Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Virology logoLink to Journal of Virology
. 1995 Oct;69(10):5988–5994. doi: 10.1128/jvi.69.10.5988-5994.1995

Potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication by inducible expression of HIV-1 PR multimers.

S J Arrigo 1, K Huffman 1
PMCID: PMC189494  PMID: 7666503

Abstract

Constructs were generated in which expression of human immunodeficiency virus type 1 (HIV-1) protease (PR) was placed under control of the HIV-1 long terminal repeat, thus requiring the HIV-1 Tat protein for expression of PR. The activity of PR was assessed by cotransfection with a construct producing a Gag substrate. Expression of PR as an intramolecular multimer resulted in a large increase in PR activity in comparison with the level obtained with the expression of PR as a monomer. A cytotoxic effect of PR expression was also exhibited by the constructs expressing PR multimers. CD4+ T-cell lines were generated with a construct producing PR as a linked tetramer and screened for PR activity and inducibility. The replication of HIV-1 in these cell lines was several orders of magnitude reduced in comparison with that in cell lines not expressing PR. Infection in these cell lines could be detected early after infection but disappeared over time. Infection of the PR-expressing cell lines could be increased several orders of magnitude by the addition of a specific inhibitor of PR, U75875 (Upjohn), after infection of the cells, demonstrating that the potent inhibition of HIV-1 replication in these cells was directly due to the expression of PR.

Full Text

The Full Text of this article is available as a PDF (306.6 KB).

Selected References

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

  1. Adams L. D., Tomasselli A. G., Robbins P., Moss B., Heinrikson R. L. HIV-1 protease cleaves actin during acute infection of human T-lymphocytes. AIDS Res Hum Retroviruses. 1992 Feb;8(2):291–295. doi: 10.1089/aid.1992.8.291. [DOI] [PubMed] [Google Scholar]
  2. Arrigo S. J., Chen I. S. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991 May;5(5):808–819. doi: 10.1101/gad.5.5.808. [DOI] [PubMed] [Google Scholar]
  3. Arrigo S. J., Haines J. K., Huffman K. M. Intrinsic activity of human immunodeficiency virus type 1 protease heterologous fusion proteins in mammalian cells. DNA Cell Biol. 1995 Jan;14(1):15–23. doi: 10.1089/dna.1995.14.15. [DOI] [PubMed] [Google Scholar]
  4. Arrigo S. J., Heaphy S., Haines J. K. In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function. J Virol. 1992 Sep;66(9):5569–5575. doi: 10.1128/jvi.66.9.5569-5575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Darke P. L., Nutt R. F., Brady S. F., Garsky V. M., Ciccarone T. M., Leu C. T., Lumma P. K., Freidinger R. M., Veber D. F., Sigal I. S. HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochem Biophys Res Commun. 1988 Oct 14;156(1):297–303. doi: 10.1016/s0006-291x(88)80839-8. [DOI] [PubMed] [Google Scholar]
  7. Erickson-Viitanen S., Manfredi J., Viitanen P., Tribe D. E., Tritch R., Hutchison C. A., 3rd, Loeb D. D., Swanstrom R. Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res Hum Retroviruses. 1989 Dec;5(6):577–591. doi: 10.1089/aid.1989.5.577. [DOI] [PubMed] [Google Scholar]
  8. Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  9. Hoshikawa N., Kojima A., Yasuda A., Takayashiki E., Masuko S., Chiba J., Sata T., Kurata T. Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study. J Gen Virol. 1991 Oct;72(Pt 10):2509–2517. doi: 10.1099/0022-1317-72-10-2509. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kaplan A. H., Swanstrom R. The HIV-1 gag precursor is processed via two pathways: implications for cytotoxicity. Biomed Biochim Acta. 1991;50(4-6):647–653. [PubMed] [Google Scholar]
  12. Karacostas V., Wolffe E. J., Nagashima K., Gonda M. A., Moss B. Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. Virology. 1993 Apr;193(2):661–671. doi: 10.1006/viro.1993.1174. [DOI] [PubMed] [Google Scholar]
  13. Katoh I., Ikawa Y., Yoshinaka Y. Retrovirus protease characterized as a dimeric aspartic proteinase. J Virol. 1989 May;63(5):2226–2232. doi: 10.1128/jvi.63.5.2226-2232.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Kramer R. A., Schaber M. D., Skalka A. M., Ganguly K., Wong-Staal F., Reddy E. P. HTLV-III gag protein is processed in yeast cells by the virus pol-protease. Science. 1986 Mar 28;231(4745):1580–1584. doi: 10.1126/science.2420008. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kräusslich H. G., Ingraham R. H., Skoog M. T., Wimmer E., Pallai P. V., Carter C. A. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. Proc Natl Acad Sci U S A. 1989 Feb;86(3):807–811. doi: 10.1073/pnas.86.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kräusslich H. G. Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation. J Virol. 1992 Jan;66(1):567–572. doi: 10.1128/jvi.66.1.567-572.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  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. Miller M., Jaskólski M., Rao J. K., Leis J., Wlodawer A. Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature. 1989 Feb 9;337(6207):576–579. doi: 10.1038/337576a0. [DOI] [PubMed] [Google Scholar]
  23. Park J., Morrow C. D. Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. J Virol. 1991 Sep;65(9):5111–5117. doi: 10.1128/jvi.65.9.5111-5117.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rivière Y., Blank V., Kourilsky P., Israël A. Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection. Nature. 1991 Apr 18;350(6319):625–626. doi: 10.1038/350625a0. [DOI] [PubMed] [Google Scholar]
  25. Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
  26. Sarver N., Rossi J. Gene therapy: a bold direction for HIV-1 treatment. AIDS Res Hum Retroviruses. 1993 May;9(5):483–487. doi: 10.1089/aid.1993.9.483. [DOI] [PubMed] [Google Scholar]
  27. Shoeman R. L., Sachse C., Höner B., Mothes E., Kaufmann M., Traub P. Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. Actin, desmin, myosin, and tropomyosin. Am J Pathol. 1993 Jan;142(1):221–230. [PMC free article] [PubMed] [Google Scholar]
  28. Sodroski J., Rosen C., Wong-Staal F., Salahuddin S. Z., Popovic M., Arya S., Gallo R. C., Haseltine W. A. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science. 1985 Jan 11;227(4683):171–173. doi: 10.1126/science.2981427. [DOI] [PubMed] [Google Scholar]
  29. Tomasselli A. G., Howe W. J., Hui J. O., Sawyer T. K., Reardon I. M., DeCamp D. L., Craik C. S., Heinrikson R. L. Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2. Proteins. 1991;10(1):1–9. doi: 10.1002/prot.340100102. [DOI] [PubMed] [Google Scholar]
  30. Tomasselli A. G., Hui J. O., Adams L., Chosay J., Lowery D., Greenberg B., Yem A., Deibel M. R., Zürcher-Neely H., Heinrikson R. L. Actin, troponin C, Alzheimer amyloid precursor protein and pro-interleukin 1 beta as substrates of the protease from human immunodeficiency virus. J Biol Chem. 1991 Aug 5;266(22):14548–14553. [PubMed] [Google Scholar]
  31. Wills J. W., Craven R. C. Form, function, and use of retroviral gag proteins. AIDS. 1991 Jun;5(6):639–654. doi: 10.1097/00002030-199106000-00002. [DOI] [PubMed] [Google Scholar]
  32. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]

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

RESOURCES