Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Jun;70(6):3870–3875. doi: 10.1128/jvi.70.6.3870-3875.1996

Expression of human immunodeficiency virus type 1 reverse transcriptase in trans during virion release and after infection.

M A Ansari-Lari 1, R A Gibbs 1
PMCID: PMC190264  PMID: 8648723

Abstract

The normal reverse transcription of retroviral RNA is a complex process which depends on the orchestration of several steps throughout the virus life cycle. During the assembly of retroviruses, reverse transcriptase (RT) is directed into the virion as a component of the Gag-Pol polyprotein. In the maturation of the Gag-Pol polyprotein of human immunodeficiency virus type 1 (HIV-1), cleavage by the viral protease occurs during viral budding. After infection, reverse transcription of viral RNA into double-stranded DNA is completed in the cytoplasm of the infected cell. In this study, the processing and reverse transcription of HIV-1 have been examined by separate expression of mature HIV-1 RT and proviral molecules bearing RT mutations. The effects of RT expression in trans during virion release and after viral entry were investigated. Constitutive expression of HIV-1 RT was established in CD4- and non-CD4-expressing cells via the coexpression of its individual subunits, and three HIV-1 RT mutant constructs were generated. The results indicate that a bona fide RT trans complementation does not occur during virion release or after infection. However, after infection of an RT-expressing cell with a high titer RT-defective virus, intracellular reverse transcription can be detected.

Full Text

The Full Text of this article is available as a PDF (324.0 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. Ahmad N., Maitra R. K., Venkatesan S. Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6111–6115. doi: 10.1073/pnas.86.16.6111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ansari-Lari M. A., Donehower L. A., Gibbs R. A. Analysis of human immunodeficiency virus type 1 integrase mutants. Virology. 1995 Aug 1;211(1):332–335. doi: 10.1006/viro.1995.1412. [DOI] [PubMed] [Google Scholar]
  4. Ansari-Lari M. A., Gibbs R. A. Analysis of HIV type 1 reverse transcriptase expression in a human cell line. AIDS Res Hum Retroviruses. 1994 Sep;10(9):1117–1124. doi: 10.1089/aid.1994.10.1117. [DOI] [PubMed] [Google Scholar]
  5. Chen H., Boyle T. J., Malim M. H., Cullen B. R., Lyerly H. K. Derivation of a biologically contained replication system for human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7678–7682. doi: 10.1073/pnas.89.16.7678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connor R. I., Chen B. K., Choe S., Landau N. R. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology. 1995 Feb 1;206(2):935–944. doi: 10.1006/viro.1995.1016. [DOI] [PubMed] [Google Scholar]
  7. Farmerie W. G., Loeb D. D., Casavant N. C., Hutchison C. A., 3rd, Edgell M. H., Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science. 1987 Apr 17;236(4799):305–308. doi: 10.1126/science.2436298. [DOI] [PubMed] [Google Scholar]
  8. Farnet C. M., Haseltine W. A. Circularization of human immunodeficiency virus type 1 DNA in vitro. J Virol. 1991 Dec;65(12):6942–6952. doi: 10.1128/jvi.65.12.6942-6952.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gibbs R. A., Nguyen P. N., Edwards A., Civitello A. B., Caskey C. T. Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families. Genomics. 1990 Jun;7(2):235–244. doi: 10.1016/0888-7543(90)90545-6. [DOI] [PubMed] [Google Scholar]
  10. Haseltine W. A. Molecular biology of the human immunodeficiency virus type 1. FASEB J. 1991 Jul;5(10):2349–2360. doi: 10.1096/fasebj.5.10.1829694. [DOI] [PubMed] [Google Scholar]
  11. Jiang M., Mak J., Huang Y., Kleiman L. Reverse transcriptase is an important factor for the primer tRNA selection in HIV-1. Leukemia. 1994 Apr;8 (Suppl 1):S149–S151. [PubMed] [Google Scholar]
  12. Jiang M., Mak J., Ladha A., Cohen E., Klein M., Rovinski B., Kleiman L. Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. J Virol. 1993 Jun;67(6):3246–3253. doi: 10.1128/jvi.67.6.3246-3253.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaplan A. H., Manchester M., Swanstrom R. The activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency. J Virol. 1994 Oct;68(10):6782–6786. doi: 10.1128/jvi.68.10.6782-6786.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kimpton J., Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992 Apr;66(4):2232–2239. doi: 10.1128/jvi.66.4.2232-2239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Larder B. A., Purifoy D. J., Powell K. L., Darby G. Site-specific mutagenesis of AIDS virus reverse transcriptase. 1987 Jun 25-Jul 1Nature. 327(6124):716–717. doi: 10.1038/327716a0. [DOI] [PubMed] [Google Scholar]
  16. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lori F., Hall L., Lusso P., Popovic M., Markham P., Franchini G., Reitz M. S., Jr Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence. J Virol. 1992 Sep;66(9):5553–5560. doi: 10.1128/jvi.66.9.5553-5560.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Lowe D. M., Parmar V., Kemp S. D., Larder B. A. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 1991 May 6;282(2):231–234. doi: 10.1016/0014-5793(91)80484-k. [DOI] [PubMed] [Google Scholar]
  20. MacGregor G. R., Caskey C. T. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. doi: 10.1093/nar/17.6.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mak J., Jiang M., Wainberg M. A., Hammarskjöld M. L., Rekosh D., Kleiman L. Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. J Virol. 1994 Apr;68(4):2065–2072. doi: 10.1128/jvi.68.4.2065-2072.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Page K. A., Stearns S. M., Littman D. R. Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity. J Virol. 1992 Jan;66(1):524–533. doi: 10.1128/jvi.66.1.524-533.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Park J., Morrow C. D. The nonmyristylated Pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with Pr55gag and is incorporated into viruslike particles. J Virol. 1992 Nov;66(11):6304–6313. doi: 10.1128/jvi.66.11.6304-6313.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paxton W., Connor R. I., Landau N. R. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993 Dec;67(12):7229–7237. doi: 10.1128/jvi.67.12.7229-7237.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rasheed S., Nelson-Rees W. A., Toth E. M., Arnstein P., Gardner M. B. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer. 1974 Apr;33(4):1027–1033. doi: 10.1002/1097-0142(197404)33:4<1027::aid-cncr2820330419>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  26. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  27. Sakai H., Kawamura M., Sakuragi J., Sakuragi S., Shibata R., Ishimoto A., Ono N., Ueda S., Adachi A. Integration is essential for efficient gene expression of human immunodeficiency virus type 1. J Virol. 1993 Mar;67(3):1169–1174. doi: 10.1128/jvi.67.3.1169-1174.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  29. Shank P. R., Hughes S. H., Kung H. J., Majors J. E., Quintrell N., Guntaka R. V., Bishop J. M., Varmus H. E. Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA. Cell. 1978 Dec;15(4):1383–1395. doi: 10.1016/0092-8674(78)90063-6. [DOI] [PubMed] [Google Scholar]
  30. Smith A. J., Srinivasakumar N., Hammarskjöld M. L., Rekosh D. Requirements for incorporation of Pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles. J Virol. 1993 Apr;67(4):2266–2275. doi: 10.1128/jvi.67.4.2266-2275.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stevenson M., Haggerty S., Lamonica C. A., Meier C. M., Welch S. K., Wasiak A. J. Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol. 1990 May;64(5):2421–2425. doi: 10.1128/jvi.64.5.2421-2425.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tchenio T., Heidmann T. High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay. J Virol. 1992 Mar;66(3):1571–1578. doi: 10.1128/jvi.66.3.1571-1578.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
  34. Wiskerchen M., Muesing M. A. Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J Virol. 1995 Jan;69(1):376–386. doi: 10.1128/jvi.69.1.376-386.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yunoki M., Maotani-Imai K., Kusuda H., Motoyama M., Miyake S., Imai H., Shin Y. S., Kato S., Sano K., Morita C. Production of infectious particles from defective human immunodeficiency virus type 1 (HIV-1)-producing cell clones by superinfection with infectious HIV-1. Arch Virol. 1991;116(1-4):143–158. doi: 10.1007/BF01319238. [DOI] [PubMed] [Google Scholar]
  36. Zhang H., Bagasra O., Niikura M., Poiesz B. J., Pomerantz R. J. Intravirion reverse transcripts in the peripheral blood plasma on human immunodeficiency virus type 1-infected individuals. J Virol. 1994 Nov;68(11):7591–7597. doi: 10.1128/jvi.68.11.7591-7597.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang H., Zhang Y., Spicer T. P., Abbott L. Z., Abbott M., Poiesz B. J. Reverse transcription takes place within extracellular HIV-1 virions: potential biological significance. AIDS Res Hum Retroviruses. 1993 Dec;9(12):1287–1296. doi: 10.1089/aid.1993.9.1287. [DOI] [PubMed] [Google Scholar]
  38. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]

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

RESOURCES