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

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

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

Cloning and sequence analysis of caprine N-acetylglucosamine 6-sulfatase cDNA

AbstractMucopolysaccharidosis BID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the capfne N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence