Enhanced Culture Assay for Detection and Quantitation of Latently Infected, Resting CD4+ T-Cells Carrying Replication-Competent Virus in HIV-1-Infected Individuals

  • Janet D. Siliciano
  • Robert F. Siliciano
Part of the Methods in Molecular Biology™ book series (MIMB, volume 304)


Highly active antiretroviral therapy can decrease plasma HIV-1 levels to below the limit of detection. However, HIV-1 persists in latently infected resting-memory CD4+ T-cells carrying an integrated copy of the viral genome. The pool of latently infected cells is extremely stable and represents a major barrier to HIV-1 eradication. Identification and characterization of this reservoir required the development of methods for purifying resting CD4+ T-cells from HIV-1-infected individuals, activating the cells to induce virus production, and detecting and quantitating cells capable of releasing infectious virus. The development of an enhanced viral culture assay to quantitate the number of latently infected cells carrying replication competent virus is described here.

Key Words

HIV-1 latency resting CD4+ T-cells reservoir enhanced viral culture assay replication-competent virus 



This work was supported by grants from the NIH (AI43222), the Doris Duke Charitable Foundation, and the Howard Hughes Medical Institute.


  1. 1.
    Wei, X., Ghosh, S. K., Taylor, M. E., Johnson, V. A., Emini, E. A., Deutsch, P., et al. (1995) Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373, 117–122.PubMedCrossRefGoogle Scholar
  2. 2.
    Ho, D. D., Neumann, A. U., Perelson, A. S., Chen, W., Leonard, J. M., and Markowitz, M. (1995) Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123–126.PubMedCrossRefGoogle Scholar
  3. 3.
    Perelson, A. S., Essunger, P., Cao, Y., Vesanen, M., Hurley, A., Saksela, K., Markowitz, M., and Ho, D. D. (1997) Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 387, 188–191.PubMedCrossRefGoogle Scholar
  4. 4.
    Chun, T.-W., Finzi, D., Margolick, J., Chadwick, K., Schwartz, D., and Siliciano, R. F. (1995) Fate of HIV-1-infected T cells in vivo: Rates of transition to stable latency. Nat. Med. 1, 1284–1290.PubMedCrossRefGoogle Scholar
  5. 5.
    Chun, T.-W., Carruth, L., Finzi, D., Shen, X., Digiuseppe, J. A., Taylor, H., et al. (1997) Quantitation of latent tissue reservoirs and total body load in HIV-1 infection. Nature 387, 183–188.PubMedCrossRefGoogle Scholar
  6. 6.
    Chun, T. W., Stuyver, L., Mizell, S. B., Ehler, L. A., Mican, J. M., Baseler, M., et al. (1997) Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 94, 13,193–13,197.PubMedCrossRefGoogle Scholar
  7. 7.
    Wong, J. K., Gunthard, H. F., Havlir, D. V., Zhang, Z. Q., Haase, A. T., Ignacio, C. C., et al. (1997) Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. Proc. Natl. Acad. Sci. USA 94, 12,574–12,579.PubMedCrossRefGoogle Scholar
  8. 8.
    Finzi, D., Hermankova, M., Pierson, T., Carruth, L. M., Buck, C., Chaisson, R. E., et al. (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278, 1295–1300.PubMedCrossRefGoogle Scholar
  9. 9.
    Finzi, D., Blankson, J., Siliciano, J. D., Margolick, J. B., Chadwick, K., Pierson, T., et al. (1999) Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat. Med. 5, 512–517.PubMedCrossRefGoogle Scholar
  10. 10.
    Siliciano, J. D., Kajdas, J., Finzi, D., Quinn, T. C., Chadwick, K., Margolick, J. B., et al. (2003) Long term follow-up studies confirm the extraordinary stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 9, 727–728.PubMedCrossRefGoogle Scholar
  11. 11.
    Chun, T. W., Justement, J. S., Lempicki, R. A., Yang, J., Dennis, G., Jr., Hallahan, C. W., et al. (2003) Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc. Natl. Acad. Sci. USA 100, 1908–1913.PubMedCrossRefGoogle Scholar
  12. 12.
    Persaud, D., Pierson, T., Ruff, C., Finzi, D., Chadwick, K. R., Margolick, J. B., et al. (2000) A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children. J. Clin. Invest. 105, 995–1003.PubMedCrossRefGoogle Scholar
  13. 13.
    Ho, D. D., Moudgil, T., and Alam, M. (1989) Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N. Engl. J. Med. 321, 1621–1625.PubMedCrossRefGoogle Scholar
  14. 14.
    Connor, R. I., Mohri, H., Cao, Y., and Ho, D. D. (1993) Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals. J. Virol. 67, 1772–1777.PubMedGoogle Scholar
  15. 15.
    Spina, C. A., Prince, H. E., and Richman, D. D. (1997) Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro. J. Clin. Invest. 99, 1774–1785.PubMedCrossRefGoogle Scholar
  16. 16.
    Aids Clinical Trials Group (1997) Quantitative PBMC microculture assay. In Virology Manual, National Institute of Allergy and Infectious Disease, Bethesda, MD, pp. 61–66.Google Scholar
  17. 17.
    Hermankova, M., Siliciano, J. D., Zhou, Y., Monie, D., Chadwich, K., Margolick, J. B., et al. (2003) Analysis of HIV-1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J. Virol. 77, 7383–7392.PubMedCrossRefGoogle Scholar
  18. 18.
    Blankson, J. N., Finzi, D., Pierson, T. C., Sabundayo, B. P., Chadwick, K., Margolick, J. B., et al. (2000) Biphasic decay of latently infected CD4+ T cells in acute HIV-1 infection. J. Infect. Dis. 182, 1636–1642.PubMedCrossRefGoogle Scholar
  19. 19.
    Pierson, T. C., Zhou, Y., Kieffer, T., Ruff, C. T., Buck, C., and Siliciano, R. F. (2002) Molecular characterization of preintegration latency in HIV-1 infection. J. Virol. 76, 8518–8531.PubMedCrossRefGoogle Scholar
  20. 20.
    Bukrinsky, M. I., Sharova, N., Dempsey, M. P., Stanwick, T. L., Bukrinskaya, A. G., and Stevenson, M. (1992) Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc. Natl. Acad. Sci. USA 89, 6580–6584.PubMedCrossRefGoogle Scholar
  21. 21.
    Myers, L. A., McQuay, L. J., and Hollinger, F. B. (1994) Dilution assay statistics. J. Clin. Micro. 32, 732–739.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Janet D. Siliciano
    • 1
  • Robert F. Siliciano
    • 2
  1. 1.Department of MedicineThe Johns Hopkins School of MedicineBaltimore
  2. 2.Department of MedicineThe Johns Hopkins School of Medicine and Howard Hughes Medical InstituteBaltimore

Personalised recommendations