Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Jul;69(7):4053–4059. doi: 10.1128/jvi.69.7.4053-4059.1995

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

O Schwartz 1, V Maréchal 1, O Danos 1, J M Heard 1
PMCID: PMC189139  PMID: 7539505

Abstract

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Full Text

The Full Text of this article is available as a PDF (310.9 KB).

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994 Mar 11;76(5):853–864. doi: 10.1016/0092-8674(94)90360-3. [DOI] [PubMed] [Google Scholar]
  3. Anderson S. J., Lenburg M., Landau N. R., Garcia J. V. The cytoplasmic domain of CD4 is sufficient for its down-regulation from the cell surface by human immunodeficiency virus type 1 Nef. J Virol. 1994 May;68(5):3092–3101. doi: 10.1128/jvi.68.5.3092-3101.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arthur L. O., Bess J. W., Jr, Sowder R. C., 2nd, Benveniste R. E., Mann D. L., Chermann J. C., Henderson L. E. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science. 1992 Dec 18;258(5090):1935–1938. doi: 10.1126/science.1470916. [DOI] [PubMed] [Google Scholar]
  5. Borroto-Esoda K., Boone L. R. Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction. J Virol. 1991 Apr;65(4):1952–1959. doi: 10.1128/jvi.65.4.1952-1959.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  7. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J., Richman D. D., Guatelli J. C. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol. 1994 May;68(5):2906–2914. doi: 10.1128/jvi.68.5.2906-2914.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clavel F., Charneau P. Fusion from without directed by human immunodeficiency virus particles. J Virol. 1994 Feb;68(2):1179–1185. doi: 10.1128/jvi.68.2.1179-1185.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clavel F., Orenstein J. M. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. doi: 10.1128/jvi.64.10.5230-5234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullen B. R. The role of Nef in the replication cycle of the human and simian immunodeficiency viruses. Virology. 1994 Nov 15;205(1):1–6. doi: 10.1006/viro.1994.1613. [DOI] [PubMed] [Google Scholar]
  11. Franchini G., Robert-Guroff M., Ghrayeb J., Chang N. T., Wong-Staal F. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells. Virology. 1986 Dec;155(2):593–599. doi: 10.1016/0042-6822(86)90219-9. [DOI] [PubMed] [Google Scholar]
  12. Franke E. K., Yuan H. E., Luban J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature. 1994 Nov 24;372(6504):359–362. doi: 10.1038/372359a0. [DOI] [PubMed] [Google Scholar]
  13. Garcia J. V., Miller A. D. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991 Apr 11;350(6318):508–511. doi: 10.1038/350508a0. [DOI] [PubMed] [Google Scholar]
  14. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  15. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature. 1987 Nov 19;330(6145):266–269. doi: 10.1038/330266a0. [DOI] [PubMed] [Google Scholar]
  16. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  17. Kaminchik J., Bashan N., Itach A., Sarver N., Gorecki M., Panet A. Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells. J Virol. 1991 Feb;65(2):583–588. doi: 10.1128/jvi.65.2.583-588.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kestler H. W., 3rd, Ringler D. J., Mori K., Panicali D. L., Sehgal P. K., Daniel M. D., Desrosiers R. C. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell. 1991 May 17;65(4):651–662. doi: 10.1016/0092-8674(91)90097-i. [DOI] [PubMed] [Google Scholar]
  19. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9544–9548. doi: 10.1073/pnas.86.23.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marshall W. L., Mittler E. S., Avery P., Lawrence J. P., Finberg R. W. Glycosylphosphatidylinositol-anchored CD4 supports human immunodeficiency virus type 1 replication, but not cytopathic effect, in T-cell transfectants. J Virol. 1994 Jun;68(6):4039–4042. doi: 10.1128/jvi.68.6.4039-4042.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Miller M. D., Feinberg M. B., Greene W. C. The HIV-1 nef gene acts as a positive viral infectivity factor. Trends Microbiol. 1994 Aug;2(8):294–298. doi: 10.1016/0966-842x(94)90007-8. [DOI] [PubMed] [Google Scholar]
  22. Miller M. D., Warmerdam M. T., Gaston I., Greene W. C., Feinberg M. B. The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J Exp Med. 1994 Jan 1;179(1):101–113. doi: 10.1084/jem.179.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller M. D., Warmerdam M. T., Page K. A., Feinberg M. B., Greene W. C. Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry. J Virol. 1995 Jan;69(1):579–584. doi: 10.1128/jvi.69.1.579-584.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schwartz O., Alizon M., Heard J. M., Danos O. Impairment of T cell receptor-dependent stimulation in CD4+ lymphocytes after contact with membrane-bound HIV-1 envelope glycoprotein. Virology. 1994 Jan;198(1):360–365. doi: 10.1006/viro.1994.1042. [DOI] [PubMed] [Google Scholar]
  25. Schwartz O., Dautry-Varsat A., Goud B., Maréchal V., Subtil A., Heard J. M., Danos O. Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes. J Virol. 1995 Jan;69(1):528–533. doi: 10.1128/jvi.69.1.528-533.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schwartz O., Rivière Y., Heard J. M., Danos O. Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef. J Virol. 1993 Jun;67(6):3274–3280. doi: 10.1128/jvi.67.6.3274-3280.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spina C. A., Kwoh T. J., Chowers M. Y., Guatelli J. C., Richman D. D. The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. J Exp Med. 1994 Jan 1;179(1):115–123. doi: 10.1084/jem.179.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Terwilliger E., Sodroski J. G., Rosen C. A., Haseltine W. A. Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. J Virol. 1986 Nov;60(2):754–760. doi: 10.1128/jvi.60.2.754-760.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thali M., Bukovsky A., Kondo E., Rosenwirth B., Walsh C. T., Sodroski J., Göttlinger H. G. Functional association of cyclophilin A with HIV-1 virions. Nature. 1994 Nov 24;372(6504):363–365. doi: 10.1038/372363a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Zazopoulos E., Haseltine W. A. Mutational analysis of the human immunodeficiency virus type 1 Eli Nef function. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6634–6638. doi: 10.1073/pnas.89.14.6634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. de Ronde A., Klaver B., Keulen W., Smit L., Goudsmit J. Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology. 1992 May;188(1):391–395. doi: 10.1016/0042-6822(92)90772-h. [DOI] [PubMed] [Google Scholar]

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

RESOURCES