Abstract
The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Vaishnav, Y.N. & Wong-Staal, F. Biochemistry of AIDS. Annu. Rev. Biochem. 60, 577–630 (1991).
Cullen, B.R. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56, 375– 394 (1992).
Myer, B.E. & Malim, M.H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 8 , 1538–1547 (1994).
Malim, M.H., Bohnlein, S., Hauber, J. & Cullen, B.R. –Functional dissection of the HIV-1 Rev trans-activator-derivation of a trans-dominant repressor of Rev protein. Cell 58, 205– 214 (1989).
Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. & Luhrmann, R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475–483 ( 1995).
Malim, M.H. et al. Stable expression of transdominant Rev protein in human T cells inhibits Human Immunodeficiency Virus replication. J. Exp. Med. 176, 1197–1201 ( 1992).
Woffendin, C., Ranga, U., Yang, Z., Xu, L. & Nabel, G.J. Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients. Proc. Natl. Acad. Sci. USA 93, 2889–94 (1996).
Ranga, U. et al. Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals. Proc. Natl. Acad. Sci. USA 95,1201–1206 (1998).
Fornerod, M., Ohno, M., Yoshida, M. & Mattaj. I.W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060 (1997).
Neville, M., Stutz, F., Lee, M., Davis, L.I. & Rasbach, M. The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr. Biol. 7, 767–775 ( 1997).
Bogerd, H.P., Fridell, R.A., Madore, S. & Cullen, B.R. Identification of a novel cellular factor for the Rev/Rex class of retroviral regulatory proteins. Cell 82, 485– 494 (1995).
Fritz, C.C., Zapp, M.L. & Green, M.R. A human nucleoporin-like protein that specifically interacts with Rev. Nature 376, 530-533 (1995).
Ruhl., M. et al. Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating transactivation. J. Cell Biol. 123, 1309– 1320 (1993).
Luo, Y., Yu, H. & Peterlin, B.M. Cellular protein modulates effects of human immunodefficiency virus type 1 Rev. J. Virol. 68, 3850– 3856 (1994).
Tange, T.O., Jensen, T.H. & Kjems, J. In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. J. Biol. Chem. 271, 10066– 10072 (1996).
Xu, Y., Reddy, T.R., Fischer, W. & Wong-Staal, F. A novel hnRNP specifically interacts with HIV-1 RRE RNA. J. Biomed. Sci. 3, 82–91 (1996).
Powell, D.M., Amaral, C.M., Wu, J.Y., Maniatis, T. & Greene, W.C. HIV Rev-dependent binding of SF2/ASF to the Rev response element: Possible role in Rev-mediated inhibition of HIV RNA splicing. Proc. Natl. Acad. Sci. USA. 94, 973– 978 (1997).
Taylor, S.J. & Shalloway. D. An RNA-binding protein associated with src through its SH2 and SH3 domains in mitosis. Nature 368, 867–871 (1994).
Fumagalli, S., Totty, N.F., Hsuan, J.J. & Courtneidge, S.A. A target for SRC in mitosis. Nature 368, 871– 874 (1994).
Resnick, R.J., Taylor, S.J., Lin, Q. & Shalloway, D. Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis. Oncogene 15, 1247–1253 (1997).
Chen, T., Damaj, B.B., Herrera, C., Lasko, P. & Richard, S. Self-association of the single-KH-domain family members Sam68, GRP33, GRD-1, and QK1: Role of the KH domain. Mol. Cell. Biol. 17, 5707–5718 ( 1997).
Ishidate, T. et al. Identification of a novel nuclear localization signal in Sam68. FEBS Letters 409, 237– 241 (1997).
Barlat, I. et al. A role for Sam68 in cell cycle progression antagonized by a spliced variant within the KH domain. J. Biol. Chem. 272, 3129–3132 (1997).
Bray, M. et al. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc. Natl. Acad. Sci. USA. 91, 1256– 1260 (1994).
Lavinsky, R.M. et al. Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc. Natl. Acad. Sci. USA 95, 2920–2925 (1998).
Sadaie, M.R., Benter, T. & Wong-Staal, F. Site-directed mutagenesis of two trans-regulatory genes (tat-III, trs) of HIV-1. Science 239, 910 –914 (1988).
Wolff, B., Sanglier, J-J. & Wang, Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139–147 (1997).
Diaz, J-J. et al. Post-transcriptional transactivation of human retroviral envelope glycoprotein expression by herpes simplex virus Us11 protein. Nature 379, 273–277( 1996).
Siomi, H., Matunis, M.J., Michael, W.M. & Dreyfuss, G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nuc. Acid. Res. 21, 1193– 1198 (1993).
Cruz-Alvarez, M. & Pellicer, A. Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins. J. Biol. Chem . 262, 13377–13380 ( 1987).
Siomi, H., Matunis, M.J., Nussbaum, R.L. & Dreyfuss, G. The protein product of fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell 74, 291–298 (1993).
Jones, A.R. & Schedl, T. Mutations in gld-1, a female germ cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. Genes Dev. 9, 1491–1504 (1995).
Lin, Q., Taylor, S.J. & Shalloway, D. Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains. J. Biol. Chem . 272, 27274–27280 ( 1997).
Yu, M., Poeschla, E. & Wong-Staal, F. Progress towards gene therapy for HIV infection. Gene Ther. 1, 13–26 ( 1994).
Woffendin, C. et al. Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells. Proc. Natl. Acad. Sci. USA 91, 11581–11585 ( 1994).
Junker, U. et al. Inhibition of human immunodeficiency virus type 1 replication in myelomonocytic cells derived from retroviral vector-transduced peripheral blood progenitor cells. Hum. Gene Ther. 9, 333–340 (1998).
Seed, B. & Sheen, J.-Y. A simple phase-extraction assay for chloramphenicol acetyltransferase activity. Gene 67, 271–277 (1988).
Torchia, J. et al. The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387, 677 –684 (1997).
Reddy, T.R., Tang, H., Li, X. & Wong-Staal. F. Functional interaction of HTLV-1 transactivator Tax with activating transcription factor-4 (ATF4). Oncogene 14, 2785– 2792 (1997).
Acknowledgements
We also thank D. Looney, C. Barroga and K. Kuhen for critical review of the manuscript, and C. Westberg for editorial assistance. Recombinant Rev was obtained through the AIDS Research and Reference Reagents Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (D. Rekosh, M.-L. Hammerskjold and M. Orsini). This work was supported in part by NIH grant GM056089 to F.W.-S. We also acknowledge the support of the Center for AIDS Research (NIH Grant P30AI 36214-06) and National Center for Microscopy and Imaging Research at UCSD (NIH grant # RR04050; PI, M. H. Ellisman).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Reddy, T., Xu, W., Mau, J. et al. Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev. Nat Med 5, 635–642 (1999). https://doi.org/10.1038/9479
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/9479
This article is cited by
-
Association of N6-methyladenosine with viruses and related diseases
Virology Journal (2019)
-
Mechanism of mRNA-STAR domain interaction: Molecular dynamics simulations of Mammalian Quaking STAR protein
Scientific Reports (2017)
-
Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68
Nature Communications (2016)
-
Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections
Virology Journal (2015)
-
Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev
Retrovirology (2014)