fewer amino acid residues than APJ. Such peptides or peptide derivatives can represent research and diagnostic tools with the study of HIV infection and the development of therapeutics against HIV more effective. Peptides and peptide derivatives of APJ, according to the present invention, include those which correspond to the extracellular regions of APJ, for example, the first extracellular region, (amino acids 1-25), the second extracellular region (amino acids 91- 103), the third extracellular region (amino acids 168-198) or the fourth extracellular region (amino acids 272-284), as shown in Figure 9. The peptides corresponding to the extracellular loops of another HIV co-receptor, co-receptor C .CR5, it has previously been shown to inhibit fusion between HIV-1 env expressing cells and murine cells co-expressing CD4 and CCR5 (Combadiere et al., PCT / US97 / 09586, publication number WO 97/45543). Preferred peptides and peptide derivatives are those which correspond to a portion of either the first extracellular region or the second extracellular region. Peptides or peptide derivatives particularly preferred are those which comprise the amino acid sequence Asn-Tyr-Tyr-Gly (SEQ ID NO: 3) contained within the first extracellular region. The APJ variants useful for the present invention comprise APJ analogs, homologs, muteins and mimetics. The variants can be generated directly from APJ itself by chemical modification, by digestion with proteolytic enzymes or by combinations thereof. Additionally, genetic engineering techniques can also be used, as well as methods for the synthesis of polypeptides directly from amino acid sequences. The peptides of the invention can be synthesized by such commonly used methods as protection with t-BOC or FMOC of alpha-amino groups. Both methods involve gradual synthesis so that a single amino acid is added at each step from the C-terminal part of the peptide (see, Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). The peptides of the invention can also be synthesized by well-known solid-phase peptide synthesis methods (described in Merrifield, J. Am. Chem. Soc. 85: 2149 (1962) and Stewart and Young, Solid Phase Peptide Syn thesis (Freeman, San Francisco, 1969) pp. 27-62), using copoly (styrene-divinylbenzene) containing 0.1-1.0 mmoles of amines / g of polymer. Upon completion of the chemical synthesis, the peptides can be deprotected and separated from the polymer by treatment with liquid HF-10% anisole for about 1 / 4-1 hours at 0 ° C. After evaporation of the reagents, the peptides are extracted from the polymer with a 1% acetic acid solution which is then lyophilized to provide the crude material. The crude material can usually be purified by standard techniques, such as, for example, by gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of the appropriate fractions of the column will provide the peptide or homogeneous peptide derivatives, which can then be characterized by standard techniques such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation , solubility and can be quantified by analysis of Edman degradation in solid phase. Alternatively, the peptides can be reproduced by recombinant methods which are well known - by those skilled in the art. The term "substantially purified", as used herein, refers to a molecule, such as a peptide that is substantially free of other proteins, lipids, carbohydrates, nucleic acids and other biological materials with which it is naturally associated. For example, a substantially pure molecule, such as a polypeptide, can be at least 60% dry weight of the molecule of interest. A person skilled in the art can purify APJ peptides using standard protein purification methods and the purity of the polypeptides can be determined using standard methods including, for example, polyacrylamide gel electrophoresis (e.g., SDS-PAGE), chromatography Column (for example, high resolution liquid chromatography (CLAP) and amino-terminal amino acid sequence analysis.) Non-peptidic compounds that mimic the binding and function of APJ ("mimetics") can be produced by the solution indicated in Saragovi et al. ., Science 253: 792-95 (1991) .Mimetics are molecules which mimic elements of the secondary structure of proteins See, for example, Johnson et al., Peptide Turn Mimetics, in Biotechnology and Pharmacy, Pezzuto et al. al., Eds., (Chapman and Hall, New York 1993) The reasoning behind the use of peptidomimetics is that the main peptide structure of proteins nas exists chiefly to orient the side chains of amino acids such that facilitate molecular interactions. For the purposes of the present invention, it can be considered that the appropriate mimetics are equivalent to APJ themselves. Larger peptides can be produced by the "native chemical" ligation technique which binds peptides (Dawson, et al., Science 266: 776 (1994)). Variants can be generated by recombinant techniques using genomic or cDNA cloning methods. Site-specific and site-directed mutagenesis techniques can be used. See Current Protocols in Molecular Biology, Vol. 1, chapter 8 (Ausubel et al., Eds., J. Wiley & amp; amp;; Sons 1989 Supp. 1990-93); Protein Engineering (Oxender &Fox, eds., Liss, Inc. 1987). In addition, linker and PCR-mediated scanning techniques can be used for mutagenesis. See PCR Technology (Erlich ed., Stockton Press 1989); Curren t Protocols in Molecular Biology, Vols. 1 and 2, supra. Protein sequencing, structure and modeling solutions for use with any of the above techniques are described in Protein Engineering, loe. ci t. and Curren t Protocols in Molecular Biology, Vols. 1 and 2, supra. If the compounds described above are used, those skilled in the art can systematically ensure that such compounds are susceptible for use. with the present invention in view of the cell-cell fusion assay systems and the infectivity assay systems described herein. The invention also includes various pharmaceutical compositions that inhibit the entry of HIV into a target cell that expresses the CD4 and APJ polypeptides. The pharmaceutical compositions according to the invention are prepared by placing a variant of APJ or an antibody against APJ, according to the present invention, in a form suitable for administration to a subject, by the use of carriers, excipients and additives or auxiliaries. . Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk, proteins, gelatin, starch, vitamins, cellulose and their derivatives, animal and vegetable oils, polyethylene glycols and solvents such as water sterile, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include substances for replenishing fluids and nutrients. The preservatives include antimicrobial agents, antioxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers and the like, as described, for example, in Remington's Pharmaceutical Science, 15th ed. Eaton: Mack Publishing Co., 1405-1412, 1461-1487 (1975), the content of which is incorporated herein by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to the systematic skills in the art. See Goodman and Gilman's The Pharma cologi cal Basis for Therapeutics (7th ed.). In another embodiment, the invention relates to a method for inhibiting the entry of HIV into a target cell. This method involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier. "Administering" the pharmaceutical composition of the present invention can be carried out by any means known to those skilled in the art. By "subject" is meant any mammal, preferably a human. For example, neuropathy has been observed in the brains of newborn infants born to HIV-1 seropositive mothers (Kolson et al., 1998, Adv. Res. 50: 1-47). Therefore, a particularly preferred method is a method of treating a fetal subject having or at risk of presenting an HIV infection by administering an antibody with APJ or a fragment of the APJ peptide. The antibody to APJ and the peptide fragment APJ are preferably administered to the fetal subject by means of administration to the mother. The pharmaceutical compositions are preferably prepared and administered in dosage units. The solid dosage units are tablets, capsules and suppositories. For treatment of a subject, depending on the activity of the compound, the manner of administration, nature and disorder, age and body weight of the patient, different daily doses are necessary. Nevertheless, under certain circumstances, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out either by single administration in the form of a single dose unit or in addition to several smaller dose units and also by multiple administration of subdivided doses at specific intervals. The pharmaceutical compositions according to the invention are generally administered topically, intravenously, orally or parenterally or as implants, but rectal use is even possible in principle. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with prolonged release of active compounds, in which preparation the excipients and additives or auxiliaries, or both such as disintegrants, binders, coating agents, blowing agents, lubricants, flavors, sweeteners or solubilizers are usually used as described above. The pharmaceutical compositions are suitable for uses in various drug delivery systems. For a brief review of the present methods for drug delivery see Langer, Science 249: 1527-1533 (1990), which is incorporated herein by reference. The pharmaceutical compositions according to the invention can be administered locally or systematically. By "therapeutically effective dose" is meant the amount of a compound according to the invention necessary to prevent or cure or at least partially suppress the symptoms of the disease and its complications. The amounts effective for this use, of course, will depend on the severity of the disease and the weight and general condition of the subject. Typically, the dosages used in vi tro can provide useful guidance regarding amounts useful for in-situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders. Various considerations are described, for example, in Gilman et al., (Eds.) (1990) Goodman and Gilman's The Pharmacological Basis for Therapeutics 8th ed., Pergamon Press; and Remington's Pharma ceutical Science, 17th ed. (1990), Mack Publishing Co. , Easton, PA, each of which is incorporated herein by reference. Another preferred embodiment of this invention is the diagnosis of susceptibility to HIV infection. The nucleotide sequences encoding the APJ receptor and the antibodies to the APJ receptor may be particularly useful for the diagnosis of susceptibility to infection when higher levels of the receptors indicate an increased risk of HIV infection. For example, higher levels of the APJ receptor in tissues of the central nervous system may indicate an increased risk of neuropathogenesis associated with HIV infection. Using a suitable technique known in the art, such as Northern blot, quantitative PCR, etc., the nucleotide sequences of the receptor or fragments thereof can be used to measure the expression levels of RNA for APJ. Alternatively, antibodies to APJ can be used in standard techniques such as western blotting to detect the presence of cells expressing the APJ receptor and in standard techniques, for example FACS or ELISA, to quantify the level of expression. For any biological tissue sample, a level of APJ expression that is greater than the reference level is indicative of increased susceptibility to HIV infection. A reference level can be established when performing an analysis of a large population of individuals. In preferred embodiments, the invention relates to methods for analyzing a compound ("test compound") to determine pharmacological activity against HIV. In one embodiment, a cell fusion assay is used to perform an analysis of a compound with pharmacological activity against HIV. In the cell fusion assay, a type of eukaryotic cell that coexpresses the APJ and CD4 polypeptides with a second type of eukaryotic cell expressing an envelope protein of HIV ("env") is incubated. Then the fusion between the two different types of cells is monitored. "A test compound is added to the incubation solution before or after mixing the cells and their effect on the fusion rate of the cells is determined by any of numerous means including morphological observation or by the use of an indicator system in conjunction with a cell fusion assay.The useful indicator systems in conjunction with a cell fusion assay can be any combination of elements in which a detectable signal is produced when a first component in a first cell is contacted with a second component in a second cell by cell-cell fusion For example, the first component can be a gene encoding a polymerase such as T7 polymerase and the second component can be a gel which codes for an indicator molecule which is under the control of the T7 promoter, such as a luciferase gel. For example, a system that results in the production of an active indicator molecule of β-galactosidase before cell fusion is also considered. In another embodiment, an infectivity assay is used to perform an analysis of a compound with pharmacological activity against HIV. In an infectivity assay, a target eukaryotic cell expressing the APJ and CD4 polypeptides is incubated with a test virus expressing an HIV env protein. The infection of the target cell with the test virus is subsequently monitored. The test compound is added to the incubation solution before or after mixing the target cells with the test virus, and the effect of the compound on the infection rate of the target cells is determined by any number of means. The test viruses can be a reporter virus in which the env protein is pseudotyped on a background of indicator virus. Alternatively, the test virus can be an intact HIV virus. Infectivity is generally monitored by the use of an indicator system in conjunction with the infectivity assay. Any number of indicator systems can be used, and indicator systems are preferred which produce a detectable signal before infection. For example, the reporter virus can be constructed with a gene encoding an indicator molecule such as luciferase and β-galactosidase, which is expressed when the indicator virus infects the target cell. As another example, the target cell may contain a gene encoding an indicator molecule under the control of the LTR promoter, thereby resulting in the expression of the reporter molecule upon infection of the target cell with the HIV virus. Alternatively, a viral infection can be monitored by using a PCR to detect viral sequences within the infected cell. The cell fusion assay and the HIV infectivity assay can be used to determine the functional ability of APJ to confer env-mediated fusion competition to a diverse range of CD4 positive cell types (either recombinantly produced or occurring naturally), which include but are not limited to NIH 3T3 (murine), BS-C-1 (African green monkey), HEK 293 (human), MvlLu (mink), glioblastoma U-87 MG, SCL1 and QT6 (quail). HIV strains that may be used in conjunction with the assays, or as sources for env protein genes to be used in conjunction with the assays include strains with tropism M, tropism T or double tropism. For example, the strain with double tropism 89.6, the strain with tropism M JRFL and the strains of HIV-1 with tropism IIIB are those that can be used (Matthews et al., 1986, Proc. Na ti. Acad. Sci., USA, 83: 9709-9713; Coliman et al., 1992, J. Virol. 66: 7517-7521; Gartner et al., 1986, Science 233: 215-219). Additionally, selected primary isolates can also be used. Variations of the methods of drug analysis are known to those skilled in the art with average skill in this field. Accordingly, the cell fusion assay and the HIV infectivity assay can be used in a wide variety of formats to take advantage of the properties of the APJ receptor for analysis of drugs that are effective against HIV. Another embodiment of the invention utilizes the use of antisense technology as a specific and potent means to inhibit HIV infection of cells containing APJ, for example, by decreasing the amount of APJ expression in a cell. Antisense polynucleotides in the context of the present invention include both short sequences of known DNA and oligonucleotides usually 10-50 bases in length, as well as longer DNA sequences that can exceed the length of the APJ gene sequence itself. . The antisense polynucleotides useful for the present invention are complementary to specific regions of a corresponding target mRNA. Hybridization of antisense polynucleotides to their target transcripts can be highly specific as a result of complementary base pairing. The ability of the antisense polynucleotides to hybridize is affected by parameters such as length, chemical modification and secondary structure of the transcript which may influence access. of the polynucleotide to the target site. See Stein et al., Cancer Research 48: 2659 (1988). An antisense polynucleotide can be introduced into a cell by introducing a segment of DNA encoding the polynucleotide within the cell such that the polynucleotide is produced within the cell. An antisense polynucleotide can also be introduced into a cell by adding the polynucleotide to the environment of the cell so that the cell can directly capture the polynucleotide. This latter pathway is preferred for shorter polynucleotides up to about 20 bases in length. . When selecting the preferred length for a given polynucleotide, a balance must be established to gain the most favorable characteristics. Shorter polynucleotides such as those of 10 to 15 units, although offering greater cell penetration, have lower gene specificity. In contrast, although polynucleotides longer than 20-30 bases offer better specificity, they show decreased kinetics of uptake in cells. See Stein et al., Phosphorothioate Oligodeoxynucleotide Analogues in Oligodeoxynucleotides-Antisense Inhibitors of Gene Expression (Cohen, ed., McMillan Press, London 1988). Accessibility to target mRNA sequences are also of importance, and therefore, the ring-forming regions in the target mRNAs offer promising targets. In this description, the term "polynucleotides" encompasses oligomeric nucleic acid portions of the type found in nature, such as the deoxyrronucleotide and ribonucleotide structures of DNA and RNA, as well as man-made analogues which are capable of binding to acids nucleics found in nature. The polynucleotides of the present invention can be based on ribonucleotide or deoxyribonucleotide monomers attached to phosphodiester linkages, or by analogs linked by methyl phosphonate, phosphorothioate or other linkages. They may also comprise monomeric portions which have altered base structures or other modifications, but which will still retain the ability to bind naturally occurring DNA and RNA structures. Such polynucleotides can be prepared by methods well known in the art, for example using commercially available machines and reagents available from Perkin-Elmer / Applied Biosystems (Foster City, CA). The phosphodiester-linked polynucleotides are particularly susceptible to the action of nucleases in the serum or within the cells, and therefore in a preferred embodiment the polynucleotides of the present invention are phosphorothioate analogs or analogues attached to methyl phosphonate which are has shown that they are resistant to nuclease. Those of ordinary skill in the art will be able to select other links for use in the invention. These modifications can also be designed to improve the cellular uptake and stability of the polynucleotides.

In another embodiment of the invention, the antisense polynucleotide is an RNA molecule produced by introducing an expression construct into the target cell. The RNA molecule produced in this manner is chosen to have the ability to hybridize with APJ mRNA. Such molecules having this ability can inhibit the translation of the APJ mRNA and thus inhibit the ability of HIV to infect cells containing the RNA molecule. Polynucleotides which have the ability to hybridize with mRNA targets can inhibit the expression of corresponding gene products by multiple mechanisms. In "translation suppression", the interaction of the polynucleotides with target mRNA blocks the action of the ribosomal complex and therefore prevents translation of the messenger RNA into a protein. Haeuptle et al., Nucí. Acids Res. 14: 1427 (1986). In the case of the phosphodiester or phosphorothioate DNA polynucleotides, the intracellular RNase H can digest the directed RNA sequence once it has hybridized to the DNA oligomer. Walder and Walder, Proc. Na ti. Acad. Sci. USA 85: 5011 (1988). As an additional mechanism of action in the "suppression of transcription" it seems that some polynucleotides can form "triple" or triple helical structures with double-stranded genomic DNA that contains the gene of interest and therefore interfere with transcription by RNA polymerase . Giovannangeli et al., Proc. Na ti. Acad. Sci. 90: 10013 (1993); Ebbinghaus et al., J. Clin. Invest. 92: 2433 (1993). In a preferred embodiment, the APJ polynucleotides are synthesized according to standard methodology. DNA polynucleotides modified with phosphorothioate are typically synthesized in automated DNA synthesizers available from a variety of manufacturers. These instruments are capable of synthesizing amounts of nanomoles of polynucleotides up to 100 nucleotides long. The shorter polynucleotides synthesized by modern instruments can be modified by polyacrylamide gel electrophoresis or reverse phase chromatography. See Sambrook et al., Molecular Cloning: A Labora tory Manual, Vol. 2, Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Alternatively, an APJ polynucleotide in the form of antisense RNA can be introduced into a cell by its expression within the cell from a standard DNA expression vector. The antisense DNA sequences of APJ can be cloned from standard plasmids into expression vectors, expression vectors which have characteristics that allow higher levels of, or more efficient expression of, resident polynucleotides. At a minimum, these constructs require a prokaryotic or eukaryotic promoter sequence which initiates the transcription of the inserted DNA sequences. A preferred expression vector is one in which the expression is inducible at high concentrations. This is accomplished by the addition of a regulatory region which provides enhanced transcription of the sequences towards the 3 'end in the appropriate host cell. See Sambrook et al., Molecular Cloning: A Labora tory Manual Vol. 3, Chapter 16, Cold-Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). For example, APJ antisense expression vectors can be constructed using the polymerase chain reaction (PCR) to amplify the appropriate fragments of single-stranded cDNA from a plasmid such as pRc in which APJ cDNA has been incorporated. Fang et al., J. Biol. Chem. 267: 25889-25897 (1992). Polynucleotide synthesis and purification techniques are described in Sambrook et al., And Ausubel et al. (eds.), Curren t Protocols in Molecular Biology (Wiley Interscience 1987) (below "Ausubel"), respectively. The PCR procedure is carried out by well-known methodology. See, for example Ausubel and Bangham, The Polymerase Chain Reaction: Getting Started, in Protocols in Human Molecular Genetics (Humana Press 1991). In addition, PCR equipment can be purchased from companies such as Stratagene Cloning System (La Jolla, CA) and Invitrogen (San Diego, CA). The PCR products are subcloned into cloning vectors. In this context, a "cloning vector" is an -DNA molecule, such as a plasmid, cosmid or bacteriophage that can be replicated autonomously in a prokaryotic host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites in which DNA sequences can be inserted in a determinable manner without loss of an essential biological function of the vector, as well as a frame that is suitable for use in the identification and selection of cells transformed with the cloning vector. Suitable cloning vectors are described by Sambrook et al., Ausubel, and Brown (ed.), Molecular Biology Labfax (Academic Press 1991). Cloning vectors can be obtained, for example, from GIBCO / BRL (Gaitherburg, MD), Clontech Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI), Stratagene Cloning Systems (La Jolla, CA) , Invitrogen (San Diego, CA), and American Type Culture Collection (Rockville, MD). Preferably, the PCR products are ligated to a "TA" cloning vector. Methods for generating PCR products with a thymidine or saline adenine are well known to those skilled in the art. See, for example, Ausubel on pages 15.7.1-15.7.6. In addition, equipment can be purchased to perform the TA cloning of companies such as Invitrogen (San Diego, CA). The cloned antisense fragments are exemplified by transforming competent bacterial cells with a cloning vector and growing the bacterial host cells in the presence of the appropriate antibiotic. See, for example, Sambrook et al., And Ausubel. Then PCR is used to analyze the bacterial host cells to determine APJ antisense orientation clones. The use of PCR by bacterial host cells is described, for example, by Hoffman et al., Sequencing DNA Amplified Directly from a Bacterial Colony, in PCR Protocols: Methods and Applications, White (ed.), Pages 205-210 (Humana Press 1993), and by Cooper et al., PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adapter / Specific DOS Primer-Pair Strategy, Id, on pages 305-316. The cloned antisense fragments are separated from the cloning vector and inserted into an expression vector. For example, HindIII and Xbal can be used to separate the antisense fragment from the cloning vector TA pCRMR-II (Invitrogen, San Diego, CA). Suitable expression vectors typically contain: (1) DNA elements Prokaryotic - which code for a bacterial origin of replication and a marker of antibiotic resistance to provide for the amplification and selection of the expression vector in a bacterial host; (2) DNA elements that control the initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription / polyadenylation termination sequence. For a mammalian host, the transcriptional and translational regulatory signals are preferably derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus or the like, in which the regulatory signals are associated with a particular gene which has a high level expression. Transcriptional and translational regulatory sequences can also be obtained from mammalian genes, such as the genes of actin, collagen, myokin and metallothionin. The transcriptional regulatory sequences include a sufficient promoter region to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the mouse metallothionein I gene promoter (Hamer et al., J. Mol. Appl. Genes, 1: 273 (1982)); the Herpes virus TK promoter (McKnight, Cell 31: 355 (1982)); the SV40 early promoter (Benoist et al., Na ture 290: 304 (1981); the Rous sarcoma virus promoter (Gorman et al., Proc.

Na t 'l. Acad. Sci. USA, 79: 6777 (198Z)); and the cytomegalovirus promoter (Foecking et al., Gene 45: 101 (1980)). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used to control the expression of the fusion gene if the prokaryotic promoter is regulated by a eukaryotic promoter. Zhou et al., Mol. Cell. Biol. 10: 4529 (1990); Kaufman et al., Nucí. Acids Res. 19: 44-85 (1991). A vector for introducing at least one antisense polynucleotide into a cell by expression from a DNA is the pRc / CMV vector (Invitrogen, San Diego, CA), which provides a high level of constitutive transcription from elongation sequences. mammal promoters. The cloned APJ antisense vectors are amplified in bacterial host cells, isolated from cells, and analyzed as described above. Another possible method by which antisense sequences can be exploited is via gene therapy. Virus-like vectors, usually derived from retroviruses, can demonstrate utility as vehicles for the import and expression of antisense constructs in human cells. Generally, such vectors are non-replicable in vivo, preventing any unwanted infection of non-target cells. In such cases, auxiliary cell lines (helper) are provided which provide the replicative functions lost in vivo, thereby allowing the amplification and packaging of the antisense vector. An additional precaution against accidental infection of non-target cells involves the use of regulatory sequences specific to the target cell. When they are under the control of such sequences, the antisense constructs are not expressed in normal tissues. Two previous studies have explored the feasibility of using antisense polynucleotides to inhibit the expression of a growth factor that binds to heparin. Kouhara et al., Oncogene 9: 455-462 (1994); Morrision, J. Biol. Chem. 266: 728 (1991). Kouhara et al. show that androgen-dependent growth of mouse mammary carcinoma cells (SC-3) is mediated by induction of an androgen-induced heparin-binding growth factor (AIGF). A sequence of 15 antisense units corresponding to the translation start of the AIGF site is measured to determine its ability to interfere with androgen induction of SC-3 cells. At concentrations of 5 μM, the antisense polynucleotide effectively inhibits DNA synthesis. Morrision shows that antisense polynucleotides directed against basic fibroblast growth factor can inhibit the growth of astrocytes in culture. Therefore, the general feasibility of directing an individual gene product in a mammalian cell has been established.

The antisense polynucleotides according to the present invention are derived from any portion of the open reading frame of the cDNA for APJ. Preferably, the mRNA sequences (i) surround the translation initiation site, and (ii) form curl structures that are directed. In-.base in the size of the human genome, statistical studies show that a DNA segment of approximately 14-15 pairs in length will have a unique sequence in the genome. To ensure specificity of direction of the RNA for APJ, therefore, it is preferred that the antisense polynucleotides have a length of at least 15 nucleotides. Therefore, the shorter polynucleotides contemplated by the present invention encompass nucleotides corresponding to positions 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 2-16, 3- 17, etc. of the cDNA sequence for APJ. Position 1 refers to the first nucleotide of the region that codes for APJ. Not every antisense polynucleotide will provide a sufficient degree of inhibition or a sufficient level of specificity for the target APJ. Therefore, it will be necessary to analyze "polynucleotides to determine which ones have the appropriate antisense characteristics." A preferred test method for a useful antisense polynucleotide is the inhibition of cell fusion between (1) cells containing CD4 and APJ, and (2) cells containing env.

The administration of an antisense polynucleotide to a subject, either a synthetic naked polynucleotide or as part of an expression vector, can be carried out by any common route (oral, nasal, buccal, rectal, vaginal or topical), or by subcutaneous, intramuscular, interperitoneal or intravenous injection. However, the pharmaceutical compositions of the present invention are advantageously administered in the form of injectable compositions. A typical composition for such a purpose comprises a pharmaceutically acceptable solvent or diluent, and other suitable physiological compounds. For example, the composition may contain a polynucleotide and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl. They have been administered intravenously until 700 milligrams of antisense polynucleotide to a patient during the course of 10 days (i.e., 0.05 mg / kg / hour) without signs of toxicity. Sterling, "Systemic Antisense Treatment Reported," Genetic Engineering News 12: 1, 28 (1992). Other pharmaceutically acceptable excipients include non-aqueous or aqueous solutions and non-toxic compositions including salts, preservatives, buffers and the like. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous solutions include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Conservatives include antimicrobial substances, antioxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to the skills common to those skilled in the art. A preferred pharmaceutical composition for topical administration is a dermal cream or a transdermal patch. The antisense polynucleotides or their expression vectors can be administered by injection or as an oil suspension. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. In addition, the antisense polynucleotides or vectors can be combined with a lipophilic carrier such as any of the many sterols that include cholesterol, cholate and deoxycholic acid. A preferred sterol is cholesterol. Suspensions for aqueous injection may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension also contains stabilizers.

An alternative formulation for the administration of antisense polynucleotides for APJ involves liposomes. The encapsulation in liposome provides an alternative formulation for the administration of polynucleotides antisense for APJ and expression vectors. Liposomes are microscopic vesicles that consist of one or more lipid dicaps that surround the aqueous compartments. See, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.

Infect. Dis. 12 (Suppl 1): S61 (1993), and Kim, Drugs 46: 618 W 10 (1993). The liposomes are similar in composition to cell membranes and, as a result, liposomes can be administered safely and are biodegradable. Based on the method of preparation, the liposomes can be unilamellar or multilamellar, and the liposomes can vary in size with diameters ranging from 0.01 μM to more than 10 μM. Various agents can be encapsulated in the liposomes: hydrophobic cleavage agents in the bilayers and hydrophilic cleavage agents within the interior aqueous space or spaces. See, for example, Machy et al., Liposomes in Cell Biology And Pharmacology (John Libby 1987), and Ostro et al., American J. Hosp. Pharm. 46: 1576 (1989). In addition, it is possible to control the therapeutic availability of the encapsulated agent by varying the size of the liposome, the number of bilayers, the lipid composition, as well as the charge and characteristics of the liposome. surface of the liposomes.

Liposomes can be absorbed in virtually any cell type and then slowly release the encapsulated agent. Alternatively, an absorbed liposome can be subjected to endocytosis by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents. Scherphof et al., Ann. N. Y. Acad. Sci. 446: 368 (1985). After intravenous administration, conventional liposomes are preferably phagocytosed in the reticuloendothelial system. However, a reticular endothelial system can be avoided by several methods that influence saturation with large doses of liposome particles, or selective inactivation of macrophages by pharmacological means. Claassen et al., Biochim. Biophys. Acta 802: 428 (1984). In addition, the incorporation of phospholipids derivatized with glycolipids or polyethylene glycol within the liposome membranes has been shown to result in significantly reduced uptake by the reticuloendothelial system. Alien et al., Biochim. Biophys. Minutes 1068: 133 (1991); Alien et al., Biochim. Biophys. Acta 1150: 9 (1993). These Stealth ™ Liposomes have increased circulation time and improved targeting of tumors in animals. Woodle et al., Proc. Ameri. Assoc. Cancer Res. 33: 2672 (1992); Gregoriadis et al., Drugs 45:15 (1993).

Antisense polynucleotides and expression vectors can be encapsulated within liposomes using standard techniques. A variety of different compositions of liposomes and methods for synthesis are known to those skilled in the art. See, for example, the US patent. No. 4,844,904, the US patent. No. 5,000,959, the US patent. No. 4,863,740 and the US patent. No. 4,975,282, all of which are incorporated herein by reference. Liposomes can be prepared to target particular cells or organs by varying the phospholipid composition or by inserting receptors or ligands into the liposomes. For example, antibodies specific for tumors associated with antigens may be incorporated within the liposomes, together with antisense polynucleotides or expression vectors, to direct the liposome more effectively to the tumor cells. See, for example, Zelphati et al., Antisense Research and Developmen t 3: 323-338 (1993), which describes the use of "immunoliposomes" containing antisense polynucleotides for therapy in humans. In general, the dosage of antisense polynucleotides encapsulated in liposome that are administered as well as the vectors will vary based on factors such as the age, weight, health, sex and general medical condition of the patient as well as the previous medical history. Dose ranges for particular formulations can be determined by using a suitable animal model. The above solutions can also be used not only with antisense nucleic acids, but also with ribosims or triplex agents to block the transcription or translation of a mRNA for specific APJ, either by masking that mRNA with an antisense nucleic acid or by a triplex agent, or separating it with a ribosim. The use of an oligonucleotide to stop transcription is a process known as a triplex strategy since the oligomer winds around the double helical DNA, forming a three-stranded helix. Therefore, these triplex compounds can be designed to recognize a single site in a chosen gene (Maher, et al., Antisense Res. And Dev., 1 (3) 1227, 1991; Helene, C, Anticancer Drug Design, 6 (6): 569, 1991). 'Ribosomes are RNA molecules that have the ability to specifically separate other single-stranded RNAs in a manner analogous to DNA restriction endonucleases. By modifying the nucleotide sequences which code for these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and divide them (Cech, J. Amer. Med. Assn., 260: 3030, 1988). . A major advantage of these solutions is that, because they are sequence specific, only mRNAs with particular sequences are inactivated. There are two basic types of ribosomes, specifically of the tetrahymena type (Hassellhoff, Na ture, 334: 585, 1988) and of the "hammer head" type. The ribshymes of the tetrahymena type recognize sequences which are four bases in length, while the ribosimuses of the "hammerhead" type are known sequences of 11-18 bases in length. The longer the recognition sequence, the greater the probability that the sequence occurs exclusively in the target mRNA species. Accordingly, ribosimals of the hammerhead type are preferable to ribhysemas of the tetrahymena type to inactivate a specific species of mRNA, and 18-base recognition sequences are preferable in comparison to shorter recognition sequences. Without further elaboration, it is considered that a person skilled in the art can, using the preceding description, use the present invention to its fullest extent. The following examples should be considered as illustrative and are therefore limiting for the rest of the description. cell by measuring luciferase activity in cell lysates 7-8 hours after mixing. In this assay, cell-cell fusion results in cytoplasmic mixing by introducing luciferase, which can be easily quantified. As shown in Figure 1-and the coexpression of either the CCR5 or CXCR4 co-receptor with CD4 results in efficient fusion by Env proteins R5 and X4, respectively. The R5-X4 env proteins such as HIV-1 89.6 mediated fusion with cells that have either the CCR5 or CXCR co-receptor. No fusion is observed when CD4 is expressed alone. When APJ is coexpressed with CD4 in QT6 cells, the cell-cell fusion is mediated by the 89.6 env protein R5-X4 and by several env X4 proteins at levels = 10% of that observed with CXCR4 (Figure 1). For a primary env X4 protein, ZR001.3, fusion with cells expressing APJ is more efficient than with CXCR4. Most of the R5 env proteins mediate fusion with cells that express APJ, but only at very low concentrations in relation to that observed with CCR5 (figure 1 and table 1). However, ADA and the primary isolate TH 22-4 show fusion mediated by APJ generally at half the level observed when CCR5 serves as the viral co-receptor. The HIV-2 env ST protein also mediates a very inefficient fusion with cells that express both CD4 and APJ. The ability of APJ to sustain fusion by some viral env proteins X4 and R5-X4 almost as efficiently as the main coreceptors is remarkable, since most of the other alternative HIV-1 coreceptors are typically • support cell-cell fusion much less efficiently than CCR5 or CXCR4. 5 The ability of APJ to support fusion by a panel of SIV envelope proteins was also examined. Unlike HIV-1, tropic SIV strains for M and for T use CCR5 as a co-receptor, whereas CXCR4 is rarely used or used by SIV (Chen et al., 1997, J.

Virol. , 71: 2705-2714; Edinger et al., 1997, Proceedings of the National Academy of Sciences, USA, 94: 4005-4010; and Marcon et al., 1997, J. Virol. , 71: 2522-2527). In addition, orphan receptors STRL33, GPR15 and GPR1 can be used as co-receptors for trophic SIV strains for T and M (Deng et al., 1997, Na ture, 388: 296-300 and Farzan et al., 1997, J. Exp. Med., 186: 405-411). In the present experiments, it was determined that APJ supports fusion for several trophic SIV env proteins for M and T at levels that are less efficient than those granted with CCR5, with the exception of SIV mac316 trophic for M and a variant of this env protein (316mut) which efficiently used APJ as a co-receptor in the cell-cell fusion assays (figure 2 and table 1). Additionally, APJ typically supports the section less efficiently compared to orphan receivers GPR1, GPR15 / BOB, and STRL33 / Bonzo. Finally, because it is previously determined that many strains of SIV can infect cells in a CD4-independent and CCR5-dependent manner (Edinger et al., 1997, Proc. Na ti. Acad. Sci., USA, 94: 14742- 14747), the ability of HIV-1, HIV-2 and SIV env proteins to mediate fusion with cells expressing only APJ was also tested - The results show that the APJ coreceptor activity is strictly dependent on CD4, since cells expressing APJ alone do not support cell-cell pressure with any of the env proteins tested.

TABLE 1 Example 2. Determination of. the ability of APJ to sustain virus infection.

The ability of APJ to sustain virus infection was determined in order to more rigorously determine the ability of APJ to function as a coreceptor.

A first assay system used a luciferase reporter virus assay in which the various env proteins were pseudotyped on the major structure of the luciferase reporter virus. Luciferase reporter viruses were prepared by transfecting human HEK 293 cells with a plasmid expressing env under the control of the CMV promoter or SV40, and with a plasmid containing the proviral genome with an inactive env gene and the luciferase gene instead of nef (eg, the main structure of luciferase virus NL4-3 (pNL-Luc-ER-)) (Chen et al., 1994, J. Virol., 68: 654-660 and Connor et al., 1995, Virology, 206: 935-944). The target cells for infection were HEK 293 or CCCS cells with CD4 and the coreceptors introduced by transfection of calcium phosphate. Infections were carried out in medium containing 8 μg / ml DEAE dextran. The cells were used 3-4 days after infection by resuspension in NP-40 0.5% in PBS and assayed for luciferase activity. Unfortunately, most env proteins which efficiently catalyze fusion with cells expressing CD4 and APJ (such as HIV-1 89.6) can not be pseudotyped successfully. Vi env proteins that can be pseudotyped, judging by infection of CCR5 or CXCR4 positive cells, fail to infect CD4 expressing cells and the APJ co-receptor, or do so inefficiently (see figure 3). In some cases, env proteins that mediate fusion with cells that express APJ at intermediate levels do not sustain viral infection. For example, the seudatype virus with the ADA env protein does not infect APJ positive cells even though the cells express the ADA env protein mediated by fusion with APJ positive cells at half efficiency compared to CCR5 positive cells. The reasons for these test-dependent discrepancies are not clear, but may reflect the efficiencies in which various env proteins can be pseudotyped.

Another test system was used in order to test envelope proteins which could not have been pseudotyped but which were able to mediate cell-cell fusion with cells expressing APJ in a significant way. Objective HEK 293 cells that have been "transfected with plasmids expressing CD4, the desired receptor and luciferase under the control of viral LTR were infected with HIV-1 89.6 intact or HIV-1 IIIB (Figure 4). Two days after infection, the results show that APJ positive cells infected with HIV-1 89.6 are almost as efficient as cells expressing CXCR 4. HIV-1 IIIB (HxB3) also infects APJ positive cells at levels well above Finally, a PCR-based entry assay was also used to determine whether APJ can support infection with HIV-1 89.6 and IIIB.QT6 cells stably express human CD4 and transiently express the co-receptor and coinfected with 50 ng of p24 of cell-free virus, treated with DNase After two days, the cells were washed and used, and HIV-1 specific LTR DNA sequences were detected by PCR using primers LTR-plus / LTR-minus (5 '-ACAAGCTAGTACCCAGTTGAGCC-3' (SEC. FROM IDENT. NO .: 4), 5t-CACACACTACTTGAAGCACTCA-3 '(SEQ ID NO: 5)). The products are separated by electrophoresis on 2% agarose gels, transferred to Hybond N + (Amersham), and detected using the Biotin kit labeled at the 3 'end (DuPont, 5'-ATCTACAAGGGACTTTCCCGC-3' probe (SEQ. DE IDENT NO .: 6), followed by exposure As shown in Figure 5, both HIV-1 IIIB and 89.6 can enter QT6 cells expressing both CD4 and APJ, although entry into the cell is less efficient than with " main coreceptors of HIV-1.

Example 3: Examination of the distribution of APJ in human brain by northern transfer analysis APJ was originally cloned from human genomic DNA and analysis of rat tissues using a probe based on a rat homologue shows that APJ is widely expressed in the brain (O 'Dowd et al., 1993, Gene, 136: 355- 360). It has also been shown that APJ is expressed in certain areas of the human brain (Matsumoto et al., 1996, Neurosci, Lett., 219: 119-122). Due to the efficient use of APJ as a co-receptor by some virus strains, the distribution of APJ in the human brain has been further examined by northern blot analysis. Membranes containing poly A + RNA from various regions of the human brain are obtained from Clontech. Prime-It II random primer labeling equipment (Stratagene, La Jolla, CA) was used to label the cDNA probe with a-32P-dATP (3,000 Ci / mmoles) using the Klenow enzyme. The cDNA probe labeled with a-32P is quantified using Quick Spin columns (Boehringer Mannheim, Indianapolis, IN). The membranes are hybridized overnight with 107 cpms of the labeled probe in hybridization buffer containing 25 mM Na / Na2P04, 50 mM Tris pH 7.4, 6X SSPE, 0.1% SDS, 100 μg / ml of chain DNA simple and Denhart IX solution. The membranes are washed twice in IX SSPE, 0.1% SDS at 42 ° C for 10 minutes and changed to a high stringency wash solution of 0.2X SSPE, 0.1% SDS at 42 ° C for 10 minutes. The membranes are then exposed to a Fuji image plate for 4 hours. The images on the plate are captured on a BASlOOOMac Bio-Imaging (Fuji) analyzer and processed with Mac BAS software. The images are printed on a digital printer Pictography 3000 (Fuji). The results show that high levels of APJ transcripts are present in the corpus callosum, spinal cord and medulla. Lower levels of APJ transcripts are detected in other regions of the human brain (Figure 6). In peripheral tissues, the APJ transcript is easily detected in the spleen but is absent in the PBL (figure 7). Minor levels of transcript are detected in other peripheral tissues. To investigate the distribution of APJ in cells commonly used to propagate HIV-1, RT-PCR analysis is performed on a large number of cell lines and on some types of primary cells. A U87 cell line that stably expresses APJ is generated and used as a positive control. Primary cells are isolated as follows. Human blood mononuclear cells (PBMC) are harvested from blood of normal volunteers using Ficoll-Hypaque, suppressed from monocytes by serial adhesion to plastic, stimulated with phytohemagglutinin (PHA-L, 5 μg / ml; Sigma) for 3 days and then resuspended with interleukin 2 (20 U / ml, (Boehringer Mannheim Biochemicals) .The RNA is extracted after three days of PHA'y stimulation and also after one week in IL-2. they are purified from PBMC by selective adhesion to gelatin followed by plastic and then maintained in culture to maintain differentiation into monocyte-derived macrophages (MDM) as previously described (Coliman et al., 1989, J. Exp. med., 170: 1149-1163) RNA is extracted from undifferentiated monocytes immediately after purification and from MDM after one week in culture For the isolation of total cellular RNA for RT-PCR, 5-10 x 10 6 cells are resuspended in 1 ml of Trizol (GIBCO-BRL) and processed as directed by the manufacturer, then the total RNA is treated with 1 μl (10-50 units) of DNase (RNase-free) (Boehringer Mannheim) per 10 μg of RNA for 30 minutes at 37 ° C in the presence of 5 mM MgCl 2 , with subsequent inactivation at 65 ° C for 10 minutes in the presence of 5 mM EDTA; the concentration of RNA based on DO260 is calculated. The Titan RT-PCR system (Boehringer Mannheim) is used to evaluate RNA expression patterns. The specific internal primers are used towards the 5 'end and towards the 3' end which result in an amplified product of 481 base pairs. The primers used were the following: direct 5'-TACACAGACTGGAAATCCTCG-3 '(SEQ ID NO: 7) and reverse 5'-TGCACCTTAGTGGTGTTCTCC-3' (SEQ ID NO: 8). In order to control the contamination of the RNA sample with genomic DNA in spite of the DNase treatment, all the RNA samples were also amplified with a Titan enzyme mixture in which the RT activity was destroyed but not the PCR activity. treatment at 95 ° C for 10 minutes (this inactivation protocol was found to eliminate the ability to amplify an RNA but not a DNA template). In each RT-PCR reaction, the RNA isolated from U87-APJ from stably transfected cells is included as a positive RNA control and the plasmid DNA is included as a second positive control. The results of the investigation of the distribution of APJ in cell lines and cell types shows that APJ is expressed in C8166 cells, but that the reaction products specific for APJ can not be detected in the other cell lines examined, which include Jurkat, Hut78, CEMxl74 and PMl cells. Additionally, the expression of APJ is not detected in PBMC stimulated with PHA, PHA with IL-2 or anti-CD3 and IL-2, or in monocytes or monocyte-derived macrophages (Figure 8). For other aspects of nucleic acids, polypeptides, antibodies, etc., reference is made to the standard textbooks of molecular biology, protein science and immunology. See, Davis et al., (1986), Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York; Hames et al., (1985), Nucleic Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al .; Curren t Protocols in Molecular Biology, edited by F.M. Ausubel et al., John Wiley & Sons, Inc .; Curren t Protocols in Human Genetics, edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc .; Current Protocols in Protein Science, - edited by John E. Coligan et al., John Wiley & Sons, Inc .; Curren t Protocols in Immunology; edited by John E. Coligan et al., John Wiley & Sons, Inc. The entire description of all patent applications, patents and publications mentioned herein are incorporated herein by reference. From the above description, a person skilled in the art can easily determine the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention and adapt it to the various uses and conditions.

Claims (35)

1. A recombinant human cell transformed with a polynucleotide that encodes an APJ polypeptide or a polynucleotide that encodes a CD4 polypeptide, in which the human cell coexpresses the APJ and CD4 polypeptides.

2. A recombinant human cell transformed with a polynucleotide encoding an APJ polypeptide and a polynucleotide encoding a CD4 polypeptide, wherein the cell coexpresses the APJ and CD4 polypeptides.

3. The recombinant human cell, as described in claim 1, wherein the cell is stably transformed.

4. The recombinant human cell, as described in claim 2, wherein the cell is stably transformed with both polynucleotides.

5. An antibody, characterized in that it binds specifically to an extracellular domain of APJ, wherein the antibody inhibits HIV infection of a target cell that coexpresses the APJ and CD polypeptides.

6. An antibody, characterized in that it binds specifically to an extracellular APJ domain, wherein the antibody inhibits membrane fusion between a first cell and expressing the APJ and CD4 polypeptides, and a second cell expressing an HIV env protein.

7. The antibody as described in claim 5 or 6, wherein the antibody is a monoclonal antibody.

8. The antibody as described in the claim 7, wherein the antibody recognizes an epitope comprising an amino acid sequence which is a portion of the first extracellular domain of APJ.

9. The antibody as described in the claim 8, wherein the amino acid sequence corresponding to a portion of the first extracellular APJ domain comprises the amino acid sequence Asn-Tyr-Tyr-Gly (SEQ ID NO: 3).

10. The antibody as described in claim 7, wherein the antibody recognizes an epitope comprising an amino acid sequence that corresponds to a portion of the second extracellular APJ domain.

11. A peptide fragment of APJ, substantially purified, in which the peptide inhibits HIV infection of a target cell that coexpresses the APJ and CD4 polypeptides.

12. A peptide fragment of APJ, substantially purified, wherein the peptide inhibits cell fusion between a first cell that coexpresses the APJ and CD4 polypeptides, and a second cell expressing an HIV env protein.

13. A peptide fragment of APJ, substantially purified, as described in claim 11 or 12, wherein the peptide fragment comprises an amino acid sequence which is a portion of the first extracellular domain of APJ.

14. A substantially purified peptide fragment of APJ, as described in claim 13, wherein the amino acid sequence corresponds to a portion of the first extracellular domain of APJ, comprises the amino acid sequence Asn-Tyr-Tyr-Gly (SEQ. IDENT. NO .: 3).

15. The substantially purified peptide fragment of APJ, as described in claim 11 or 12, wherein the peptide fragment comprises an amino acid sequence corresponding to a portion of the second extracellular APJ domain.

16. A method to indetify a compound that modulates the interaction between an HIV virus and an APJ receptor ", which comprises incubating a first human cell line which coexpresses the CD4 and APJ polypeptides with a second human cell line which expresses an env protein under conditions which promote cell fusion, in the presence and absence of a test compound, and determine whether the presence of the test compound inhibits cell fusion between the human cell line and the second line of human cells.

17. The method as described in the claim 16, in which cell fusion is determined by the detection of an indicator molecule.

18. The method as recited in claim 17, wherein the indicator molecule is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.

19. The method as described in claim 17, wherein the reporter molecule is B-galactosidase or luciferase. to

20. A method for identifying a compound that modulates the interaction between an HIV virus and an APJ receptor, which comprises incubating a line of human cells which express the CD4 and APJ polypeptides with a test virus presenting a protein. env, in the presence and absence of a test compound, and determine whether the presence of the test compound inhibits infection of the human cell line by the test virus.

21. The method as described in claim 20, wherein the infection is determined by detection of an indicator molecule.

22. The method as described in the claim 21, wherein the indicator molecule is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme.

23. The method as described in claim 21, wherein the reporter molecule is B-galactosidase or luciferase.

24. The use of an effective amount of an APJ binding agent or blocker for the production of a medicament for the treatment of HIV infection by inhibiting HIV infection of a target cell expressing the APJ and CD4 polypeptides.

25. The method as defined in claim 24, wherein the agent is an antibody to APJ or an epitope that binds a fragment thereof.

26. The method as defined in claim 25, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

27. The method as defined in claim 24, wherein the agent is a pepetidic fragment of APJ.

28. The use of an agent that suppresses APJ in a subject, for the production of a medicament for treating a subject having an HIV-related disorder associated with the expression of APJ.

29. The method as defined in claim 28, wherein the agent is an antibody to APJ. •

30. The method as defined in claim 28, wherein the agent is an "antisense polynucleotide" that hybridizes to an APJ polynucleotide.

31. The method as defined in claim 28, wherein the agent is introduced into a cell using a • 10 carrier.

32. The method as defined in claim 31, wherein the carrier is a vector.

33. The use of a therapeutically effective amount of an antibody to APJ or a peptide fragment, wherein it is used for the production of a medicament for the treatment of a subject who has or is at risk of having an HIV infection. or a related disorder.

34. The use as described in claim 33, wherein the subject is a fetus.

35. A transgenic non-human animal, which has a phenotype defined by the expression of APJ polypeptide and the CD4 polypeptide otherwise does not occur naturally in the animal, in which the phenotype is conferred by a transgene contained in the cells somatic and germ cells of the animal, and wherein the transgene comprises a polynucleotide encoding an APJ polypeptide or a polynucleotide "that encodes a CD4 polypeptide.