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. 1994 Feb 15;13(4):774–783. doi: 10.1002/j.1460-2075.1994.tb06320.x

Association of p56lck with the cytoplasmic domain of CD4 modulates HIV-1 expression.

M Tremblay 1, S Meloche 1, S Gratton 1, M A Wainberg 1, R P Sékaly 1
PMCID: PMC394876  PMID: 8112293

Abstract

To investigate the role played by the cytoplasmic domain of the CD4 glycoprotein in the process of HIV infection, we have transfected two CD4-negative human T cell lines with cDNAs encoding the full-length CD4 and a truncated form of the molecule, lacking most of the cytoplasmic domain. Levels of viral replication were significantly higher in cells carrying the truncated version of CD4, in comparison with cells expressing the full-length CD4, as measured by the percentage of cells expressing viral p24 protein and the number of infectious particles released into culture supernatants. The extent of viral entry and reverse transcription was similar in each case, as monitored by an enzymatic test and quantitative PCR. Quantitative differences at RNA and protein levels were responsible for changes in viral production. To further characterize the mechanisms responsible for decreased rates of HIV replication in CD4-expressing cells we have treated the different cell lines, very early after HIV infection, with azidothymidine and soluble CD4, two antiviral agents that inhibit replication of HIV at different stages in the virus replicative cycle. Results from these experiments indicate that a cellular signal is mediated by the CD4 molecule, which negatively regulates the expression of viral DNA already present in such cells. This signal would be initiated following oligomerization of the CD4 molecule by the virus itself. Results from experiments with a CD4 construct containing mutations of the cysteine residues which are responsible for association of CD4 with p56lck demonstrate that p56lck is implicated in the transduction of the signal negatively regulating HIV replication.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abraham N., Miceli M. C., Parnes J. R., Veillette A. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature. 1991 Mar 7;350(6313):62–66. doi: 10.1038/350062a0. [DOI] [PubMed] [Google Scholar]
  2. Arthos J., Deen K. C., Chaikin M. A., Fornwald J. A., Sathe G., Sattentau Q. J., Clapham P. R., Weiss R. A., McDougal J. S., Pietropaolo C. Identification of the residues in human CD4 critical for the binding of HIV. Cell. 1989 May 5;57(3):469–481. doi: 10.1016/0092-8674(89)90922-7. [DOI] [PubMed] [Google Scholar]
  3. Banda N. K., Bernier J., Kurahara D. K., Kurrle R., Haigwood N., Sekaly R. P., Finkel T. H. Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med. 1992 Oct 1;176(4):1099–1106. doi: 10.1084/jem.176.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bank I., Chess L. Perturbation of the T4 molecule transmits a negative signal to T cells. J Exp Med. 1985 Oct 1;162(4):1294–1303. doi: 10.1084/jem.162.4.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bedinger P., Moriarty A., von Borstel R. C., 2nd, Donovan N. J., Steimer K. S., Littman D. R. Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4. Nature. 1988 Jul 14;334(6178):162–165. doi: 10.1038/334162a0. [DOI] [PubMed] [Google Scholar]
  6. Böhnlein E., Lowenthal J. W., Siekevitz M., Ballard D. W., Franza B. R., Greene W. C. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell. 1988 Jun 3;53(5):827–836. doi: 10.1016/0092-8674(88)90099-2. [DOI] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Coombs R. W., Collier A. C., Allain J. P., Nikora B., Leuther M., Gjerset G. F., Corey L. Plasma viremia in human immunodeficiency virus infection. N Engl J Med. 1989 Dec 14;321(24):1626–1631. doi: 10.1056/NEJM198912143212402. [DOI] [PubMed] [Google Scholar]
  9. De Lean A., Hancock A. A., Lefkowitz R. J. Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol Pharmacol. 1982 Jan;21(1):5–16. [PubMed] [Google Scholar]
  10. Diamond D. C., Finberg R., Chaudhuri S., Sleckman B. P., Burakoff S. J. Human immunodeficiency virus infection is efficiently mediated by a glycolipid-anchored form of CD4. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5001–5005. doi: 10.1073/pnas.87.13.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drysdale C. M., Pavlakis G. N. Rapid activation and subsequent down-regulation of the human immunodeficiency virus type 1 promoter in the presence of Tat: possible mechanisms contributing to latency. J Virol. 1991 Jun;65(6):3044–3051. doi: 10.1128/jvi.65.6.3044-3051.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fleury S., Lamarre D., Meloche S., Ryu S. E., Cantin C., Hendrickson W. A., Sekaly R. P. Mutational analysis of the interaction between CD4 and class II MHC: class II antigens contact CD4 on a surface opposite the gp120-binding site. Cell. 1991 Sep 6;66(5):1037–1049. doi: 10.1016/0092-8674(91)90447-7. [DOI] [PubMed] [Google Scholar]
  13. Folks T., Powell D. M., Lightfoote M. M., Benn S., Martin M. A., Fauci A. S. Induction of HTLV-III/LAV from a nonvirus-producing T-cell line: implications for latency. Science. 1986 Feb 7;231(4738):600–602. doi: 10.1126/science.3003906. [DOI] [PubMed] [Google Scholar]
  14. Gallo R. C., Salahuddin S. Z., Popovic M., Shearer G. M., Kaplan M., Haynes B. F., Palker T. J., Redfield R., Oleske J., Safai B. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science. 1984 May 4;224(4648):500–503. doi: 10.1126/science.6200936. [DOI] [PubMed] [Google Scholar]
  15. Gay D., Maddon P., Sekaly R., Talle M. A., Godfrey M., Long E., Goldstein G., Chess L., Axel R., Kappler J. Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature. 1987 Aug 13;328(6131):626–629. doi: 10.1038/328626a0. [DOI] [PubMed] [Google Scholar]
  16. Geppert T. D., Lipsky P. E. Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3. J Immunol. 1987 Mar 15;138(6):1660–1666. [PubMed] [Google Scholar]
  17. Giacca M., Gutierrez M. I., Menzo S., d'Adda di Fagagna F., Falaschi A. A human binding site for transcription factor USF/MLTF mimics the negative regulatory element of human immunodeficiency virus type 1. Virology. 1992 Jan;186(1):133–147. doi: 10.1016/0042-6822(92)90067-y. [DOI] [PubMed] [Google Scholar]
  18. Glaichenhaus N., Shastri N., Littman D. R., Turner J. M. Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells. Cell. 1991 Feb 8;64(3):511–520. doi: 10.1016/0092-8674(91)90235-q. [DOI] [PubMed] [Google Scholar]
  19. Goodenow M., Huet T., Saurin W., Kwok S., Sninsky J., Wain-Hobson S. HIV-1 isolates are rapidly evolving quasispecies: evidence for viral mixtures and preferred nucleotide substitutions. J Acquir Immune Defic Syndr. 1989;2(4):344–352. [PubMed] [Google Scholar]
  20. Haffar O. K., Moran P. A., Smithgall M. D., Diegel M. L., Sridhar P., Ledbetter J. A., Zarling J. M., Hu S. L. Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles. J Virol. 1992 Jul;66(7):4279–4287. doi: 10.1128/jvi.66.7.4279-4287.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hara J., Benedict S. H., Champagne E., Mak T. W., Minden M., Gelfand E. W. Comparison of T cell receptor alpha, beta, and gamma gene rearrangement and expression in T cell acute lymphoblastic leukemia. J Clin Invest. 1988 Apr;81(4):989–996. doi: 10.1172/JCI113453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harada S., Koyanagi Y., Yamamoto N. Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay. Science. 1985 Aug 9;229(4713):563–566. doi: 10.1126/science.2992081. [DOI] [PubMed] [Google Scholar]
  23. Harper M. E., Marselle L. M., Gallo R. C., Wong-Staal F. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci U S A. 1986 Feb;83(3):772–776. doi: 10.1073/pnas.83.3.772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Haughn L., Gratton S., Caron L., Sékaly R. P., Veillette A., Julius M. Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor. Nature. 1992 Jul 23;358(6384):328–331. doi: 10.1038/358328a0. [DOI] [PubMed] [Google Scholar]
  25. Hivroz C., Mazerolles F., Soula M., Fagard R., Graton S., Meloche S., Sekaly R. P., Fischer A. Human immunodeficiency virus gp120 and derived peptides activate protein tyrosine kinase p56lck in human CD4 T lymphocytes. Eur J Immunol. 1993 Mar;23(3):600–607. doi: 10.1002/eji.1830230303. [DOI] [PubMed] [Google Scholar]
  26. Ho D. D., Moudgil T., Alam M. Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N Engl J Med. 1989 Dec 14;321(24):1621–1625. doi: 10.1056/NEJM198912143212401. [DOI] [PubMed] [Google Scholar]
  27. Hoffman A. D., Banapour B., Levy J. A. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology. 1985 Dec;147(2):326–335. doi: 10.1016/0042-6822(85)90135-7. [DOI] [PubMed] [Google Scholar]
  28. Juszczak R. J., Turchin H., Truneh A., Culp J., Kassis S. Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes. J Biol Chem. 1991 Jun 15;266(17):11176–11183. [PubMed] [Google Scholar]
  29. Kato H., Horikoshi M., Roeder R. G. Repression of HIV-1 transcription by a cellular protein. Science. 1991 Mar 22;251(5000):1476–1479. doi: 10.1126/science.2006421. [DOI] [PubMed] [Google Scholar]
  30. Koretzky G. A., Picus J., Schultz T., Weiss A. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2037–2041. doi: 10.1073/pnas.88.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamarre D., Ashkenazi A., Fleury S., Smith D. H., Sekaly R. P., Capon D. J. The MHC-binding and gp120-binding functions of CD4 are separable. Science. 1989 Aug 18;245(4919):743–746. doi: 10.1126/science.2549633. [DOI] [PubMed] [Google Scholar]
  32. Landau N. R., Warton M., Littman D. R. The envelope glycoprotein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4. Nature. 1988 Jul 14;334(6178):159–162. doi: 10.1038/334159a0. [DOI] [PubMed] [Google Scholar]
  33. Lori F., di Marzo Veronese F., de Vico A. L., Lusso P., Reitz M. S., Jr, Gallo R. C. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. doi: 10.1128/jvi.66.8.5067-5074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Luo K. X., Sefton B. M. Cross-linking of T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site. Mol Cell Biol. 1990 Oct;10(10):5305–5313. doi: 10.1128/mcb.10.10.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maddon P. J., McDougal J. S., Clapham P. R., Dalgleish A. G., Jamal S., Weiss R. A., Axel R. HIV infection does not require endocytosis of its receptor, CD4. Cell. 1988 Sep 9;54(6):865–874. doi: 10.1016/s0092-8674(88)91241-x. [DOI] [PubMed] [Google Scholar]
  36. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marshall W. L., Diamond D. C., Kowalski M. M., Finberg R. W. High level of surface CD4 prevents stable human immunodeficiency virus infection of T-cell transfectants. J Virol. 1992 Sep;66(9):5492–5499. doi: 10.1128/jvi.66.9.5492-5499.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Minty A. J., Caravatti M., Robert B., Cohen A., Daubas P., Weydert A., Gros F., Buckingham M. E. Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. J Biol Chem. 1981 Jan 25;256(2):1008–1014. [PubMed] [Google Scholar]
  39. Mizukami T., Fuerst T. R., Berger E. A., Moss B. Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9273–9277. doi: 10.1073/pnas.85.23.9273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Molina T. J., Kishihara K., Siderovski D. P., van Ewijk W., Narendran A., Timms E., Wakeham A., Paige C. J., Hartmann K. U., Veillette A. Profound block in thymocyte development in mice lacking p56lck. Nature. 1992 May 14;357(6374):161–164. doi: 10.1038/357161a0. [DOI] [PubMed] [Google Scholar]
  41. Newell M. K., Haughn L. J., Maroun C. R., Julius M. H. Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature. 1990 Sep 20;347(6290):286–289. doi: 10.1038/347286a0. [DOI] [PubMed] [Google Scholar]
  42. Peterson A., Seed B. Genetic analysis of monoclonal antibody and HIV binding sites on the human lymphocyte antigen CD4. Cell. 1988 Jul 1;54(1):65–72. doi: 10.1016/0092-8674(88)90180-8. [DOI] [PubMed] [Google Scholar]
  43. Poulin L., Evans L. A., Tang S. B., Barboza A., Legg H., Littman D. R., Levy J. A. Several CD4 domains can play a role in human immunodeficiency virus infection in cells. J Virol. 1991 Sep;65(9):4893–4901. doi: 10.1128/jvi.65.9.4893-4901.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Psallidopoulos M. C., Schnittman S. M., Thompson L. M., 3rd, Baseler M., Fauci A. S., Lane H. C., Salzman N. P. Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals. J Virol. 1989 Nov;63(11):4626–4631. doi: 10.1128/jvi.63.11.4626-4631.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Raziuddin, Mikovits J. A., Calvert I., Ghosh S., Kung H. F., Ruscetti F. W. Negative regulation of human immunodeficiency virus type 1 expression in monocytes: role of the 65-kDa plus 50-kDa NF-kappa B dimer. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9426–9430. doi: 10.1073/pnas.88.21.9426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ryu S. E., Kwong P. D., Truneh A., Porter T. G., Arthos J., Rosenberg M., Dai X. P., Xuong N. H., Axel R., Sweet R. W. Crystal structure of an HIV-binding recombinant fragment of human CD4. Nature. 1990 Nov 29;348(6300):419–426. doi: 10.1038/348419a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sattentau Q. J., Weiss R. A. The CD4 antigen: physiological ligand and HIV receptor. Cell. 1988 Mar 11;52(5):631–633. doi: 10.1016/0092-8674(88)90397-2. [DOI] [PubMed] [Google Scholar]
  49. Schnittman S. M., Psallidopoulos M. C., Lane H. C., Thompson L., Baseler M., Massari F., Fox C. H., Salzman N. P., Fauci A. S. The reservoir for HIV-1 in human peripheral blood is a T cell that maintains expression of CD4. Science. 1989 Jul 21;245(4915):305–308. doi: 10.1126/science.2665081. [DOI] [PubMed] [Google Scholar]
  50. Shaw A. S., Chalupny J., Whitney J. A., Hammond C., Amrein K. E., Kavathas P., Sefton B. M., Rose J. K. Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol Cell Biol. 1990 May;10(5):1853–1862. doi: 10.1128/mcb.10.5.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shaw G. M., Hahn B. H., Arya S. K., Groopman J. E., Gallo R. C., Wong-Staal F. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science. 1984 Dec 7;226(4679):1165–1171. doi: 10.1126/science.6095449. [DOI] [PubMed] [Google Scholar]
  52. Sleckman B. P., Peterson A., Foran J. A., Gorga J. C., Kara C. J., Strominger J. L., Burakoff S. J., Greenstein J. L. Functional analysis of a cytoplasmic domain-deleted mutant of the CD4 molecule. J Immunol. 1988 Jul 1;141(1):49–54. [PubMed] [Google Scholar]
  53. Swain S. L. T cell subsets and the recognition of MHC class. Immunol Rev. 1983;74:129–142. doi: 10.1111/j.1600-065x.1983.tb01087.x. [DOI] [PubMed] [Google Scholar]
  54. Tjøtta E., Hungnes O., Grinde B. An assay for quantifying infectious HIV particles. J Virol Methods. 1990 Feb;27(2):169–174. doi: 10.1016/0166-0934(90)90133-z. [DOI] [PubMed] [Google Scholar]
  55. Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J Virol. 1992 Aug;66(8):4893–4900. doi: 10.1128/jvi.66.8.4893-4900.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Turner J. M., Brodsky M. H., Irving B. A., Levin S. D., Perlmutter R. M., Littman D. R. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 1990 Mar 9;60(5):755–765. doi: 10.1016/0092-8674(90)90090-2. [DOI] [PubMed] [Google Scholar]
  57. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  58. Veillette A., Bookman M. A., Horak E. M., Samelson L. E., Bolen J. B. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989 Mar 16;338(6212):257–259. doi: 10.1038/338257a0. [DOI] [PubMed] [Google Scholar]
  59. Wang J. H., Yan Y. W., Garrett T. P., Liu J. H., Rodgers D. W., Garlick R. L., Tarr G. E., Husain Y., Reinherz E. L., Harrison S. C. Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains. Nature. 1990 Nov 29;348(6300):411–418. doi: 10.1038/348411a0. [DOI] [PubMed] [Google Scholar]
  60. Wilde D. B., Marrack P., Kappler J., Dialynas D. P., Fitch F. W. Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GK1.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation, release of lymphokines, and binding by cloned murine helper T lymphocyte lines. J Immunol. 1983 Nov;131(5):2178–2183. [PubMed] [Google Scholar]
  61. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]

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