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. 1995 Oct 24;92(22):10069–10073. doi: 10.1073/pnas.92.22.10069

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

L M Babé 1, J Rosé 1, C S Craik 1
PMCID: PMC40737  PMID: 7479728

Abstract

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

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Selected References

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

  1. Babé L. M., Craik C. S. Constitutive production of nonenveloped human immunodeficiency virus type 1 particles by a mammalian cell line and effects of a protease inhibitor on particle maturation. Antimicrob Agents Chemother. 1994 Oct;38(10):2430–2439. doi: 10.1128/aac.38.10.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babé L. M., Pichuantes S., Craik C. S. Inhibition of HIV protease activity by heterodimer formation. Biochemistry. 1991 Jan 8;30(1):106–111. doi: 10.1021/bi00215a016. [DOI] [PubMed] [Google Scholar]
  3. Bahner I., Zhou C., Yu X. J., Hao Q. L., Guatelli J. C., Kohn D. B. Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes. J Virol. 1993 Jun;67(6):3199–3207. doi: 10.1128/jvi.67.6.3199-3207.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng Y. S., Yin F. H., Foundling S., Blomstrom D., Kettner C. A. Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9660–9664. doi: 10.1073/pnas.87.24.9660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Debouck C., Gorniak J. G., Strickler J. E., Meek T. D., Metcalf B. W., Rosenberg M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8903–8906. doi: 10.1073/pnas.84.24.8903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg M. B., Trono D. Intracellular immunization: trans-dominant mutants of HIV gene products as tools for the study and interruption of viral replication. AIDS Res Hum Retroviruses. 1992 Jun;8(6):1013–1022. doi: 10.1089/aid.1992.8.1013. [DOI] [PubMed] [Google Scholar]
  7. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  8. Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995 Jan 12;373(6510):123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
  9. Ho D. D., Toyoshima T., Mo H., Kempf D. J., Norbeck D., Chen C. M., Wideburg N. E., Burt S. K., Erickson J. W., Singh M. K. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor. J Virol. 1994 Mar;68(3):2016–2020. doi: 10.1128/jvi.68.3.2016-2020.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hope T. J., Klein N. P., Elder M. E., Parslow T. G. trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes. J Virol. 1992 Apr;66(4):1849–1855. doi: 10.1128/jvi.66.4.1849-1855.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnston M. I., Hoth D. F. Present status and future prospects for HIV therapies. Science. 1993 May 28;260(5112):1286–1293. doi: 10.1126/science.7684163. [DOI] [PubMed] [Google Scholar]
  12. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A., Scolnick E. M., Sigal I. S. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. doi: 10.1073/pnas.85.13.4686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kräusslich H. G. Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3213–3217. doi: 10.1073/pnas.88.8.3213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Malim M. H., Böhnlein S., Hauber J., Cullen B. R. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell. 1989 Jul 14;58(1):205–214. doi: 10.1016/0092-8674(89)90416-9. [DOI] [PubMed] [Google Scholar]
  15. Navia M. A., Fitzgerald P. M., McKeever B. M., Leu C. T., Heimbach J. C., Herber W. K., Sigal I. S., Darke P. L., Springer J. P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989 Feb 16;337(6208):615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Pearl L. H., Taylor W. R. A structural model for the retroviral proteases. Nature. 1987 Sep 24;329(6137):351–354. doi: 10.1038/329351a0. [DOI] [PubMed] [Google Scholar]
  18. Rosé J. R., Babé L. M., Craik C. S. Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J Virol. 1995 May;69(5):2751–2758. doi: 10.1128/jvi.69.5.2751-2758.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smythe J. A., Sun D., Thomson M., Markham P. D., Reitz M. S., Jr, Gallo R. C., Lisziewicz J. A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3657–3661. doi: 10.1073/pnas.91.9.3657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Trono D., Feinberg M. B., Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. doi: 10.1016/0092-8674(89)90874-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]
  24. Zhang Z. Y., Poorman R. A., Maggiora L. L., Heinrikson R. L., Kézdy F. J. Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. J Biol Chem. 1991 Aug 25;266(24):15591–15594. [PubMed] [Google Scholar]
  25. el-Farrash M. A., Kuroda M. J., Kitazaki T., Masuda T., Kato K., Hatanaka M., Harada S. Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor. J Virol. 1994 Jan;68(1):233–239. doi: 10.1128/jvi.68.1.233-239.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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