Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Nov;67(11):6487–6498. doi: 10.1128/jvi.67.11.6487-6498.1993

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

R P Bennett 1, T D Nelle 1, J W Wills 1
PMCID: PMC238085  PMID: 8411352

Abstract

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.

Full text

PDF
6489

Images in this article

6490Image
on p.6490
6493Image
on p.6493
6494Image
on p.6494
6494Image
on p.6494
6494Image
on p.6494
6495Image
on p.6495

Selected References

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

  1. Bennett R. P., Rhee S., Craven R. C., Hunter E., Wills J. W. Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly. J Virol. 1991 Jan;65(1):272–280. doi: 10.1128/jvi.65.1.272-280.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bess J. W., Jr, Powell P. J., Issaq H. J., Schumack L. J., Grimes M. K., Henderson L. E., Arthur L. O. Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. J Virol. 1992 Feb;66(2):840–847. doi: 10.1128/jvi.66.2.840-847.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bryant M., Ratner L. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci U S A. 1990 Jan;87(2):523–527. doi: 10.1073/pnas.87.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burstein H., Bizub D., Skalka A. M. Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers. J Virol. 1991 Nov;65(11):6165–6172. doi: 10.1128/jvi.65.11.6165-6172.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cameron C. E., Grinde B., Jentoft J., Leis J., Weber I. T., Copeland T. D., Wlodawer A. Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins. J Biol Chem. 1992 Nov 25;267(33):23735–23741. [PubMed] [Google Scholar]
  6. Craven R. C., Bennett R. P., Wills J. W. Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly. J Virol. 1991 Nov;65(11):6205–6217. doi: 10.1128/jvi.65.11.6205-6217.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Rocquigny H., Ficheux D., Gabus C., Allain B., Fournie-Zaluski M. C., Darlix J. L., Roques B. P. Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):823–829. doi: 10.1093/nar/21.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Rocquigny H., Gabus C., Vincent A., Fournié-Zaluski M. C., Roques B., Darlix J. L. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6472–6476. doi: 10.1073/pnas.89.14.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dupraz P., Oertle S., Meric C., Damay P., Spahr P. F. Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. J Virol. 1990 Oct;64(10):4978–4987. doi: 10.1128/jvi.64.10.4978-4987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dupraz P., Spahr P. F. Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging. J Virol. 1992 Aug;66(8):4662–4670. doi: 10.1128/jvi.66.8.4662-4670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erdie C. R., Wills J. W. Myristylation of Rous sarcoma virus Gag protein does not prevent replication in avian cells. J Virol. 1990 Oct;64(10):5204–5208. doi: 10.1128/jvi.64.10.5204-5208.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallis B., Linial M., Eisenman R. An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA. Virology. 1979 Apr 15;94(1):146–161. doi: 10.1016/0042-6822(79)90445-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gorelick R. J., Henderson L. E., Hanser J. P., Rein A. Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8420–8424. doi: 10.1073/pnas.85.22.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Göttlinger H. G., Dorfman T., Sodroski J. G., Haseltine W. A. Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3195–3199. doi: 10.1073/pnas.88.8.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Housset V., De Rocquigny H., Roques B. P., Darlix J. L. Basic amino acids flanking the zinc finger of Moloney murine leukemia virus nucleocapsid protein NCp10 are critical for virus infectivity. J Virol. 1993 May;67(5):2537–2545. doi: 10.1128/jvi.67.5.2537-2545.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988 Nov 4;55(3):447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  22. Jones T. A., Blaug G., Hansen M., Barklis E. Assembly of gag-beta-galactosidase proteins into retrovirus particles. J Virol. 1990 May;64(5):2265–2279. doi: 10.1128/jvi.64.5.2265-2279.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kotler M., Katz R. A., Danho W., Leis J., Skalka A. M. Synthetic peptides as substrates and inhibitors of a retroviral protease. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4185–4189. doi: 10.1073/pnas.85.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Leis J., Baltimore D., Bishop J. M., Coffin J., Fleissner E., Goff S. P., Oroszlan S., Robinson H., Skalka A. M., Temin H. M. Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol. 1988 May;62(5):1808–1809. doi: 10.1128/jvi.62.5.1808-1809.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Levin J. G., Seidman J. G. Effect of polymerase mutations on packaging of primer tRNAPro during murine leukemia virus assembly. J Virol. 1981 Apr;38(1):403–408. doi: 10.1128/jvi.38.1.403-408.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levin J. G., Seidman J. G. Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA. J Virol. 1979 Jan;29(1):328–335. doi: 10.1128/jvi.29.1.328-335.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Linial M. L., Miller A. D. Retroviral RNA packaging: sequence requirements and implications. Curr Top Microbiol Immunol. 1990;157:125–152. doi: 10.1007/978-3-642-75218-6_5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Moscufo N., Simons J., Chow M. Myristoylation is important at multiple stages in poliovirus assembly. J Virol. 1991 May;65(5):2372–2380. doi: 10.1128/jvi.65.5.2372-2380.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Méric C., Goff S. P. Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol. 1989 Apr;63(4):1558–1568. doi: 10.1128/jvi.63.4.1558-1568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Méric C., Gouilloud E., Spahr P. F. Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes. J Virol. 1988 Sep;62(9):3328–3333. doi: 10.1128/jvi.62.9.3328-3333.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pal R., Reitz M. S., Jr, Tschachler E., Gallo R. C., Sarngadharan M. G., Veronese F. D. Myristoylation of gag proteins of HIV-1 plays an important role in virus assembly. AIDS Res Hum Retroviruses. 1990 Jun;6(6):721–730. doi: 10.1089/aid.1990.6.721. [DOI] [PubMed] [Google Scholar]
  36. Pepinsky R. B., Vogt V. M. Identification of retrovirus matrix proteins by lipid-protein cross-linking. J Mol Biol. 1979 Jul 15;131(4):819–837. doi: 10.1016/0022-2836(79)90203-1. [DOI] [PubMed] [Google Scholar]
  37. Rein A., McClure M. R., Rice N. R., Luftig R. B., Schultz A. M. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7246–7250. doi: 10.1073/pnas.83.19.7246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Resh M. D., Ling H. P. Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60v-src. Nature. 1990 Jul 5;346(6279):84–86. doi: 10.1038/346084a0. [DOI] [PubMed] [Google Scholar]
  39. Resh M. D. Specific and saturable binding of pp60v-src to plasma membranes: evidence for a myristyl-src receptor. Cell. 1989 Jul 28;58(2):281–286. doi: 10.1016/0092-8674(89)90842-8. [DOI] [PubMed] [Google Scholar]
  40. Rhee S. S., Hunter E. A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus. Cell. 1990 Oct 5;63(1):77–86. doi: 10.1016/0092-8674(90)90289-q. [DOI] [PubMed] [Google Scholar]
  41. Rhee S. S., Hunter E. Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids. J Virol. 1987 Apr;61(4):1045–1053. doi: 10.1128/jvi.61.4.1045-1053.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Salmeen I., Rimai L., Liebes L., Rich M. A., McCormick J. J. Hydrodynamic diameters of RNA tumor viruses. Studies by laser beat frequency light scattering spectroscopy of avian myeloblastosis and Rauscher murine leukemia viruses. Biochemistry. 1975 Jan 14;14(1):134–141. doi: 10.1021/bi00672a023. [DOI] [PubMed] [Google Scholar]
  43. Schultz A. M., Rein A. Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events. J Virol. 1989 May;63(5):2370–2373. doi: 10.1128/jvi.63.5.2370-2373.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schwartz S., Felber B. K., Pavlakis G. N. Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol. 1992 Jan;66(1):150–159. doi: 10.1128/jvi.66.1.150-159.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Skalka A. M. Retroviral proteases: first glimpses at the anatomy of a processing machine. Cell. 1989 Mar 24;56(6):911–913. doi: 10.1016/0092-8674(89)90621-1. [DOI] [PubMed] [Google Scholar]
  46. Stewart L., Vogt V. M. trans-acting viral protease is necessary and sufficient for activation of avian leukosis virus reverse transcriptase. J Virol. 1991 Nov;65(11):6218–6231. doi: 10.1128/jvi.65.11.6218-6231.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Strambio-de-Castillia C., Hunter E. Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis. J Virol. 1992 Dec;66(12):7021–7032. doi: 10.1128/jvi.66.12.7021-7032.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weldon R. A., Jr, Erdie C. R., Oliver M. G., Wills J. W. Incorporation of chimeric gag protein into retroviral particles. J Virol. 1990 Sep;64(9):4169–4179. doi: 10.1128/jvi.64.9.4169-4179.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weldon R. A., Jr, Wills J. W. Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release. J Virol. 1993 Sep;67(9):5550–5561. doi: 10.1128/jvi.67.9.5550-5561.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wills J. W., Craven R. C., Achacoso J. A. Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells. J Virol. 1989 Oct;63(10):4331–4343. doi: 10.1128/jvi.63.10.4331-4343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Wills J. W., Craven R. C., Weldon R. A., Jr, Nelle T. D., Erdie C. R. Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src. J Virol. 1991 Jul;65(7):3804–3812. doi: 10.1128/jvi.65.7.3804-3812.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yu X., Yuan X., Matsuda Z., Lee T. H., Essex M. The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions. J Virol. 1992 Aug;66(8):4966–4971. doi: 10.1128/jvi.66.8.4966-4971.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES