Tat activates human immunodeficiency virus type 1 transcriptional elongation independent of TFIIH kinase. (73/23412)

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of Cdk9 and cyclin T1, to the HIV-1 promoter via cooperative binding to the nascent HIV-1 transactivation response RNA element. The Cdk9 kinase activity has been shown to be essential for P-TEFb to hyperphosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II and mediate Tat transactivation. Recent reports have shown that Tat can also interact with the multisubunit transcription factor TFIIH complex and increase the phosphorylation of CTD by the Cdk-activating kinase (CAK) complex associated with the core TFIIH. These observations have led to the proposal that TFIIH and P-TEFb may act sequentially and in a concerted manner to promote phosphorylation of CTD and increase polymerase processivity. Here, we show that under conditions in which a specific and efficient interaction between Tat and P-TEFb is observed, only a weak interaction between Tat and TFIIH that is independent of critical amino acid residues in the Tat transactivation domain can be detected. Furthermore, immunodepletion of CAK under high-salt conditions, which allow CAK to be dissociated from core-TFIIH, has no effect on either basal HIV-1 transcription or Tat activation of polymerase elongation in vitro. Therefore, unlike the P-TEFb kinase activity that is essential for Tat activation of HIV-1 transcriptional elongation, the CAK kinase associated with TFIIH appears to be dispensable for Tat function.  (+info)

HIV-1 incidence among opiate users in northern Thailand. (74/23412)

The incidence of human immunodeficiency virus type 1 (HIV-1) infection among opiate users was determined in a retrospective cohort of 436 patients with multiple admissions to the only inpatient drug treatment program in northern Thailand between October 1993 and September 1995. During 323.4 person-years of follow-up, 60 patients presenting for detoxification acquired HIV-1 infection, for a crude incidence rate of 18.6 per 100 person-years (95% confidence interval 14.4-23.9). All seroconverters were male. HIV-1 incidence varied by the current route of drug administration: 31.3 per 100 person-years for injectors and 2.8 per 100 person-years for noninjectors (smoking and ingestion). Significant differences were found by ethnicity: HIV-1 incidence was 29.3 per 100 person-years for Thai lowlanders and 8.5 per 100 person-years for hill tribes. Multivariate relative risk estimates showed that injecting opiates (vs. use by other routes), being unmarried, being under age 40 years, being a Thai lowlander, having a primary and secondary education, and being employed in the business sector were each independently associated with human immunodeficiency virus seroconversion. This HIV-1 incidence rate is double that reported for Bangkok and suggests that prevention and control programs for drug users need to be expanded throughout Thailand. Improved availability of more-effective treatment regimens and increased access to sterile injection equipment are needed to confront the HIV-1 epidemic among opiate users in northern Thailand.  (+info)

Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor. (75/23412)

The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.  (+info)

U937 cells overexpressing bcl-xl are resistant to human immunodeficiency virus-1-induced apoptosis and human immunodeficiency virus-1 replication. (76/23412)

Many viruses, including human immunodeficiency virus type 1 (HIV-1), induce apoptosis and are affected by cellular expression of antiapoptotic genes. We sought to examine the effect of antiapoptotic gene expression on HIV replication by transfecting the promyelomonocytic cell line U937 with the bcl-xl gene to obtain clones of U937 cells that overexpressed bcl-xl (designated U937bcl-xl), a negative control U937 clone transfected with vector alone (designated U937neo) and a clone overexpressing bcl-2 (designated U937bcl-2). After infection with HIV-1, U937neo cells underwent apoptosis four times as frequently as the U937bcl-xl cells. Furthermore, U937bcl-xl cells produced 5-fold less HIV-1 protein than U937neo, whereas U937bcl-2 produced at least 2-fold more p24 than the U937neo control. Transient coexpression of bcl-2 or bcl-xl decreased HIV production and transcription from the HIV LTR. To define the mechanism by which bcl-xl, but not bcl-2, inhibits HIV expression, we examined bcl-2 and bcl-xl expression after HIV infection and CD4 cross-linking. Although HIV-1 infection or cross-linking CD4 led to a decrease in expression of bcl-2, it had no effect on bcl-xl expression. These results provide a mechanism for the resistance of U937bcl-xl transfectants, but not U937bcl-2 transfectants, to HIV-1 replication in monocytic cells in vitro. Therapies that up-regulate bcl-xl expression potentially provide a novel means to decrease the destructiveness of HIV-1.  (+info)

Characterization of a neutralization-escape variant of SHIVKU-1, a virus that causes acquired immune deficiency syndrome in pig-tailed macaques. (77/23412)

A chimeric simian-human immunodeficiency virus (SHIV-4) containing the tat, rev, vpu, and env genes of HIV type 1 (HIV-1) in a genetic background of SIVmac239 was used to develop an animal model in which a primate lentivirus expressing the HIV-1 envelope glycoprotein caused acquired immune deficiency syndrome (AIDS) in macaques. An SHIV-infected pig-tailed macaque that died from AIDS at 24 weeks postinoculation experienced two waves of viremia: one extending from weeks 2-8 and the second extending from week 18 until death. Virus (SHIVKU-1) isolated during the first wave was neutralized by antibodies appearing at the end of the first viremic phase, but the virus (SHIVKU-1b) isolated during the second viremic phase was not neutralized by these antibodies. Inoculation of SHIVKU-1b into 4 pig-tailed macaques resulted in severe CD4(+) T cell loss by 2 weeks postinoculation, and all 4 macaques died from AIDS at 23-34 weeks postinoculation. Because this virus had a neutralization-resistant phenotype, we sequenced the env gene and compared these sequences with those of the env gene of SHIVKU-1 and parental SHIV-4. With reference to SHIV-4, SHIVKU-1b had 18 and 6 consensus amino acid substitutions in the gp120 and gp41 regions of Env, respectively. These compared with 10 and 3 amino acid substitutions in the gp120 and gp41 regions of SHIVKU-1. Our data suggested that SHIVKU-1 and SHIVKU-1b probably evolved from a common ancestor but that SHIVKU-1b did not evolve from SHIVKU-1. A chimeric virus, SHIVKU-1bMC17, constructed with the consensus env from the SHIVKU-1b on a background of SHIV-4, confirmed that amino acid substitutions in Env were responsible for the neutralization-resistant phenotype. These results are consistent with the hypothesis that neutralizing antibodies induced by SHIVKU-1 in pig-tailed macaque resulted in the selection of a neutralization-resistant virus that was responsible for the second wave of viremia.  (+info)

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences. (78/23412)

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.  (+info)

The tat protein of HIV-1 induces galectin-3 expression. (79/23412)

Animal lectins play important roles in a variety of biological processes via their recognition of glycoconjugates. Galectin-3 is a beta-galactoside-binding lectin whose expression is associated with various pathological processes including human T lymphotropic virus (HTLV)-I-infection of human T cell lines and human immunodeficiency virus (HIV) infection of T-lymphoblastic Molt-3 cell line. In the case of HIV-infected cells, it has been suggested that the increase in galectin-3 expression could be related to the expression of the viral regulatory gene tat. These results prompt us to perform more extensive analyses of the relationship between galectin-3 and HIV-1 Tat expressions. In this study, we found that Tat protein expression induces an upregulation of galectin-3 in several human cell lines. In co-transfection experiments, the 5'-regulatory sequences of the galectin-3 gene were significantly upregulated by expression vectors encoding the Tat protein. Analysis performed with 5'-regulatory deleted sequences suggested that galectin-3 induction by Tat is dependent on activation of the Sp-1 binding transcription factor.  (+info)

Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. (80/23412)

Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors.  (+info)