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. 1994 Oct;38(10):2430–2439. doi: 10.1128/aac.38.10.2430

Constitutive production of nonenveloped human immunodeficiency virus type 1 particles by a mammalian cell line and effects of a protease inhibitor on particle maturation.

L M Babé 1, C S Craik 1
PMCID: PMC284757  PMID: 7840583

Abstract

A stable cell line encoding the sequences of all the human immunodeficiency virus type 1 proteins, with the exception of the gp160 envelope glycoprotein, was derived from transfection of monkey COS-7 cells. This cell line, referred to as CH-1, produces active viral protease that correctly processes its natural substrates and yields capsid particles. These particles contain reverse transcriptase activity and packaged viral RNA but are noninfectious. The level of expression of viral proteins is not toxic to the cells, yet it is comparable to that observed for chronically infected lymphocytes. These constitutively synthesized viral proteins provide a consistent system for the analysis of potential inhibitors of late viral functions. The lack of gp160 increases the biosafety of this assay system, while it allows the measurement of the effects on the production and release of capsid particles. A human immunodeficiency virus type 1 protease inhibitor was used to confirm the viral polyprotein maturation pathway in this system. Particles from cells treated with this protease inhibitor contain unprocessed p55gag precursor and have the same density as the mature particles. These immature particles contain viral RNA, but reverse transcriptase activity is significantly reduced. This cell line may serve to identify compounds that are able to affect viral assembly and maturation as well as to identify the interactions between the viral and cellular proteins involved in these essential processes.

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Selected References

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

  1. Ashorn P., McQuade T. J., Thaisrivongs S., Tomasselli A. G., Tarpley W. G., Moss B. An inhibitor of the protease blocks maturation of human and simian immunodeficiency viruses and spread of infection. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7472–7476. doi: 10.1073/pnas.87.19.7472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gelderblom H. R. Assembly and morphology of HIV: potential effect of structure on viral function. AIDS. 1991 Jun;5(6):617–637. [PubMed] [Google Scholar]
  3. Gelderblom H. R., Ozel M., Pauli G. Morphogenesis and morphology of HIV. Structure-function relations. Arch Virol. 1989;106(1-2):1–13. doi: 10.1007/BF01311033. [DOI] [PubMed] [Google Scholar]
  4. Gheysen D., Jacobs E., de Foresta F., Thiriart C., Francotte M., Thines D., De Wilde M. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 1989 Oct 6;59(1):103–112. doi: 10.1016/0092-8674(89)90873-8. [DOI] [PubMed] [Google Scholar]
  5. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  6. Gluzman Y., Sambrook J. F., Frisque R. J. Expression of early genes of origin-defective mutants of simian virus 40. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3898–3902. doi: 10.1073/pnas.77.7.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  8. Haynes J. R., Cao S. X., Rovinski B., Sia C., James O., Dekaban G. A., Klein M. H. Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines. AIDS Res Hum Retroviruses. 1991 Jan;7(1):17–27. doi: 10.1089/aid.1991.7.17. [DOI] [PubMed] [Google Scholar]
  9. Höner B., Shoeman R. L., Traub P. Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture. J Cell Sci. 1991 Dec;100(Pt 4):799–807. doi: 10.1242/jcs.100.4.799. [DOI] [PubMed] [Google Scholar]
  10. Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  11. Japour A. J., Mayers D. L., Johnson V. A., Kuritzkes D. R., Beckett L. A., Arduino J. M., Lane J., Black R. J., Reichelderfer P. S., D'Aquila R. T. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group. Antimicrob Agents Chemother. 1993 May;37(5):1095–1101. doi: 10.1128/aac.37.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaplan A. H., Swanstrom R. Human immunodeficiency virus type 1 Gag proteins are processed in two cellular compartments. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4528–4532. doi: 10.1073/pnas.88.10.4528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karacostas V., Nagashima K., Gonda M. A., Moss B. Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8964–8967. doi: 10.1073/pnas.86.22.8964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kohl N. E., Diehl R. E., Rands E., Davis L. J., Hanobik M. G., Wolanski B., Dixon R. A. Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles. J Virol. 1991 Jun;65(6):3007–3014. doi: 10.1128/jvi.65.6.3007-3014.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kräusslich H. G., Ochsenbauer C., Traenckner A. M., Mergener K., Fäcke M., Gelderblom H. R., Bosch V. Analysis of protein expression and virus-like particle formation in mammalian cell lines stably expressing HIV-1 gag and env gene products with or without active HIV proteinase. Virology. 1993 Feb;192(2):605–617. doi: 10.1006/viro.1993.1077. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Laurent A. G., Krust B., Rey M. A., Montagnier L., Hovanessian A. G. Cell surface expression of several species of human immunodeficiency virus type 1 major core protein. J Virol. 1989 Sep;63(9):4074–4078. doi: 10.1128/jvi.63.9.4074-4078.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Luthman H., Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 1983 Mar 11;11(5):1295–1308. doi: 10.1093/nar/11.5.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McQuade T. J., Tomasselli A. G., Liu L., Karacostas V., Moss B., Sawyer T. K., Heinrikson R. L., Tarpley W. G. A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science. 1990 Jan 26;247(4941):454–456. doi: 10.1126/science.2405486. [DOI] [PubMed] [Google Scholar]
  20. Meek T. D., Lambert D. M., Dreyer G. B., Carr T. J., Tomaszek T. A., Jr, Moore M. L., Strickler J. E., Debouck C., Hyland L. J., Matthews T. J. Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. Nature. 1990 Jan 4;343(6253):90–92. doi: 10.1038/343090a0. [DOI] [PubMed] [Google Scholar]
  21. Mergener K., Fäcke M., Welker R., Brinkmann V., Gelderblom H. R., Kräusslich H. G. Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells. Virology. 1992 Jan;186(1):25–39. doi: 10.1016/0042-6822(92)90058-w. [DOI] [PubMed] [Google Scholar]
  22. Mervis R. J., Ahmad N., Lillehoj E. P., Raum M. G., Salazar F. H., Chan H. W., Venkatesan S. The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol. 1988 Nov;62(11):3993–4002. doi: 10.1128/jvi.62.11.3993-4002.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  24. Overton H. A., McMillan D. J., Gridley S. J., Brenner J., Redshaw S., Mills J. S. Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins. Virology. 1990 Nov;179(1):508–511. doi: 10.1016/0042-6822(90)90326-m. [DOI] [PubMed] [Google Scholar]
  25. Page K. A., Landau N. R., Littman D. R. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J Virol. 1990 Nov;64(11):5270–5276. doi: 10.1128/jvi.64.11.5270-5276.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pepin R. A., Lucas D. J., Lang R. B., Lee N., Liao M. J., Testa D. Detection of picogram amounts of nucleic acid by dot blot hybridization. Biotechniques. 1990 Jun;8(6):628–632. [PubMed] [Google Scholar]
  27. Popovic M., Sarngadharan M. G., Read E., Gallo R. C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984 May 4;224(4648):497–500. doi: 10.1126/science.6200935. [DOI] [PubMed] [Google Scholar]
  28. Roberts N. A., Martin J. A., Kinchington D., Broadhurst A. V., Craig J. C., Duncan I. B., Galpin S. A., Handa B. K., Kay J., Kröhn A. Rational design of peptide-based HIV proteinase inhibitors. Science. 1990 Apr 20;248(4953):358–361. doi: 10.1126/science.2183354. [DOI] [PubMed] [Google Scholar]
  29. Rosenberg Z. F., Fauci A. S. Immunopathogenic mechanisms of HIV infection: cytokine induction of HIV expression. Immunol Today. 1990 May;11(5):176–180. doi: 10.1016/0167-5699(90)90070-p. [DOI] [PubMed] [Google Scholar]
  30. Stephens E. B., Compans R. W. Assembly of animal viruses at cellular membranes. Annu Rev Microbiol. 1988;42:489–516. doi: 10.1146/annurev.mi.42.100188.002421. [DOI] [PubMed] [Google Scholar]
  31. Stewart L., Schatz G., Vogt V. M. Properties of avian retrovirus particles defective in viral protease. J Virol. 1990 Oct;64(10):5076–5092. doi: 10.1128/jvi.64.10.5076-5092.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tan C. K., Zhang J., Li Z. Y., Tarpley W. G., Downey K. M., So A. G. Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase. Biochemistry. 1991 Mar 12;30(10):2651–2655. doi: 10.1021/bi00224a013. [DOI] [PubMed] [Google Scholar]
  33. Wlodawer A., Erickson J. W. Structure-based inhibitors of HIV-1 protease. Annu Rev Biochem. 1993;62:543–585. doi: 10.1146/annurev.bi.62.070193.002551. [DOI] [PubMed] [Google Scholar]

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