Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Jul;69(7):4316–4322. doi: 10.1128/jvi.69.7.4316-4322.1995

Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in situ hybridization: evidence of epitope masking of the CD4 cell surface molecule in vivo.

B K Patterson 1, C Goolsby 1, V Hodara 1, K L Lohman 1, S M Wolinsky 1
PMCID: PMC189171  PMID: 7539507

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection of T cells and cells of the monocyte/macrophage lineage requires a specific interaction between the CD4 antigen expressed on the cell surface and the HIV-1 external envelope glycoprotein (gp120). To study the association between HIV-1 infection and modulation of cell surface expression of the CD4 molecule in vivo, we examined the CD4+ T cells harboring proviral DNA obtained from HIV-1-infected individuals who had received no antiretroviral therapy for at least 90 days. Simultaneous immunophenotyping of CD4 cell surface expression and PCR-driven in situ hybridization for HIV-1 DNA were used to resolve the CD4+ T cells into distinct populations predicted upon the presence or absence of proviral DNA. Among the HIV-1-infected study subjects, the percentage of CD4+ T cells harboring proviral DNA ranged from 17.3 to 55.5%, with a mean of 40.5%. Cell surface fluorescent staining with anti-CD4 antibody directed against a non-gp120 binding site-related epitope (L120) or a conformation-dependent epitope of the gp120 binding site (Leu 3A) demonstrated either an equivalent or a 1.5- to 3-fold-lower cell surface staining intensity for the HIV-1 DNA-positive subpopulation relative to the HIV-1 DNA-negative subpopulation, respectively. These data suggest that masking or alteration of specific epitopes on the CD4 molecule occurs after viral infection.

Full Text

The Full Text of this article is available as a PDF (240.4 KB).

Selected References

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

  1. Bagasra O., Seshamma T., Oakes J. W., Pomerantz R. J. High percentages of CD4-positive lymphocytes harbor the HIV-1 provirus in the blood of certain infected individuals. AIDS. 1993 Nov;7(11):1419–1425. doi: 10.1097/00002030-199311000-00003. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Butera S. T., Roberts B. D., Folks T. M. Regulation of HIV-1 expression by cytokine networks in a CD4+ model of chronic infection. J Immunol. 1993 Jan 15;150(2):625–634. [PubMed] [Google Scholar]
  5. Clerici M., Lucey D. R., Berzofsky J. A., Pinto L. A., Wynn T. A., Blatt S. P., Dolan M. J., Hendrix C. W., Wolf S. F., Shearer G. M. Restoration of HIV-specific cell-mediated immune responses by interleukin-12 in vitro. Science. 1993 Dec 10;262(5140):1721–1724. doi: 10.1126/science.7903123. [DOI] [PubMed] [Google Scholar]
  6. Crise B., Rose J. K. Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum. J Virol. 1992 Apr;66(4):2296–2301. doi: 10.1128/jvi.66.4.2296-2301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doyle C., Strominger J. L. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature. 1987 Nov 19;330(6145):256–259. doi: 10.1038/330256a0. [DOI] [PubMed] [Google Scholar]
  8. Embretson J., Zupancic M., Beneke J., Till M., Wolinsky S., Ribas J. L., Burke A., Haase A. T. Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):357–361. doi: 10.1073/pnas.90.1.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Embretson J., Zupancic M., Ribas J. L., Burke A., Racz P., Tenner-Racz K., Haase A. T. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature. 1993 Mar 25;362(6418):359–362. doi: 10.1038/362359a0. [DOI] [PubMed] [Google Scholar]
  10. Fauci A. S. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science. 1988 Feb 5;239(4840):617–622. doi: 10.1126/science.3277274. [DOI] [PubMed] [Google Scholar]
  11. Fox C. H., Tenner-Rácz K., Rácz P., Firpo A., Pizzo P. A., Fauci A. S. Lymphoid germinal centers are reservoirs of human immunodeficiency virus type 1 RNA. J Infect Dis. 1991 Dec;164(6):1051–1057. doi: 10.1093/infdis/164.6.1051. [DOI] [PubMed] [Google Scholar]
  12. Garcia J. V., Alfano J., Miller A. D. The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. J Virol. 1993 Mar;67(3):1511–1516. doi: 10.1128/jvi.67.3.1511-1516.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Groux H., Torpier G., Monté D., Mouton Y., Capron A., Ameisen J. C. Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med. 1992 Feb 1;175(2):331–340. doi: 10.1084/jem.175.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gruters R. A., Terpstra F. G., De Jong R., Van Noesel C. J., Van Lier R. A., Miedema F. Selective loss of T cell functions in different stages of HIV infection. Early loss of anti-CD3-induced T cell proliferation followed by decreased anti-CD3-induced cytotoxic T lymphocyte generation in AIDS-related complex and AIDS. Eur J Immunol. 1990 May;20(5):1039–1044. doi: 10.1002/eji.1830200514. [DOI] [PubMed] [Google Scholar]
  17. Healey D., Dianda L., Moore J. P., McDougal J. S., Moore M. J., Estess P., Buck D., Kwong P. D., Beverley P. C., Sattentau Q. J. Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding. J Exp Med. 1990 Oct 1;172(4):1233–1242. doi: 10.1084/jem.172.4.1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hofmann B., Jakobsen K. D., Odum N., Dickmeiss E., Platz P., Ryder L. P., Pedersen C., Mathiesen L., Bygbjerg I. B., Faber V. HIV-induced immunodeficiency. Relatively preserved phytohemagglutinin as opposed to decreased pokeweed mitogen responses may be due to possibly preserved responses via CD2/phytohemagglutinin pathway. J Immunol. 1989 Mar 15;142(6):1874–1880. [PubMed] [Google Scholar]
  19. Hoxie J. A., Alpers J. D., Rackowski J. L., Huebner K., Haggarty B. S., Cedarbaum A. J., Reed J. C. Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV. Science. 1986 Nov 28;234(4780):1123–1127. doi: 10.1126/science.3095925. [DOI] [PubMed] [Google Scholar]
  20. Hsia K., Spector S. A. Human immunodeficiency virus DNA is present in a high percentage of CD4+ lymphocytes of seropositive individuals. J Infect Dis. 1991 Sep;164(3):470–475. doi: 10.1093/infdis/164.3.470. [DOI] [PubMed] [Google Scholar]
  21. Jabbar M. A., Nayak D. P. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane. J Virol. 1990 Dec;64(12):6297–6304. doi: 10.1128/jvi.64.12.6297-6304.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koga Y., Sasaki M., Yoshida H., Wigzell H., Kimura G., Nomoto K. Cytopathic effect determined by the amount of CD4 molecules in human cell lines expressing envelope glycoprotein of HIV. J Immunol. 1990 Jan 1;144(1):94–102. [PubMed] [Google Scholar]
  23. Lifson J. D., Reyes G. R., McGrath M. S., Stein B. S., Engleman E. G. AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen. Science. 1986 May 30;232(4754):1123–1127. doi: 10.1126/science.3010463. [DOI] [PubMed] [Google Scholar]
  24. Linette G. P., Hartzman R. J., Ledbetter J. A., June C. H. HIV-1-infected T cells show a selective signaling defect after perturbation of CD3/antigen receptor. Science. 1988 Jul 29;241(4865):573–576. doi: 10.1126/science.2899908. [DOI] [PubMed] [Google Scholar]
  25. Maddon P. J., Littman D. R., Godfrey M., Maddon D. E., Chess L., Axel R. The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell. 1985 Aug;42(1):93–104. doi: 10.1016/s0092-8674(85)80105-7. [DOI] [PubMed] [Google Scholar]
  26. Mariani R., Skowronski J. CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5549–5553. doi: 10.1073/pnas.90.12.5549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McDougal J. S., Kennedy M. S., Sligh J. M., Cort S. P., Mawle A., Nicholson J. K. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. Science. 1986 Jan 24;231(4736):382–385. doi: 10.1126/science.3001934. [DOI] [PubMed] [Google Scholar]
  28. McKnight A., Clapham P. R., Goudsmit J., Cheingsong-Popov R., Weber J. N., Weiss R. A. Development of HIV-1 group-specific neutralizing antibodies after seroconversion. AIDS. 1992 Aug;6(8):799–802. doi: 10.1097/00002030-199208000-00005. [DOI] [PubMed] [Google Scholar]
  29. Miedema F., Petit A. J., Terpstra F. G., Schattenkerk J. K., de Wolf F., Al B. J., Roos M., Lange J. M., Danner S. A., Goudsmit J. Immunological abnormalities in human immunodeficiency virus (HIV)-infected asymptomatic homosexual men. HIV affects the immune system before CD4+ T helper cell depletion occurs. J Clin Invest. 1988 Dec;82(6):1908–1914. doi: 10.1172/JCI113809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oyaizu N., McCloskey T. W., Coronesi M., Chirmule N., Kalyanaraman V. S., Pahwa S. Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals. Blood. 1993 Dec 1;82(11):3392–3400. [PubMed] [Google Scholar]
  31. Pantaleo G., Graziosi C., Demarest J. F., Butini L., Montroni M., Fox C. H., Orenstein J. M., Kotler D. P., Fauci A. S. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature. 1993 Mar 25;362(6418):355–358. doi: 10.1038/362355a0. [DOI] [PubMed] [Google Scholar]
  32. Pantaleo G., Graziosi C., Fauci A. S. New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med. 1993 Feb 4;328(5):327–335. doi: 10.1056/NEJM199302043280508. [DOI] [PubMed] [Google Scholar]
  33. Patterson B. K., Till M., Otto P., Goolsby C., Furtado M. R., McBride L. J., Wolinsky S. M. Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science. 1993 May 14;260(5110):976–979. doi: 10.1126/science.8493534. [DOI] [PubMed] [Google Scholar]
  34. Piatak M., Jr, Saag M. S., Yang L. C., Clark S. J., Kappes J. C., Luk K. C., Hahn B. H., Shaw G. M., Lifson J. D. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science. 1993 Mar 19;259(5102):1749–1754. doi: 10.1126/science.8096089. [DOI] [PubMed] [Google Scholar]
  35. Poli G., Bressler P., Kinter A., Duh E., Timmer W. C., Rabson A., Justement J. S., Stanley S., Fauci A. S. Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor alpha by transcriptional and post-transcriptional mechanisms. J Exp Med. 1990 Jul 1;172(1):151–158. doi: 10.1084/jem.172.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rosenberg Z. F., Fauci A. S. The immunopathogenesis of HIV infection. Adv Immunol. 1989;47:377–431. doi: 10.1016/s0065-2776(08)60665-3. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Salmon P., Olivier R., Riviere Y., Brisson E., Gluckman J. C., Kieny M. P., Montagnier L., Klatzmann D. Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication. J Exp Med. 1988 Dec 1;168(6):1953–1969. doi: 10.1084/jem.168.6.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sattentau Q. J., Dalgleish A. G., Weiss R. A., Beverley P. C. Epitopes of the CD4 antigen and HIV infection. Science. 1986 Nov 28;234(4780):1120–1123. doi: 10.1126/science.2430333. [DOI] [PubMed] [Google Scholar]
  40. Schnittman S. M., Greenhouse J. J., Psallidopoulos M. C., Baseler M., Salzman N. P., Fauci A. S., Lane H. C. Increasing viral burden in CD4+ T cells from patients with human immunodeficiency virus (HIV) infection reflects rapidly progressive immunosuppression and clinical disease. Ann Intern Med. 1990 Sep 15;113(6):438–443. doi: 10.7326/0003-4819-113-6-438. [DOI] [PubMed] [Google Scholar]
  41. Seshamma T., Bagasra O., Trono D., Baltimore D., Pomerantz R. J. Blocked early-stage latency in the peripheral blood cells of certain individuals infected with human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10663–10667. doi: 10.1073/pnas.89.22.10663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shaw A. S., Amrein K. E., Hammond C., Stern D. F., Sefton B. M., Rose J. K. The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain. Cell. 1989 Nov 17;59(4):627–636. doi: 10.1016/0092-8674(89)90008-1. [DOI] [PubMed] [Google Scholar]
  43. Siliciano R. F., Lawton T., Knall C., Karr R. W., Berman P., Gregory T., Reinherz E. L. Analysis of host-virus interactions in AIDS with anti-gp120 T cell clones: effect of HIV sequence variation and a mechanism for CD4+ cell depletion. Cell. 1988 Aug 12;54(4):561–575. doi: 10.1016/0092-8674(88)90078-5. [DOI] [PubMed] [Google Scholar]
  44. Sodroski J., Goh W. C., Rosen C., Dayton A., Terwilliger E., Haseltine W. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986 May 22;321(6068):412–417. doi: 10.1038/321412a0. [DOI] [PubMed] [Google Scholar]
  45. Stevenson M., Haggerty S., Lamonica C., Mann A. M., Meier C., Wasiak A. Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis. J Virol. 1990 Aug;64(8):3792–3803. doi: 10.1128/jvi.64.8.3792-3803.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stevenson M., Zhang X. H., Volsky D. J. Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus. J Virol. 1987 Dec;61(12):3741–3748. doi: 10.1128/jvi.61.12.3741-3748.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Terai C., Kornbluth R. S., Pauza C. D., Richman D. D., Carson D. A. Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1. J Clin Invest. 1991 May;87(5):1710–1715. doi: 10.1172/JCI115188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tyler D. S., Stanley S. D., Zolla-Pazner S., Gorny M. K., Shadduck P. P., Langlois A. J., Matthews T. J., Bolognesi D. P., Palker T. J., Weinhold K. J. Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies. J Immunol. 1990 Nov 15;145(10):3276–3282. [PubMed] [Google Scholar]
  49. Tyler D. S., Stanley S. D., Zolla-Pazner S., Gorny M. K., Shadduck P. P., Langlois A. J., Matthews T. J., Bolognesi D. P., Palker T. J., Weinhold K. J. Identification of sites within gp41 that serve as targets for antibody-dependent cellular cytotoxicity by using human monoclonal antibodies. J Immunol. 1990 Nov 15;145(10):3276–3282. [PubMed] [Google Scholar]
  50. Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995 Jan 12;373(6510):117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]
  51. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  52. Willard-Gallo K. E., Van de Keere F., Kettmann R. A specific defect in CD3 gamma-chain gene transcription results in loss of T-cell receptor/CD3 expression late after human immunodeficiency virus infection of a CD4+ T-cell line. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6713–6717. doi: 10.1073/pnas.87.17.6713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yssel H., de Waal Malefyt R., Duc Dodon M. D., Blanchard D., Gazzolo L., de Vries J. E., Spits H. Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells. J Immunol. 1989 Apr 1;142(7):2279–2289. [PubMed] [Google Scholar]
  54. van Noesel C. J., Gruters R. A., Terpstra F. G., Schellekens P. T., van Lier R. A., Miedema F. Functional and phenotypic evidence for a selective loss of memory T cells in asymptomatic human immunodeficiency virus-infected men. J Clin Invest. 1990 Jul;86(1):293–299. doi: 10.1172/JCI114698. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES