Abstract
A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function.
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Selected References
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- Arya S. K., Guo C., Josephs S. F., Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. doi: 10.1126/science.2990040. [DOI] [PubMed] [Google Scholar]
- Bukhari A. I., Zipser D. Mutants of Escherichia coli with a defect in the degradation of nonsense fragments. Nat New Biol. 1973 Jun 20;243(129):238–241. doi: 10.1038/newbio243238a0. [DOI] [PubMed] [Google Scholar]
- Cochrane A. W., Chen C. H., Kramer R., Tomchak L., Rosen C. A. Purification of biologically active human immunodeficiency virus rev protein from Escherichia coli. Virology. 1989 Nov;173(1):335–337. doi: 10.1016/0042-6822(89)90252-3. [DOI] [PubMed] [Google Scholar]
- Cochrane A., Kramer R., Ruben S., Levine J., Rosen C. A. The human immunodeficiency virus rev protein is a nuclear phosphoprotein. Virology. 1989 Jul;171(1):264–266. doi: 10.1016/0042-6822(89)90535-7. [DOI] [PubMed] [Google Scholar]
- Colbère-Garapin F., Ryhiner M. L., Stephany I., Kourilsky P., Garapin A. C. Patterns of integration of exogenous DNA sequences transfected into mammalian cells of primate and rodent origin. Gene. 1986;50(1-3):279–288. doi: 10.1016/0378-1119(86)90332-x. [DOI] [PubMed] [Google Scholar]
- Cullen B. R., Hauber J., Campbell K., Sodroski J. G., Haseltine W. A., Rosen C. A. Subcellular localization of the human immunodeficiency virus trans-acting art gene product. J Virol. 1988 Jul;62(7):2498–2501. doi: 10.1128/jvi.62.7.2498-2501.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
- Dayton A. I., Terwilliger E. F., Potz J., Kowalski M., Sodroski J. G., Haseltine W. A. Cis-acting sequences responsive to the rev gene product of the human immunodeficiency virus. J Acquir Immune Defic Syndr. 1988;1(5):441–452. [PubMed] [Google Scholar]
- Emerman M., Vazeux R., Peden K. The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell. 1989 Jun 30;57(7):1155–1165. doi: 10.1016/0092-8674(89)90053-6. [DOI] [PubMed] [Google Scholar]
- Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
- Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felber B. K., Paskalis H., Kleinman-Ewing C., Wong-Staal F., Pavlakis G. N. The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats. Science. 1985 Aug 16;229(4714):675–679. doi: 10.1126/science.2992082. [DOI] [PubMed] [Google Scholar]
- Friedman A. D., Triezenberg S. J., McKnight S. L. Expression of a truncated viral trans-activator selectively impedes lytic infection by its cognate virus. Nature. 1988 Sep 29;335(6189):452–454. doi: 10.1038/335452a0. [DOI] [PubMed] [Google Scholar]
- Giam C. Z., Nerenberg M., Khoury G., Jay G. Expression of the complete human T-cell leukemia virus type I pX coding sequence as a functional protein in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7192–7196. doi: 10.1073/pnas.83.19.7192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goh W. C., Rosen C., Sodroski J., Ho D. D., Haseltine W. A. Identification of a protein encoded by the trans activator gene tatIII of human T-cell lymphotropic retrovirus type III. J Virol. 1986 Jul;59(1):181–184. doi: 10.1128/jvi.59.1.181-184.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Hadzopoulou-Cladaras M., Felber B. K., Cladaras C., Athanassopoulos A., Tse A., Pavlakis G. N. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol. 1989 Mar;63(3):1265–1274. doi: 10.1128/jvi.63.3.1265-1274.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hammarskjöld M. L., Heimer J., Hammarskjöld B., Sangwan I., Albert L., Rekosh D. Regulation of human immunodeficiency virus env expression by the rev gene product. J Virol. 1989 May;63(5):1959–1966. doi: 10.1128/jvi.63.5.1959-1966.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hauber J., Perkins A., Heimer E. P., Cullen B. R. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6364–6368. doi: 10.1073/pnas.84.18.6364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hizi A., McGill C., Hughes S. H. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1218–1222. doi: 10.1073/pnas.85.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Malim M. H., Böhnlein S., Hauber J., Cullen B. R. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell. 1989 Jul 14;58(1):205–214. doi: 10.1016/0092-8674(89)90416-9. [DOI] [PubMed] [Google Scholar]
- Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
- Mermer B., Malamy M., Coffin J. M. Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli. Mol Cell Biol. 1983 Oct;3(10):1746–1758. doi: 10.1128/mcb.3.10.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
- Perkins A., Cochrane A. W., Ruben S. M., Rosen C. A. Structural and functional characterization of the human immunodeficiency virus rev protein. J Acquir Immune Defic Syndr. 1989;2(3):256–263. [PubMed] [Google Scholar]
- Peterlin B. M., Luciw P. A., Barr P. J., Walker M. D. Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9734–9738. doi: 10.1073/pnas.83.24.9734. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
- Rassoulzadegan M., Binetruy B., Cuzin F. High frequency of gene transfer after fusion between bacteria and eukaryotic cells. Nature. 1982 Jan 21;295(5846):257–259. doi: 10.1038/295257a0. [DOI] [PubMed] [Google Scholar]
- Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
- Rosen C. A., Terwilliger E., Dayton A., Sodroski J. G., Haseltine W. A. Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2071–2075. doi: 10.1073/pnas.85.7.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sandri-Goldin R. M., Goldin A. L., Levine M., Glorioso J. C. High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion. Mol Cell Biol. 1981 Aug;1(8):743–752. doi: 10.1128/mcb.1.8.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schaffner W. Direct transfer of cloned genes from bacteria to mammalian cells. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2163–2167. doi: 10.1073/pnas.77.4.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sodroski J., Goh W. C., Rosen C., Campbell K., Haseltine W. A. Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. 1986 Jul 31-Aug 6Nature. 322(6078):470–474. doi: 10.1038/322470a0. [DOI] [PubMed] [Google Scholar]
- Sodroski J., Goh W. C., Rosen C., Dayton A., Terwilliger E., Haseltine W. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986 May 22;321(6068):412–417. doi: 10.1038/321412a0. [DOI] [PubMed] [Google Scholar]
- Sodroski J., Patarca R., Rosen C., Wong-Staal F., Haseltine W. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science. 1985 Jul 5;229(4708):74–77. doi: 10.1126/science.2990041. [DOI] [PubMed] [Google Scholar]
- Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
- Weiss R. L. Protoplast formation in Escherichia coli. J Bacteriol. 1976 Nov;128(2):668–670. doi: 10.1128/jb.128.2.668-670.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wright C. M., Felber B. K., Paskalis H., Pavlakis G. N. Expression and characterization of the trans-activator of HTLV-III/LAV virus. Science. 1986 Nov 21;234(4779):988–992. doi: 10.1126/science.3490693. [DOI] [PubMed] [Google Scholar]
- Yoakum G. H., Korba B. E., Lechner J. F., Tokiwa T., Gazdar A. F., Seeley T., Siegel M., Leeman L., Autrup H., Harris C. C. High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells. Science. 1983 Oct 28;222(4622):385–389. doi: 10.1126/science.6194563. [DOI] [PubMed] [Google Scholar]