Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1997 May;41(5):1017–1023. doi: 10.1128/aac.41.5.1017

Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

R W Humphrey 1, A Ohagen 1, D A Davis 1, T Fukazawa 1, H Hayashi 1, S Höglund 1, H Mitsuya 1, R Yarchoan 1
PMCID: PMC163843  PMID: 9145862

Abstract

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

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. Bugelski P. J., Kirsh R., Hart T. K. HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages. J Leukoc Biol. 1994 Sep;56(3):374–380. doi: 10.1002/jlb.56.3.374. [DOI] [PubMed] [Google Scholar]
  3. Collier A. C., Coombs R. W., Schoenfeld D. A., Bassett R. L., Timpone J., Baruch A., Jones M., Facey K., Whitacre C., McAuliffe V. J. Treatment of human immunodeficiency virus infection with saquinavir, zidovudine, and zalcitabine. AIDS Clinical Trials Group. N Engl J Med. 1996 Apr 18;334(16):1011–1017. doi: 10.1056/NEJM199604183341602. [DOI] [PubMed] [Google Scholar]
  4. Danner S. A., Carr A., Leonard J. M., Lehman L. M., Gudiol F., Gonzales J., Raventos A., Rubio R., Bouza E., Pintado V. A short-term study of the safety, pharmacokinetics, and efficacy of ritonavir, an inhibitor of HIV-1 protease. European-Australian Collaborative Ritonavir Study Group. N Engl J Med. 1995 Dec 7;333(23):1528–1533. doi: 10.1056/NEJM199512073332303. [DOI] [PubMed] [Google Scholar]
  5. Davis D. A., Dorsey K., Wingfield P. T., Stahl S. J., Kaufman J., Fales H. M., Levine R. L. Regulation of HIV-1 protease activity through cysteine modification. Biochemistry. 1996 Feb 20;35(7):2482–2488. doi: 10.1021/bi951525k. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Göttlinger H. G., Sodroski J. G., Haseltine W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781–5785. doi: 10.1073/pnas.86.15.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kageyama S., Hoekzema D. T., Murakawa Y., Kojima E., Shirasaka T., Kempf D. J., Norbeck D. W., Erickson J., Mitsuya H. A C2 symmetry-based HIV protease inhibitor, A77003, irreversibly inhibits infectivity of HIV-1 in vitro. AIDS Res Hum Retroviruses. 1994 Jun;10(6):735–743. doi: 10.1089/aid.1994.10.735. [DOI] [PubMed] [Google Scholar]
  9. Kageyama S., Mimoto T., Murakawa Y., Nomizu M., Ford H., Jr, Shirasaka T., Gulnik S., Erickson J., Takada K., Hayashi H. In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine. Antimicrob Agents Chemother. 1993 Apr;37(4):810–817. doi: 10.1128/aac.37.4.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  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., 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]
  14. Lin H. J., Myers L. E., Yen-Lieberman B., Hollinger F. B., Henrard D., Hooper C. J., Kokka R., Kwok S., Rasheed S., Vahey M. Multicenter evaluation of quantification methods for plasma human immunodeficiency virus type 1 RNA. J Infect Dis. 1994 Sep;170(3):553–562. doi: 10.1093/infdis/170.3.553. [DOI] [PubMed] [Google Scholar]
  15. Looney D. J., Hayashi S., Nicklas M., Redfield R. R., Broder S., Wong-Staal F., Mitsuya H. Differences in the interaction of HIV-1 and HIV-2 with CD4. J Acquir Immune Defic Syndr. 1990;3(7):649–657. [PubMed] [Google Scholar]
  16. Markowitz M., Saag M., Powderly W. G., Hurley A. M., Hsu A., Valdes J. M., Henry D., Sattler F., La Marca A., Leonard J. M. A preliminary study of ritonavir, an inhibitor of HIV-1 protease, to treat HIV-1 infection. N Engl J Med. 1995 Dec 7;333(23):1534–1539. doi: 10.1056/NEJM199512073332204. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Mimoto T., Imai J., Kisanuki S., Enomoto H., Hattori N., Akaji K., Kiso Y. Kynostatin (KNI)-227 and -272, highly potent anti-HIV agents: conformationally constrained tripeptide inhibitors of HIV protease containing allophenylnorstatine. Chem Pharm Bull (Tokyo) 1992 Aug;40(8):2251–2253. doi: 10.1248/cpb.40.2251. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Rayner M. M., Cordova B. C., Meade R. P., Aldrich P. E., Jadhav P. K., Ru Y., Lam P. Y. DMP 323, a nonpeptide cyclic urea inhibitor of human immunodeficiency virus (HIV) protease, specifically and persistently blocks intracellular processing of HIV gag polyprotein. Antimicrob Agents Chemother. 1994 Jul;38(7):1635–1640. doi: 10.1128/aac.38.7.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schapiro J. M., Winters M. A., Stewart F., Efron B., Norris J., Kozal M. J., Merigan T. C. The effect of high-dose saquinavir on viral load and CD4+ T-cell counts in HIV-infected patients. Ann Intern Med. 1996 Jun 15;124(12):1039–1050. doi: 10.7326/0003-4819-124-12-199606150-00003. [DOI] [PubMed] [Google Scholar]
  22. Tritch R. J., Cheng Y. E., Yin F. H., Erickson-Viitanen S. Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. J Virol. 1991 Feb;65(2):922–930. doi: 10.1128/jvi.65.2.922-930.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vacca J. P., Dorsey B. D., Schleif W. A., Levin R. B., McDaniel S. L., Darke P. L., Zugay J., Quintero J. C., Blahy O. M., Roth E. L-735,524: an orally bioavailable human immunodeficiency virus type 1 protease inhibitor. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4096–4100. doi: 10.1073/pnas.91.9.4096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Witte O. N., Baltimore D. Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants. J Virol. 1978 Jun;26(3):750–761. doi: 10.1128/jvi.26.3.750-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES