Tatyana Ammosova

Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription

BackgroundTranscription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.ResultsWe analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.ConclusionOur results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription

BackgroundHIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo.ResultsIn vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9.ConclusionOur results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription.

Iron Chelators of the Di-2-pyridylketone Thiosemicarbazone and 2-Benzoylpyridine Thiosemicarbazone Series Inhibit HIV-1 Transcription: Identification of Novel Cellular Targets—Iron, Cyclin-Dependent Kinase (CDK) 2, and CDK9

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670–7675, 2006; J Med Chem 50:3716–3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-κB-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs.

HIV-1 Tat interacts with LIS1 protein

BackgroundHIV-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.ResultsWe 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.ConclusionOur 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.

A novel anticancer agent ARC antagonizes HIV-1 and HCV

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) pose major public health concerns worldwide. HCV is clearly associated with the occurrence of hepatocellular carcinoma, and recently HIV infection has also been linked to the development of a multitude of cancers. Previously, we identified a novel nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that exhibited proapoptotic and antiangiogenic properties in vitro. Here, we evaluated the effect of ARC on HIV-1 transcription and HCV replication. Using reporter assays, we found that ARC inhibited HIV-1 Tat-based transactivation in different cell systems. Also, using hepatoma cells that harbor subgenomic and full-length replicons of HCV, we found that ARC inhibited HCV replication. Together, our data indicate that ARC could be a promising candidate for the development of antiviral therapeutics against HIV and HCV.

Regulation of HIV‐1 transcription at 3% versus 21% oxygen concentration

HIV transcription is induced by the HIV‐1 Tat protein, in concert with cellular co‐factors including CDK9, CDK2, NF‐κB, and others. The cells of most of the bodys organs are exposed to ∼3–6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV‐1 replication may differ at 3% versus 21% O2, and that such differences may affect HIV‐1 replication. Here we show that Tat‐induced HIV‐1 transcription was reduced at 3% O2 compared to 21% O2. HIV‐1 replication was also reduced in acutely or chronically infected cells cultured at 3% O2 compared to 21% O2. This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O2, the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF‐κB was not affected. CDK9‐specific inhibitor ARC was much less efficient at 3% compared to 21% O2 and also expression of CDK9/cyclin T1‐dependent IκB inhibitor α was repressed. Our results suggest that lower HIV‐1 transcription at 3% O2 compared to 21% O2 may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O2 and that additional host cell factors such as CDK2 and NF‐κB might be major regulators of HIV‐1 transcription at low O2 concentrations. J. Cell. Physiol. 221: 469–479, 2009.

1E7-03, a low MW compound targeting host protein phosphatase-1, inhibits HIV-1 transcription

HIV‐1 transcription is activated by the Tat protein which recruits the cyclin‐dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase‐1 (PP1) through the Q35VCF38 sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV‐1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV‐1 Tat interaction with PP1 and inhibits HIV‐1 gene transcription. Here, we report our further work with the 1H4‐derived compounds and analysis of their mechanism of action.

Inhibition of PP2A by LIS1 increases HIV-1 gene expression

BackgroundLissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR.ResultsTat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity.ConclusionOur results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A.

Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

BackgroundHIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1.ResultsHere we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9’s Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1.ConclusionWe identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.

Proteomic Profile of Tears for the Diagnosis of Uveitis Associated with Juvenile Idiopathic Arthritis: Setting Targets to Achieve Results

The paper presents epidemiologic and pathophysiological aspects of the problem statement for early recognition of Uveitis (Uv) associated with Juvenile Idiopathic Arthritis (JIA) in terms of the proteomic profile of tears as well as the results of an attempt to solve this problem by means of the Tandem Mass-Spectrometry (TMS). The solution of this problem is of the highest relevance due to revolutionary changes in treatment strategies after introducing highly effective biologics. Content analysis of literature reviews reveals the following: 1. the incidence of JIA-Uv in the Northwest Federal District of Russian Federation averages 0.5-0.7 per 100 000 of children with the prevalence being ten-fold higher than incidence, 2. without Methotrexate treatment 4-7 years after the diagnosis of JIA-Uv cataract is revealed in 35-40% of children and in 5% – glaucoma as well, 3. even with Methotrexate in 28-40% of children the complications of JIA-Uv inevitably will be revealed with blurred vision in 10-36% of children, 4. timely diagnosis of JIA-Uv and adequate treatment reduce the risk of complications by 4% per year, 5. current medical care system reveals in one third of children already the complications of JIA-Uv. Revelation in tears of the motif mode for protein interaction network, triggering mobilization/inhibition of cells which moderate Uv would contradict the traditional point of view on existing natural anatomic and physiologic barriers, isolating the intraocular space, but however seems to be possible since JIA is a systemic disease and Uv leads to damage of the blood-retinal barriers. To reveal protein biomarkers of JIA-Uv tears of 31 children aged 2-17 years were studied: 17 – chronic JIA-Uv, 4 – JIA without Uv, 4 – idiopathic Uv, 3 – systemic vasculitis, 3 – healthy children. We used the current clinical guidelines and standards to diagnose the pathology and TMS with hierarchical clustering methodology for protein identification: nano C18 column attached to Shimadzu nano LC coupled in-line to LTQ Orbitrap XL tandem mass spectrometer, data-dependent 4-event scan method, a survey FT-MS parent scan followed by sequential data dependent FT-MS/MS scans on the three most abundant peptide ions. Proteins were identified from the mass spectra results with Proteome Discoverer 1.2 software for protein database search using the International Protein Index (IPI) and Human Protein Database. Quantification was conducted using SIEVE 2.0 after normalization to albumin keeping in mind the validity of proportional change of its concentration after stimulation of lacri-mation. Data from SIEVE were exported to IPA (Ingenuity Pathway Analysis) for filtering. The extracellular proteins selected in Ingenuity were further analyzed for disease relation and networks formation. TMS revealed more than 3000 proteins in tears and 300 of them have been considered to be the first row candidates to be biomarkers of JIA-Uv. The top two proteins, lactoferrin and lipocalin were upregulated over ten-fold in children with Uv. Pathway analysis placed these proteins into the inflammation-related IL-1 and TNF-α related networks which also included proteins involved in the development of endothelial dysfunction, inflammation and retinopathy. In addition, IL-23, which was previously linked to Uveitis, was found to be upregulated. Taken together, our proof-of-principle study presents a novel and yet untested approach for detection of early biomarkers of Uveitis and identified several candidate proteins.