Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription.
(73/23992)
Tat stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction. (+info)
Determinants of the natural history of human immunodeficiency virus type 1 infection.
(74/23992)
Variation in the time to AIDS and duration of survival of human immunodeficiency virus (HIV)-1-infected persons was recognized early in the epidemic. Recent studies have indicated that the rate of viral replication, as manifest by the number of copies of HIV RNA per milliliter of plasma, is a major determinant of outcome in an infected person. The predictive power of the measurement of plasma HIV RNA copy number is enhanced by combining this result with the CD4 lymphocyte number. The determinants of the rate of viral replication are less clearly defined. Recent studies suggest that polymorphism of the chemokine receptors, required for cellular infection, plays a role in regulating the rate of viral replication. The subsequent adaptive evolution of HIV-1 to the host's immune response is a consequence of this dynamic of the virus. Complicating opportunistic infections also appear to enhance HIV-1 replication, while antiviral therapy, in contrast, can and does suppress viral replication. (+info)
U937 cells overexpressing bcl-xl are resistant to human immunodeficiency virus-1-induced apoptosis and human immunodeficiency virus-1 replication.
(75/23992)
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)
Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus.
(76/23992)
A cDNA encoding the human guanylate binding protein-1 (hGBP-1) was expressed in HeLa cells using a constitutive expression vector. Stably transfected clones expressing hGBP-1 exhibited resistance to the cytopathic effect mediated by both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) and produced less viral progeny than control cells following infection with these viruses. To study the role hGBP-1 plays in the IFN-mediated antiviral effect, cells were stably transfected with a construct expressing antisense RNA for hGBP-1. VSV infection of IFN-alpha-treated antisense RNA-expressing cells produced an amount of virus comparable to that produced in the parental cell line, while EMCV infection of the IFN-alpha-treated transfected cells and VSV and EMCV infection of the IFN-gamma-treated transfected cells produced far more virus than was produced in the parental cell line. These results demonstrate that GBP-1 mediates an antiviral effect against VSV and EMCV and plays a role in the IFN-mediated antiviral response against these viruses. (+info)
The host-cell architectural protein HMG I(Y) modulates binding of herpes simplex virus type 1 ICP4 to its cognate promoter.
(77/23992)
The productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site in in vitro assays and to enhance the activity of this protein in short-term transient expression assays. (+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.
(78/23992)
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)
Kinetics of transcription of human cytomegalovirus chemokine receptor US28 in different cell types.
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In permissive cells, human cytomegalovirus encodes the protein US28, a functional CC chemokine receptor. US28 polyadenylated mRNA could be detected by RT-PCR as early as 2 h post-infection. US28 mRNA appeared after major IE1 transcripts (UL123), but before transcripts of the early genes pp65 (UL83) and gB (UL55), and the late gene pp150 (UL32). This temporal appearance indicates that US28 is transcribed earlier than previously reported. Furthermore, US28 mRNA could be detected in semi- and non-permissive cells. (+info)
Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) via increased NF-kappaB-mediated transcription.
(80/23992)
Virus infections, the majority of which are rhinovirus infections, are the major cause of asthma exacerbations. Treatment is unsatisfactory, and the pathogenesis unclear. Lower airway lymphocyte and eosinophil recruitment and activation are strongly implicated, but the mechanisms regulating these processes are unknown. Intercellular adhesion molecule-1 (ICAM-1) has a central role in inflammatory cell recruitment to the airways in asthma and is the cellular receptor for 90% of rhinoviruses. We hypothesized that rhinovirus infection of lower airway epithelium might induce ICAM-1 expression, promoting both inflammatory cell infiltration and rhinovirus infection. We therefore investigated the effect of rhinovirus infection on respiratory epithelial cell ICAM-1 expression and regulation to identify new targets for treatment of virus-induced asthma exacerbations. We observed that rhinovirus infection of primary bronchial epithelial cells and the A549 respiratory epithelial cell line increased ICAM-1 cell surface expression over 12- and 3-fold, respectively. We then investigated the mechanisms of this induction in A549 cells and observed rhinovirus-induction of ICAM-1 promoter activity and ICAM-1 mRNA transcription. Rhinovirus induction of ICAM-1 promoter activity was critically dependent upon up-regulation of NF-kappaB proteins binding to the -187/-178 NF-kappaB binding site on the ICAM-1 promoter. The principal components of the rhinovirus-induced binding proteins were NF-kappaB p65 homo- or heterodimers. These studies identify ICAM-1 and NF-kappaB as new targets for the development of therapeutic interventions for virus-induced asthma exacerbations. (+info)