Interaction of human tRNA-dihydrouridine synthase-2 with interferon-induced protein kinase PKR

PKR is an interferon (IFN)-induced protein kinase, which is involved in regulation of antiviral innate immunity, stress signaling, cell proliferation and programmed cell death. Although a low amount of PKR is expressed ubiquitously in all cell types in the absence of IFNs, PKR expression is induced at transcriptional level by IFN. PKR's enzymatic activity is activated by its binding to one of its activators. Double-stranded (ds) RNA, protein activator PACT and heparin are the three known activators of PKR. Activation of PKR in cells leads to a general block in protein synthesis due to phosphorylation of eIF2α on serine 51 by PKR. PKR activation is regulated very tightly in mammalian cells and a prolonged activation of PKR leads to apoptosis. Thus, positive and negative regulation of PKR activation is important for cell viability and function. The studies presented here describe human dihydrouridine synthase-2 (hDUS2) as a novel regulator of PKR. We originally identified hDUS2 as a protein interacting with PACT in a yeast two-hybrid screen. Further characterization revealed that hDUS2 also interacts with PKR through its dsRNA binding/dimerization domain and inhibits its kinase activity. Our results suggest that hDUS2 may act as a novel inhibitor of PKR in cells

Bryostatin Modulates Latent HIV-1 Infection via PKC and AMPK Signaling but Inhibits Acute Infection in a Receptor Independent Manner

HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -α and -δ, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs

Bryostatin is non-toxic at its active concentration either alone or in combination with other anti-HIV compounds.

<p>Magi and Jurkat cells were treated with various therapeutic drugs (MF-Metformin (1 mM), Min-Minocycline (5 µg/ml and 10 µg/ml), Sim-Simvastatin (2.5 µM), and AZT (100 µM), S.Sali-Sodium salicylate (1 mM) either alone or in combination with bryostatin for 48 and 72 h and cell survival was determined using WST8/CCK8 cytotoxicity assay. (<b>A</b>) Survival of Magi cells after treatment with bryostatin and other drugs, (<b>B</b>) bryostatin in combination with other drugs. (<b>C</b>) Jurkat cell survival after treatment with bryostatin (25 ng/ml) and other drugs, (<b>D</b>) bryostatin in combination with other drugs. Each treatment was done in triplicate. (n = 2).</p

Bryostatin ablates R5-tropic HIV infection.

<p>Magi cells (CD4⁺/CCR5⁺) were infected with 100 ng/ml p24 equivalent HIV recombinant NLENYU2 virus expressing YFP. (<b>A</b>) Productive viral infection was monitored semi-quantitatively on 3rd and 5th dpi. (<b>B</b>) GFP-positive cells per 10 fields were counted in HIV-infected cultures and plotted as mean. (<b>C</b>) Dose dependent anti-HIV effect of bryostatin was monitored by p24 assay. (<b>D</b>) p24 levels in the culture supernatants after treatment with bryostatin (25 ng/ml) and other compounds on 3rd and 5th day post infection are shown. Sodium salicylate (1 mM) was used as a negative control in each set of experiments. Degree of significance for bryostatin treatment was relative to virus control, * p<0.05, # p<0.001. (n = 4).</p

PKCs regulate reactivation of latent HIV infection downstream of AMPK.

<p>(<b>A</b> and <b>B</b>) nPKC (novel PKC) inhibitor, rottlerin abrogated viral reactivation in THP-p89 cells after AMPK activation (metformin and AICAR). Activation of AMPK alone did not affect viral reactivation. (<b>C</b>) Bryostatin- and PMA-mediated activation of PKC led to complete degradation of PKC-α and PKC-δ compared to vehicle control. H7 dihydrochloride and rottlerin (positive controls) mediated inhibition of activation of classical and novel PKCs prevented the degradation of total PKCs. Metformin an AMPK activator had no effect on the PKC, however, the inhibitor of AMPK, compound-C (inhibits AMPK activation) rescued the PKC degradation or prevented PKC activation in 24 h.</p

Effect of bryostatin on T-cell activation.

<p>Human PBMCs depleted of monocytes were cultured for 3 days and either activated with PHA and IL-2 or treatment with bryostatin for 2 days, followed by immunostaining with fluorescently labeled human anti-CD25 and anti-CD69 antibodies and monitored by flowcytometry. Representative dot plots for PHA and IL-2 activation, isotype control and bryostatin treatment are shown (n = 2).</p

AMPK mediate reactivation of latent HIV infection via PKC.

<p>(<b>A</b>) GFP-expression in THP-p89 cells upon treatment with 10 µM Compound-C (CC), an AMPK inhibitor; 1 mM Metformin (MF) and AICAR (AMPK activators) either alone or in combination with bryostatin. Activation of AMPK alone does not show viral reactivation. (<b>B</b>) Quantification of THP-p89-reactivated GFP-positive cells by flowcytometry. (<b>C</b>) Western blots for phosphorylated AMPK-alpha subunit after treatment with bryostatin (25 ng/ml) were monitored at different time points. (<b>D</b>) Quantification of phospho-alpha and -beta subunit bands on western blots. Bryostatin treatment dephosphorylates AMPK-regulatory subunit β in a time dependent manner.</p

Bryostatin inhibits HIV-1 independent of viral receptors.

<p>(<b>A</b> and <b>B</b>) Hela cells were pretreated with bryostatin (27 nM), vehicle, and AZT as positive control and thereafter infected with VSV pseudotyped-NLENY1 HIV. Productive HIV replication was monitored using YFP expression after 3dpi. Results were significant at p<0.05 (<b>C</b>) Virus production was further confirmed by p24 levels in infected culture supernatants by quantitative ELISA. (<b>D</b>) Schematic representation of the experimental design of virus infection in HeLa cells and infectivity assay in TZM-bl cells is shown. (<b>E</b> and <b>F</b>) Virus infectivity in the bryostatin treated HIV-infected culture supernatants was monitored by infecting TZM-bl cells either with YFP expression as a marker or (<b>G</b>) viral p24 production after 3dpi. (<b>H</b> and <b>I</b>) Schematic representation of single round infection experiment, bryostatin mediated HIV inhibition was monitored by p24 assay in a single round infection on Hela cells using VSV pseudotyped HIV-NLR+E- (*p<0.05, #p<0.005) (n = 2).</p

Bryostatin reactivates latent HIV infection via activation of classical and novel PKCs.

<p>(<b>A</b> and <b>B</b>) THP-p89 cells were pretreated with bryostatin either alone or with broad spectrum PKC inhibitor H7 dihydrochloride or (<b>C</b> and <b>D</b>) different concentrations of classical PKC inhibitor GF109203X and novel PKC inhibitor Rottlerin as indicated and monitored for GFP fluorescence by flowcytometry. (<b>E</b>) Total PKCα activation at different time points post-treatment with bryostatin. (<b>F</b>) PKC-δ and PKC-α in replicate experiment was degraded in a time dependent manner within 72 h of treatment with bryostatin. (<b>G</b>) Quantitative profile of PKC-α and δ after normalization with beta actin.</p

Bryostatin synergizes Tat- and Nef-mediated LTR-transactivation.

<p>SVGA-LTR-GFP reporter cells were transfected with 2 ng Tat-expression vector (suboptimal dose) and increasing concentrations of Nef-expression vector. (<b>A</b>) Quantification of GFP-positive cells. (<b>B</b>) SVGA-LTR GFP cells were transfected with 2 ng Tat-expression vector and 100 ng Nef-expression vector in the presence or absence of bryostatin (25 ng/ml) followed by GFP quantification using flowcytometry. (<b>C</b>) Mean fluorescence intensity (MFI) for the same experiment (<b>B</b>). (n = 3).</p