HIV-1 Tat interacts with LIS1 protein

BACKGROUND: HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization. RESULTS: We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system. CONCLUSION: Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation

Small Molecules Targeted to a Non-Catalytic “RVxF” Binding Site of Protein Phosphatase-1 Inhibit HIV-1

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q35VCF38, which resembles the PP1-binding “RVxF” motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the “RVxF”-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the “RVxF”-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners

Protein Phosphatase-1 Activates CDK9 by Dephosphorylating Ser175

The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription

Tricuspid regurgitation velocity and other biomarkers of mortality in children, adolescents and young adults with sickle cell disease in the United States: The PUSH study

In the US, mortality in sickle cell disease (SCD) increases after age 18- 20- years. Biomarkers of mortality risk can identify patients who need intensive follow- up and early or novel interventions. We prospectively enrolled 510 SCD patients aged 3- 20- years into an observational study in 2006- 2010 and followed 497 patients for a median of 88- months (range 1- 105). We hypothesized that elevated pulmonary artery systolic pressure as reflected in tricuspid regurgitation velocity (TRV) would be associated with mortality. Estimated survival to 18- years was 99% and to 25- years, 94%. Causes of death were known in seven of 10 patients: stroke in four (hemorrhagic two, infarctive one, unspecified one), multiorgan failure one, parvovirus B19 infection one, sudden death one. Baseline TRV - ¥2.7 m/second (>2 SD above the mean in age- matched and gender- matched non- SCD controls) was observed in 20.0% of patients who died vs 4.6% of those who survived (P =- .012 by the log rank test for equality of survival). The baseline variable most strongly associated with an elevated TRV was a high hemolytic rate. Additional biomarkers associated with mortality were ferritin - ¥2000- μg/L (observed in 60% of patients who died vs 7.8% of survivors, P <- .001), forced expiratory volume in 1 minute to forced vital capacity ratio (FEV1/FVC) <0.80 (71.4% of patients who died vs 18.8% of survivors, P <- .001), and neutrophil count - ¥10x109/L (30.0% of patients who died vs 7.9% of survivors, P =- .018). In SCD children, adolescents and young adults, steady- state elevations of TRV, ferritin and neutrophils and a low FEV1/FVC ratio may be biomarkers associated with increased risk of death.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155951/1/ajh25799_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155951/2/ajh25799.pd

Genetic determinants of telomere length from 109,122 ancestrally diverse whole-genome sequences in TOPMed

Genetic studies on telomere length are important for understanding age-related diseases. Prior GWAS for leukocyte TL have been limited to European and Asian populations. Here, we report the first sequencing-based association study for TL across ancestrally-diverse individuals (European, African, Asian and Hispanic/Latino) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. We used whole genome sequencing (WGS) of whole blood for variant genotype calling and the bioinformatic estimation of telomere length in n=109,122 individuals. We identified 59 sentinel variants (p-value OBFC1indicated the independent signals colocalized with cell-type specific eQTLs for OBFC1 (STN1). Using a multi-variant gene-based approach, we identified two genes newly implicated in telomere length, DCLRE1B (SNM1B) and PARN. In PheWAS, we demonstrated our TL polygenic trait scores (PTS) were associated with increased risk of cancer-related phenotypes

HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation

Phosphorylated VP30 of marburg virus is a repressor of transcription

The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1

Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers

In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (\u3c22%) and accuracy (85-115% of true values) were obtained over a range of 1.0-100. μM for Bp4eT and 1.5-300. μM for Bp4aT. Limits of detection were 0.3. μM and 1. μM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1. μM and 5. μM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24. h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100. μM and 300. μM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery. © 2012 Elsevier B.V