Abstract
The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.
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- Alton N. K., Vapnek D. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature. 1979 Dec 20;282(5741):864–869. doi: 10.1038/282864a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Felber B. K., Drysdale C. M., Pavlakis G. N. Feedback regulation of human immunodeficiency virus type 1 expression by the Rev protein. J Virol. 1990 Aug;64(8):3734–3741. doi: 10.1128/jvi.64.8.3734-3741.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fenrick R., Malim M. H., Hauber J., Le S. Y., Maizel J., Cullen B. R. Functional analysis of the Tat trans activator of human immunodeficiency virus type 2. J Virol. 1989 Dec;63(12):5006–5012. doi: 10.1128/jvi.63.12.5006-5012.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Ghosh S. K., Fultz P. N., Keddie E., Saag M. S., Sharp P. M., Hahn B. H., Shaw G. M. A molecular clone of HIV-1 tropic and cytopathic for human and chimpanzee lymphocytes. Virology. 1993 Jun;194(2):858–864. doi: 10.1006/viro.1993.1331. [DOI] [PubMed] [Google Scholar]
- Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
- 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]
- Heinzinger N. K., Bukrinsky M. I., Haggerty S. A., Ragland A. M., Kewalramani V., Lee M. A., Gendelman H. E., Ratner L., Stevenson M., Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7311–7315. doi: 10.1073/pnas.91.15.7311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Helseth E., Kowalski M., Gabuzda D., Olshevsky U., Haseltine W., Sodroski J. Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants. J Virol. 1990 May;64(5):2416–2420. doi: 10.1128/jvi.64.5.2416-2420.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Lewis N., Williams J., Rekosh D., Hammarskjöld M. L. Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins. J Virol. 1990 Apr;64(4):1690–1697. doi: 10.1128/jvi.64.4.1690-1697.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Malim M. H., Böhnlein S., Fenrick R., Le S. Y., Maizel J. V., Cullen B. R. Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8222–8226. doi: 10.1073/pnas.86.21.8222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
- Natsoulis G., Boeke J. D. New antiviral strategy using capsid-nuclease fusion proteins. Nature. 1991 Aug 15;352(6336):632–635. doi: 10.1038/352632a0. [DOI] [PubMed] [Google Scholar]
- Natsoulis G., Seshaiah P., Federspiel M. J., Rein A., Hughes S. H., Boeke J. D. Targeting of a nuclease to murine leukemia virus capsids inhibits viral multiplication. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):364–368. doi: 10.1073/pnas.92.2.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Subbramanian R. A., Cohen E. A. Molecular biology of the human immunodeficiency virus accessory proteins. J Virol. 1994 Nov;68(11):6831–6835. doi: 10.1128/jvi.68.11.6831-6835.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang C. T., Stegeman-Olsen J., Zhang Y., Barklis E. Assembly of HIV GAG-B-galactosidase fusion proteins into virus particles. Virology. 1994 May 1;200(2):524–534. doi: 10.1006/viro.1994.1215. [DOI] [PubMed] [Google Scholar]
- Weber D. J., Meeker A. K., Mildvan A. S. Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant. Biochemistry. 1991 Jun 25;30(25):6103–6114. doi: 10.1021/bi00239a004. [DOI] [PubMed] [Google Scholar]
- 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]
- Wu X., Chen S. Y., Iwata H., Compans R. W., Roy P. Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus. J Virol. 1992 Dec;66(12):7104–7112. doi: 10.1128/jvi.66.12.7104-7112.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu X., Conway J. A., Kim J., Kappes J. C. Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein. J Virol. 1994 Oct;68(10):6161–6169. doi: 10.1128/jvi.68.10.6161-6169.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu X. F., Matsuda M., Essex M., Lee T. H. Open reading frame vpr of simian immunodeficiency virus encodes a virion-associated protein. J Virol. 1990 Nov;64(11):5688–5693. doi: 10.1128/jvi.64.11.5688-5693.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]