Skip to main content

Advertisement

SpringerLink
Log in

Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNALys3 but promotes intracellular budding of virus-like particles

  • Original Paper
  • Published:
Journal of Biomedical Science

Abstract

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/Gag-Pol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNALys3. In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNALys3. Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(-)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(−) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(−) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(−) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Berkowitz RD, Luban J, Goff SP. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays J Virol 67:7190–7200;1993.

    PubMed  Google Scholar 

  2. Canaani E, Helm KVD, Duesberg P. Evidence for the 30–40S RNA as precursor of the 60–70S RNA of Rous sarcoma virus. Proc Natl Acad Sci USA 70:401–405;1973.

    Google Scholar 

  3. Darlix JL, Lapadat-Tapolsky M, de Rocquigny H, Roques BP. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J Mol Biol 254:523–537;1995.

    Article  PubMed  Google Scholar 

  4. Feng YX, Campbell S, Harvin D, Ehresmann B, Ehresmann C, Rein A. The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: Possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site. J Virol 73:4251–4256;1999.

    PubMed  Google Scholar 

  5. Franke EK, Yuan HEH, Luban J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature 372:359–362;1994.

    PubMed  Google Scholar 

  6. Freed EO. HIV-1 gag proteins: Diverse functions in the virus life cycle. Virology 251:1–15;1998.

    Article  PubMed  Google Scholar 

  7. Fu W, Gorelick RJ, Rein A. Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions. J Virol 68:5013–5018;1994.

    PubMed  Google Scholar 

  8. Fu W, Rein A. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J Virol 67:5443–5449;1993.

    PubMed  Google Scholar 

  9. Gorelick RJ, Chabot DJ, Rein A, Henderson LE, Arthur LO. The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent. J Virol 67:4027–4036;1993.

    PubMed  Google Scholar 

  10. Gorelick RJ, Nigida JSM, Bess JJW, Arthur LO, Henderson LE, Rein A. Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol 64:3207–3211;1990.

    PubMed  Google Scholar 

  11. Huang M, Martin MA. Incorporation of Pr160(gag-pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein. J Virol 71:4472–4478;1997.

    PubMed  Google Scholar 

  12. Huang Y, Mak J, Cao Q, Li Z, Wainberg MA, Kleiman L. Incorporation of excess wild type and mutant tRNALys3 into HIV-1. J Virol 68:7676–7683;1994.

    PubMed  Google Scholar 

  13. 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 67:3246–3253;1993.

    PubMed  Google Scholar 

  14. Jiang M, Mak J, Wainberg MA, Parniak MA, Cohen E, Kleiman L. A variable tRNA content in HIV-1 3B. Biochem Biophys Res Commun 185:1005–1015;1992.

    PubMed  Google Scholar 

  15. Karacostas V, Wolffe EJ, Nagashima K, Gonda MA, 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 193:661–671;1993.

    Article  PubMed  Google Scholar 

  16. Kung HJ, Hu S, Bender W, Bailey JM, Davidson N, Nicolson MO, McAllister RM. RD-114, baboon, and woolly monkey viral RNA's compared in size and structure. Cell 7:609–620;1976.

    Article  PubMed  Google Scholar 

  17. Li X, Mak J, Arts EJ, Kleiman L, Wainberg MA, Parniak MA. Effects of alterations of primer binding site sequences on HIV-1 replication. J Virol 68:6198–6206;1994.

    PubMed  Google Scholar 

  18. Luban J. Absconding with the chaperone: Essential cyclophilin-Gag interaction in HIV-1 virions. Cell 87:1157–1159;1996.

    Article  PubMed  Google Scholar 

  19. Mak J, Jiang M, Wainberg MA, Hammarskjold ML, Rekosh D, Kleiman L. Role of Pr160gag-pol in mediating the selective incorporation of tRNALys into human immunodeficiency virus type 1 particles. J Virol 68:2065–2072;1994.

    PubMed  Google Scholar 

  20. Mak J, Khorchid A, Cao Q, Huang Y, Lowy I, Prasad VR, Parniak MA, Wainberg MA, Kleiman L. Effects of mutations in Pr160gag-pol upon tRNALys3 and Pr160gag-pol incorporation into HIV-1. J Mol Biol 265:419–431;1997.

    PubMed  Google Scholar 

  21. Mak J, Kleiman L. Primer tRNAs for reverse transcription. J Virol 71:8087–8095;1997.

    PubMed  Google Scholar 

  22. Park J, Morrow CD. 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 65:5111–5117;1991.

    PubMed  Google Scholar 

  23. Shehu-Xhilaga M, Crowe SM, Mak J. Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J Virol 75:1834–1841;2001.

    PubMed  Google Scholar 

  24. Shehu-Xhilaga M, Kraeusslich HG, Pettit S, Swanstrom R, Lee JY, Marshall JA, Crowe SM, Mak J. Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type1 RNA dimer maturation. J Virol 75:9156–9164;2001.

    PubMed  Google Scholar 

  25. Srinivasakumar N, Hammarskjöld ML, Rekosh D. Characterization of deletion mutations in the capsid region of HIV-1 that affect particle formation and Gag-Pol precursor incorporation. J Virol 69:6106–6114;1995.

    PubMed  Google Scholar 

  26. Stoltzfus CM, Snyder PN. Structure of B77 sarcoma virus RNA: Stabilization of RNA after packaging J Virol 16:1161–1170;1975.

    PubMed  Google Scholar 

  27. Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, Sodroski J, Göttlinger HG. Functional association of cyclophilin A with HIV-1 virions. Nature 372:363–365;1994.

    PubMed  Google Scholar 

  28. Wills JW, Craven RC. Form, function, and use of retroviral Gag proteins. AIDS 5:639–654;1991.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Victorian Infectious Diseases Reference Laboratory, Melbourne

    J.-Y. Lee & J. A. Marshall

  2. National Centre of HIV Virology Research, Melbourne

    S. M. Crowe

  3. Department of Medicine, Monash University, Clayton, Australia

    M. Shehu-Xhilaga & S. M. Crowe

  4. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia

    J. Mak

  5. AIDS Pathogenesis Research Unit, The Macfarlane Burnet Institute for Medical Research and Public Health, Cnr Punt & Commercial Rds, 3004, Melbourne, Vic, Australia

    M. Shehu-Xhilaga, S. Campbell, S. M. Crowe &  J. Mak

Authors
  1. M. Shehu-Xhilaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J.-Y. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. A. Marshall
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. M. Crowe
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. Mak
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shehu-Xhilaga, M., Lee, JY., Campbell, S. et al. Overexpression and incorporation of GagPol precursor does not impede packaging of HIV-1 tRNALys3 but promotes intracellular budding of virus-like particles. J Biomed Sci 9, 697–705 (2002). https://doi.org/10.1007/BF02254998

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02254998

Key Words

Search

Navigation