HIV-1 Tat Elongates the G1 Phase and Indirectly Promotes HIV-1 Gene Expression in Cells of Glial Origin
1998; Elsevier BV; Volume: 273; Issue: 14 Linguagem: Inglês
10.1074/jbc.273.14.8130
ISSN1083-351X
AutoresMondira Kundu, Sunita Sharma, Antonio De Luca, Antonio Giordano, Jay Rappaport, Kamel Khalili, Shohreh Amini,
Tópico(s)interferon and immune responses
ResumoHuman immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-κB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex. Human immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-κB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex. Neuropathological features of HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. infection include reactive astrogliosis, neuronal loss, widespread myelin pallor, subtle alteration of neocortical dendritic processes, and formation of multinucleated giant cells (1Everall I.P. Luthert P.J. Lantos P.L. Lancet. 1991; 337: 1119-1121Abstract PubMed Scopus (436) Google Scholar, 2Ketzler S. Weis S. Haug H. Budka H. 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This apparent paradox has led to the hypothesis that indirect effects of HIV-1 infection including the release of neurotoxic viral proteins and cytokines may mediate some of the pathobiological alterations observed in CNS cells. Tat, a viral regulatory protein, may be produced by HIV-1-infected macrophages and resident microglia, as well as infected astrocytes in the brain (7Budka H. Acta Neuropathol. 1990; 76: 611-619Crossref Scopus (86) Google Scholar, 8Gyorkey F. Melnick J.L. Gyorky P.J. J. Infect. Dis. 1987; 155: 870-876Crossref PubMed Scopus (125) Google Scholar, 9Koenig S. Gendelman H.E. Orenstein J.M. DalCanto M.C. Pezeshkpour G.H. Yungbluth M. Janotta F. Aksamit A. Martin M.A. Fauci A.S. Science. 1986; 233: 1089-1093Crossref PubMed Scopus (1348) Google Scholar, 10Sharer L.R. Cho E.S. Epstein L.G. Hum. Pathol. 1985; 16: 760-765Crossref PubMed Scopus (182) Google Scholar, 11Tornatore C. Nath A. Amemiya K. Major E.O. J. 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These observations led us to the hypothesis that Tat may alter the activation and proliferation state of astrocytes and contribute to the pathogenesis of AIDS-associated dementia.The reciprocal nature of the interaction between virus and host is expected, since HIV-1 is susceptible to regulation by cellular factors and therefore by the state of the host cell. There is evidence to suggest that cellular factors, including B-myb, E2F-1, and p53, which are involved in the control of cellular proliferation, may play a role in modulation of HIV-1 gene expression (31Duan L. Ozaki I. Oakes J.W. Taylor J.P. Khalili K. Pomerantz R.J. J. Virol. 1994; 68: 4302-4313Crossref PubMed Google Scholar, 32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar, 33Sala A. Kundu M. Casela I. Engelhard A. Calabretta B. Grasso L. Paggi M.G. Giordano A. Watson R.J. Khalili K. Peschle C. Proc. Natl. Acad. Sci. U. S. 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Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Phosphorylation of Rb in late G1 by the G1 cyclin-Cdk complexes results in the release of free E2F-1, which activates transcription of several genes involved in S phase (45Slansky J.E. Farnham P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar).In this study, we sought to further examine the interplay between virus and host cell cycle progression. We demonstrate that the viral transactivator protein, Tat, is able to arrest human astrocytic cells, U-87MG, in the G1 phase of the cell cycle by dysregulating the expression and activity of cyclin E and Cdk2. This results in accumulation of Rb in its underphosphorylated form, which in turn augments transcription directed by the HIV-1 LTR. We propose that in addition to its ability to directly transactivate the HIV-1 LTR, Tat alters the proliferation state of astrocytes and facilitates expression and replication of the HIV-1 genome.DISCUSSIONAIDS dementia complex is one of the most prevalent neurological complications of HIV-1 infection of the central nervous system. ADC affects almost 10% of AIDS patients (73Bacellar H. Munoz A. Miller E.N. Cohen B.A. Besley D. Selnes O.A. Becker J.T. McArthur J.C. Neurology. 1994; 44: 1892-1900Crossref PubMed Google Scholar). Productive infection in the CNS occurs primarily in macrophages and resident microglia (74Lipton S.A. Gendelman H.E. N. Engl. J. Med. 1995; 332: 934-940Crossref PubMed Scopus (441) Google Scholar, 75Simpson D.M. Tagliati M. Ann. Intern. Med. 1994; 121 (Correction (1995) Ann. Intern. Med. 122, 317): 769-785Crossref PubMed Scopus (195) Google Scholar). However, there is evidence of a “restricted” infection in astrocytes (76Blumberg B.M. Gelbard H.A. Epstein L.G. Virus Res. 1994; 32: 253-267Crossref PubMed Scopus (80) Google Scholar). The term “restricted” is used to describe the restriction of viral gene expression to the regulatory proteins, Tat, Rev, and Nef, which are derived from the multiply spliced viral mRNA species commonly found in infected astrocytes (62Tornatore C. Meyers K. Atwood W. Conant K. Major E.O. J. Virol. 1994; 68: 93-102Crossref PubMed Google Scholar). To account for the discrepancy between the small infected cell population and the widespread pathology, the current models regarding the neuropathogenesis of ADC propose a major role for indirect effects of HIV-1 infection (77Dewhurst S. Gelbard H.A. Fine S.M. Mol. Med. Today. 1996; 2: 16-23Abstract Full Text PDF PubMed Scopus (39) Google Scholar). In this respect, the infection of astrocytes may play a central role in the pathogenesis of ADC. Infected astrocytes and microglial cells can release the viral protein Tat. Tat can be taken up by neighboring cells in a biologically active form that can stimulate the expression of cytokines, including IL-1, IL-6, tumor necrosis factor-α, and transforming growth factor-β and several extracellular matrix proteins in the CNS (25Zauli G. Furlini G. Re M.C. Milani D. Capitani S. La Placa M. New Microbiol. 1993; 16: 115-120PubMed Google Scholar, 27Cupp C. Taylor J.P. Khalili K. Amini S. Oncogene. 1993; 8: 2231-2236PubMed Google Scholar, 29Rasty S. Thatikunta P. Gordon J. Khalili K. Amini S. Glorioso J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6073-6078Crossref PubMed Scopus (37) Google Scholar, 53Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 66: 7159-7167Crossref PubMed Google Scholar, 54Gibellini D. Zauli G. Re M.C. Milani D. Furlini G. Caramelli E. Capitani S. La Placa M. Br. J. Haematol. 1994; 88: 261-267Crossref PubMed Scopus (73) Google Scholar, 55Hofman F.M. Wright A.D. Dohadwala M.M. 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Knudsen B. Magnuson D.S. Haughey N. Geiger J.D. J. Virol. 1996; 70: 1475-1480Crossref PubMed Google Scholar, 80Strijbos P.J. Zamani M.R. Rothwell N.J. Arbuthnott G. Harkiss G. Neurosci Lett. 1995; 197: 215-218Crossref PubMed Scopus (24) Google Scholar, 81Weeks B.S. Lieberman D.M. Johnson B. Roque E. Green M. Loewenstein P. Oldfield E.H. Kleinman H.K. J. Neurosci. Res. 1995; 42: 34-40Crossref PubMed Scopus (67) Google Scholar). When injected into the brains of mice, Tat can stimulate edema and gliosis (82Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-529Crossref PubMed Scopus (133) Google Scholar). Here, we provide the first evidence that Tat inhibits the proliferation of glioblastoma cells, which are similar in many respects to activated astrocytes. By altering the cellular pathways involved in regulating astrocyte proliferation, Tat has the potential to disrupt the supportive function of astrocytes and contribute to the neuronal loss associated with ADC.Cellular proliferation is regulated by a series of positive and negative phosphorylation events, many of which involve cyclins and cyclin-dependent kinases. The progression of cells from G1 to S phase relies primarily on the cyclin D-Cdk4, cyclin D-Cdk6, and cyclin E-Cdk2 complex subunits (34Hunt T. Semin. Cell. Biol. 1991; 2: 213-222PubMed Google Scholar, 35Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 36Pines J. Trends Biochem. Sci. 1993; 18: 195-197Abstract Full Text PDF PubMed Scopus (405) Google Scholar, 37Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1986) Google Scholar). In this study, we demonstrate that Tat may block cells in the G1 phase by inhibiting the kinase activity of Cdk2 in astrocytic cells. The dissociation between the cyclin E- and Cdk2-associated kinase activity may also contribute to the disruption in the cell cycle. Other groups have demonstrated that Tat may inhibit the proliferative response of T-lymphocytes to antigenic and mitogenic stimuli (16Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Crossref PubMed Google Scholar, 18Lachgar A. Bernard J. Bizzini B. Astgen A. Le Coq H. Fouchard M. Chams V. Feldman M. Burny A. Zagury J.F. Biomed. Pharmacother. 1996; 50: 13-18Crossref PubMed Scopus (6) Google Scholar, 20Patki A.H. Lederman M.M. Cell. Immunol. 1996; 169: 40-46Crossref PubMed Scopus (31) Google Scholar, 23Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (317) Google Scholar). This negative response is associated with decreased IL-2 production (21Puri R.K. Leland P. Aggarwal B.B. Aids Res. Hum. Retroviruses. 1995; 11: 31-40Crossref PubMed Scopus (35) Google Scholar). IL-2 decreases the expression of the Cdk inhibitor p27 that inactivates the kinase activity of the cyclin E-Cdk2 complex (83Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (903) Google Scholar). Thus, it appears that Tat targets the same cellular pathway in cells of astrocytic and lymphocytic origin to inhibit cell growth. It should be noted that Tat has been associated with increased cellular proliferation in T cells (78Zauli G. La Placa M. Vignoli M. Re M.C. Gibellini D. Furlini G. Milani D. Marchisio M. Mazzoni M. Capitani S. J. Acquired Immune Defic. Syndrome Hum. Retrovirol. 1995; 10: 306-316Crossref PubMed Scopus (68) Google Scholar). Although the reasons for this discrepancy have not been elucidated, it is possible that it may depend on the culture conditions and amount of Tat used in the assays.The retinoblastoma susceptibility gene product is one of the targets of the cyclin E-Cdk2 complex (39Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (221) Google Scholar). The decrease in phosphorylated Rb detected in Tat-treated glioblastoma cells is therefore likely to be associated with the diminished cyclin E-Cdk2 kinase activity in these cells. The decreased phosphorylation of Rb in Tat-treated cells has implications for HIV-1 gene expression, since the underphosphorylated form of Rb stimulates HIV-1 transcription. When Rb exists in this form, it can interact with E2F-1 and prevent it from modulating transcription (41Buchkovich K. Duffy L.A. Harlow E. Cell. 1989; 58: 1097-1105Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 42Flemington E.K. Speck S.H. Kaelin Jr., W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6914-6918Crossref PubMed Scopus (285) Google Scholar, 43Hiebert S.W. Chellappan S.P. Horowitz J.M. Nevins J.R. Genes Dev. 1992; 6: 177-185Crossref PubMed Scopus (466) Google Scholar, 44Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Earlier studies demonstrated that the cell cycle regulatory protein, E2F-1, represses the activity of the HIV-1 promoter by binding to a site within the HIV-1 enhancer region and interacting with the 50-kDa subunit of NF-κB (p50) (32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). 4Kundu, M., Guermah, M., Roeder, R. G., Amini, S., and Khalili, K. (1997) J. Biol. Chem. 27229468–29474. Since the same region of the promoter is targeted by Rb, it is possible that Rb modulates HIV-1 transcription by binding to E2F-1 and inhibiting its repressive activity. It appears that by arresting cells in the G1 phase, Tat is able to maintain cells in a state that is favorable for HIV-1 transcription (Fig. 6).The observations presented in this study provide the first evidence that HIV-1, akin to the DNA tumor viruses, encodes regulatory proteins that manipulate host cell proliferation to promote viral advantage. Both HIV-1 and the DNA tumor viruses encode proteins that target the activity of the retinoblastoma protein, although to different ends. By preventing Rb phosphorylation, Tat blocks cellular proliferation at the G1 phase. By contrast, E1A, T-antigen, and E7 bind Rb and disrupt its interaction with E2F-1, which promotes entry of cells into the S phase (84Levine A.J. Annu. Rev. Biochem. 1993; 62: 623-651Crossref PubMed Scopus (475) Google Scholar). The phase of the cell cycle favored by HIV-1versus the DNA tumor viruses reflects one of the fundamental differences between these viruses: the nature of the genome. HIV-1 relies on the host cell transcription machinery for replication, because its genome consists of RNA. The genomes of the adenovirus, Simian virus 40, and human papilloma virus consist of DNA, so these viruses rely on the host cell DNA synthesis machinery for replication. Another interesting parallel is that both HIV-1 and adenovirus modulate the function of E2F-1 on their respective promoters to promote viral transcription. E1A promotes the release of E2F-1, which activates transcription of the adenovirus E2 promoter (44Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 85Cress W.D. Nevins J.R. Curr. Top. Microbiol. Immunol. 1996; 208: 63-78Crossref PubMed Scopus (55) Google Scholar). Tat, on the other hand, promotes sequestration of E2F-1, a negative regulator of HIV-1 transcription (32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). 4Kundu, M., Guermah, M., Roeder, R. G., Amini, S., and Khalili, K. (1997) J. Biol. Chem. 27229468–29474.Taken together, our data suggest that the complex interplay between virus and host, with respect to host cell cycle and HIV-1 replication, may promote HIV-1 gene expression in astrocytic cells. Furthermore, these interactions, by altering the state of astrocytes and stimulating the production of neurotoxic factors, could contribute to the pathogenesis of ADC. Neuropathological features of HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. infection include reactive astrogliosis, neuronal loss, widespread myelin pallor, subtle alteration of neocortical dendritic processes, and formation of multinucleated giant cells (1Everall I.P. Luthert P.J. Lantos P.L. Lancet. 1991; 337: 1119-1121Abstract PubMed Scopus (436) Google Scholar, 2Ketzler S. Weis S. Haug H. Budka H. 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