Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Oct;68(10):6161–6169. doi: 10.1128/jvi.68.10.6161-6169.1994

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

X Wu 1, J A Conway 1, J Kim 1, J C Kappes 1
PMCID: PMC237035  PMID: 8083957

Abstract

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.

Full text

PDF
6161

Images in this article

6162Image
on p.6162
6164Image
on p.6164
6164Image
on p.6164
6165Image
on p.6165
6166Image
on p.6166
6167Image
on p.6167
6167Image
on p.6167

Selected References

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

  1. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernstein D. I., Stanberry L. R., Harrison C. J., Kappes J. C., Myers M. G. Antibody response, recurrence patterns and subsequent herpes simplex virus type 2 (HSV-2) re-infection following initial HSV-2 infection of guinea-pigs: effects of acyclovir. J Gen Virol. 1986 Aug;67(Pt 8):1601–1612. doi: 10.1099/0022-1317-67-8-1601. [DOI] [PubMed] [Google Scholar]
  3. Clavel F., Orenstein J. M. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. doi: 10.1128/jvi.64.10.5230-5234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen E. A., Dehni G., Sodroski J. G., Haseltine W. A. Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol. 1990 Jun;64(6):3097–3099. doi: 10.1128/jvi.64.6.3097-3099.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen E. A., Terwilliger E. F., Jalinoos Y., Proulx J., Sodroski J. G., Haseltine W. A. Identification of HIV-1 vpr product and function. J Acquir Immune Defic Syndr. 1990;3(1):11–18. [PubMed] [Google Scholar]
  6. Delchambre M., Gheysen D., Thines D., Thiriart C., Jacobs E., Verdin E., Horth M., Burny A., Bex F. The GAG precursor of simian immunodeficiency virus assembles into virus-like particles. EMBO J. 1989 Sep;8(9):2653–2660. doi: 10.1002/j.1460-2075.1989.tb08405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dong J., Hunter E. Analysis of retroviral assembly using a vaccinia/T7-polymerase complementation system. Virology. 1993 May;194(1):192–199. doi: 10.1006/viro.1993.1249. [DOI] [PubMed] [Google Scholar]
  8. Fuerst T. R., Earl P. L., Moss B. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol Cell Biol. 1987 Jul;7(7):2538–2544. doi: 10.1128/mcb.7.7.2538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gorelick R. J., Nigida S. M., Jr, Bess J. W., Jr, Arthur L. O., Henderson L. E., Rein A. Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol. 1990 Jul;64(7):3207–3211. doi: 10.1128/jvi.64.7.3207-3211.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guyader M., Emerman M., Montagnier L., Peden K. VPX mutants of HIV-2 are infectious in established cell lines but display a severe defect in peripheral blood lymphocytes. EMBO J. 1989 Apr;8(4):1169–1175. doi: 10.1002/j.1460-2075.1989.tb03488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guyader M., Emerman M., Sonigo P., Clavel F., Montagnier L., Alizon M. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature. 1987 Apr 16;326(6114):662–669. doi: 10.1038/326662a0. [DOI] [PubMed] [Google Scholar]
  14. Hayashi T., Shioda T., Iwakura Y., Shibuta H. RNA packaging signal of human immunodeficiency virus type 1. Virology. 1992 Jun;188(2):590–599. doi: 10.1016/0042-6822(92)90513-o. [DOI] [PubMed] [Google Scholar]
  15. Henderson L. E., Benveniste R. E., Sowder R., Copeland T. D., Schultz A. M., Oroszlan S. Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne). J Virol. 1988 Aug;62(8):2587–2595. doi: 10.1128/jvi.62.8.2587-2595.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henderson L. E., Bowers M. A., Sowder R. C., 2nd, Serabyn S. A., Johnson D. G., Bess J. W., Jr, Arthur L. O., Bryant D. K., Fenselau C. Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences. J Virol. 1992 Apr;66(4):1856–1865. doi: 10.1128/jvi.66.4.1856-1865.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henderson L. E., Sowder R. C., Copeland T. D., Benveniste R. E., Oroszlan S. Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2. Science. 1988 Jul 8;241(4862):199–201. doi: 10.1126/science.3388031. [DOI] [PubMed] [Google Scholar]
  18. Horton R., Spearman P., Ratner L. HIV-2 viral protein X association with the GAG p27 capsid protein. Virology. 1994 Mar;199(2):453–457. doi: 10.1006/viro.1994.1144. [DOI] [PubMed] [Google Scholar]
  19. Jowett J. B., Hockley D. J., Nermut M. V., Jones I. M. Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation. J Gen Virol. 1992 Dec;73(Pt 12):3079–3086. doi: 10.1099/0022-1317-73-12-3079. [DOI] [PubMed] [Google Scholar]
  20. Kappes J. C., Conway J. A., Lee S. W., Shaw G. M., Hahn B. H. Human immunodeficiency virus type 2 vpx protein augments viral infectivity. Virology. 1991 Sep;184(1):197–209. doi: 10.1016/0042-6822(91)90836-z. [DOI] [PubMed] [Google Scholar]
  21. Kappes J. C., Morrow C. D., Lee S. W., Jameson B. A., Kent S. B., Hood L. E., Shaw G. M., Hahn B. H. Identification of a novel retroviral gene unique to human immunodeficiency virus type 2 and simian immunodeficiency virus SIVMAC. J Virol. 1988 Sep;62(9):3501–3505. doi: 10.1128/jvi.62.9.3501-3505.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kappes J. C., Parkin J. S., Conway J. A., Kim J., Brouillette C. G., Shaw G. M., Hahn B. H. Intracellular transport and virion incorporation of vpx requires interaction with other virus type-specific components. Virology. 1993 Mar;193(1):222–233. doi: 10.1006/viro.1993.1118. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kumar P., Hui H. X., Kappes J. C., Haggarty B. S., Hoxie J. A., Arya S. K., Shaw G. M., Hahn B. H. Molecular characterization of an attenuated human immunodeficiency virus type 2 isolate. J Virol. 1990 Feb;64(2):890–901. doi: 10.1128/jvi.64.2.890-901.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lavallée C., Yao X. J., Ladha A., Göttlinger H., Haseltine W. A., Cohen E. A. Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles. J Virol. 1994 Mar;68(3):1926–1934. doi: 10.1128/jvi.68.3.1926-1934.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lever A., Gottlinger H., Haseltine W., Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. doi: 10.1128/jvi.63.9.4085-4087.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li Y., Hui H., Burgess C. J., Price R. W., Sharp P. M., Hahn B. H., Shaw G. M. Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation. J Virol. 1992 Nov;66(11):6587–6600. doi: 10.1128/jvi.66.11.6587-6600.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lu Y. L., Spearman P., Ratner L. Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol. 1993 Nov;67(11):6542–6550. doi: 10.1128/jvi.67.11.6542-6550.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marcon L., Michaels F., Hattori N., Fargnoli K., Gallo R. C., Franchini G. Dispensable role of the human immunodeficiency virus type 2 Vpx protein in viral replication. J Virol. 1991 Jul;65(7):3938–3942. doi: 10.1128/jvi.65.7.3938-3942.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matsuda Z., Yu X., Yu Q. C., Lee T. H., Essex M. A virion-specific inhibitory molecule with therapeutic potential for human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3544–3548. doi: 10.1073/pnas.90.8.3544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Minassian A. A., Kalyanaraman V. S., Gallo R. C., Popovic M. Monoclonal antibodies against human immunodeficiency virus (HIV) type 2 core proteins: cross-reactivity with HIV type 1 and simian immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6939–6943. doi: 10.1073/pnas.85.18.6939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ogawa K., Shibata R., Kiyomasu T., Higuchi I., Kishida Y., Ishimoto A., Adachi A. Mutational analysis of the human immunodeficiency virus vpr open reading frame. J Virol. 1989 Sep;63(9):4110–4114. doi: 10.1128/jvi.63.9.4110-4114.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Paxton W., Connor R. I., Landau N. R. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993 Dec;67(12):7229–7237. doi: 10.1128/jvi.67.12.7229-7237.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
  35. Shioda T., Shibuta H. Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV. Virology. 1990 Mar;175(1):139–148. doi: 10.1016/0042-6822(90)90194-v. [DOI] [PubMed] [Google Scholar]
  36. Tousson A., Zeng C., Brinkley B. R., Valdivia M. M. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation. J Cell Biol. 1991 Feb;112(3):427–440. doi: 10.1083/jcb.112.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tristem M., Marshall C., Karpas A., Hill F. Evolution of the primate lentiviruses: evidence from vpx and vpr. EMBO J. 1992 Sep;11(9):3405–3412. doi: 10.1002/j.1460-2075.1992.tb05419.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tristem M., Marshall C., Karpas A., Petrik J., Hill F. Origin of vpx in lentiviruses. Nature. 1990 Sep 27;347(6291):341–342. doi: 10.1038/347341b0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Yu X. F., Ito S., Essex M., Lee T. H. A naturally immunogenic virion-associated protein specific for HIV-2 and SIV. Nature. 1988 Sep 15;335(6187):262–265. doi: 10.1038/335262a0. [DOI] [PubMed] [Google Scholar]
  41. Yu X., Matsuda Z., Yu Q. C., Lee T. H., Essex M. Vpx of simian immunodeficiency virus is localized primarily outside the virus core in mature virions. J Virol. 1993 Jul;67(7):4386–4390. doi: 10.1128/jvi.67.7.4386-4390.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yuan X., Matsuda Z., Matsuda M., Essex M., Lee T. H. Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res Hum Retroviruses. 1990 Nov;6(11):1265–1271. doi: 10.1089/aid.1990.6.1265. [DOI] [PubMed] [Google Scholar]

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

RESOURCES