Inhibition of human immunodeficiency virus type-1 by cdk inhibitors

Current therapy for human immunodeficiency virus (HIV-1) infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI) r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202) in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

<p>Abstract</p> <p>Background</p> <p>Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression.</p> <p>Results</p> <p>Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction <it>in vitro</it>. Analysis of the effect of NF90ctv-TAR RNA interaction <it>in vivo </it>showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene.</p> <p>Conclusion</p> <p>Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1.</p

The structure of the wild type TAR RNA and the TAR mutants used in this assay are illustrated

<p><b>Copyright information:</b></p><p>Taken from "Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1"</p><p>http://www.retrovirology.com/content/4/1/41</p><p>Retrovirology 2007;4():41-41.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1910605.</p><p></p> 500 ng of protein from the TAR Fraction containing NF90 was incubated with 0.2 pmole radiolabeled TAR RNA (lanes 2, 6, 10, 14, 18). Competition for radiolabeled TAR RNA binding was done with increasing amounts of unlabeled TAR RNA (lanes 3, 5), TM12 RNA (lanes 7, 9), TM18 RNA (lanes 11–13), TM27 RNA (lanes 15–17), or TM12+TM27 RNAs (lanes 19–21). Samples were run on a 10%PAGE

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1-3

<p><b>Copyright information:</b></p><p>Taken from "Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1"</p><p>http://www.retrovirology.com/content/4/1/41</p><p>Retrovirology 2007;4():41-41.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1910605.</p><p></p>lls transduced with empty vector (pOZ, lanes 2,3) infected with HIV-1pNL4-3 or pseudotyped VSVG-HIV-1 for single round infection (lanes 6,7), was fractionated on a 1% forlmadehyde-agarose gel and probed with [32p]-labeled HIV-1LTR probe. Shown in Figure 4A is a Northern blot of HIV-1 p NL4-3 and VSVG-HIV infected and non-infected cells (designated as plus (+) or minus (-) respectively. Lane 1 is positive control HIV RNA from ACH2 cells. Figure 4B shows the same gel probed with β-actin. Figure 4C illustrates the ethidium bromide-stained gel shown in Figure 4A and 4B

Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1-4

<p><b>Copyright information:</b></p><p>Taken from "Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1"</p><p>http://www.retrovirology.com/content/4/1/41</p><p>Retrovirology 2007;4():41-41.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1910605.</p><p></p> Tat protein for 10 minutes on ice. Next, 100 μl of 30% strepavidin sepharose beads in binding buffer (50 mM Tris-HCl, pH7.8; 5 mM DTT, 100 μg of BSA, 60 mM KCl and 5 mM MgCl) were added to the reaction for a final volume of 200 μl. The TAR/Tat complex was incubated with beads for an additional 1 hr on ice. Next, purified NF90c at various concentrations (0.1, 1, and 5 μg) were added to the mixture. All samples were further incubated on ice for additional one hour. Finally, samples were centrifuged at 4°c for 5 minutes, and washed (3×) with TNE+ 0.1% NP-40 (50 mM Tris-HCl, pH7.8, 300 mM NaCl, 1 mM ETDA, plus 0.1% NP-40). A final wash was with TNE+ 0.1% NP-40 was performed. Bound complexes were separated on a 4–20% SDS/PAGE and western blotted with anti-Tat mAb or anti-NF90c antibodies. Same Blot was cut in half for either Tat or NF90c western blot. Lane 1: C protein molecular weight marker, Lane 2 is with no Tat, Lane 3 with Tat, Lane 4 and 5 are with addition of five microgram of either wild type TAR or a mutant TAR RNA (TM26) as specific and non-specific competitors, respectively. Lanes 6–8 represent addition of purified NF90ctv protein in presence of constant amount of Tat

Cannabinoids Reduce Extracellular Vesicle Release from HIV-1 Infected Myeloid Cells and Inhibit Viral Transcription

Of the 37.9 million individuals infected with human immunodeficiency virus type 1 (HIV-1), approximately 50% exhibit HIV-associated neurocognitive disorders (HAND). We and others previously showed that HIV-1 viral RNAs, such as trans-activating response (TAR) RNA, are incorporated into extracellular vesicles (EVs) and elicit an inflammatory response in recipient na&iuml;ve cells. Cannabidiol (CBD) and &Delta;9-tetrahydrocannabinol (THC), the primary cannabinoids present in cannabis, are effective in reducing inflammation. Studies show that cannabis use in people living with HIV-1 is associated with lower viral load, lower circulating CD16+ monocytes and high CD4+ T-cell counts, suggesting a potentially therapeutic application. Here, HIV-1 infected U1 monocytes and primary macrophages were used to assess the effects of CBD. Post-CBD treatment, EV concentrations were analyzed using nanoparticle tracking analysis. Changes in intracellular and EV-associated viral RNA were quantified using RT-qPCR, and changes in viral proteins, EV markers, and autophagy proteins were assessed by Western blot. Our data suggest that CBD significantly reduces the number of EVs released from infected cells and that this may be mediated by reducing viral transcription and autophagy activation. Therefore, CBD may exert a protective effect by alleviating the pathogenic effects of EVs in HIV-1 and CNS-related infections

Anti-Vpr Activity of a Yeast Chaperone Protein

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G(2) arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients