Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

Increasing evidence indicates that RNA interference (RNAi) may be used to provide antiviral immunity in mammalian cells. Human micro (mi)RNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN)-induced protein kinase R (PKR) but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si) and micro (mi)RNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit

Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro . Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages

Multiple levels of Protein Kinase R inhibition during Human Immunodeficiency Virus replication by double-stranded RNA binding proteins and its relationship to the weak innate cell response to viral infection

Following infection by most viruses, an antiviral state is induced in the host cells characterized by the expression of interferon (IFN) and several IFN-stimulated genes (ISGs). IFN treatment is effective to inhibit HIV replication in infected cells, but shows no significant improvement of HIV-infected patients. Currently, the discrepancy between the in vitro and the in vivo findings remains largely unresolved. The IFN-induced RNA-dependent protein kinase PKR is activated via trans-phosphorylation and plays a central role in the IFN-induced antiviral pathway. Our results show that PKR is transiently activated following HIV-1 infection of Jurkat and peripheral blood mononuclear cells. The kinase is then inactivated at the viral peak, when HIV replication is highly active. By immunoprecipitation, we found that PKR forms a ribonucleoprotein complex with cellular double-stranded RNA binding proteins (dsRBPs), the TAR RNA Binding Protein (TRBP), the adenosine deaminase acting on RNA (ADAR)1 and the PKR Activator (PACT) during HIV replication. Over-expression of PKR is sufficient to inhibit HIV production in HEK 293T cells. This inhibition is reversed by expression of the ADAR1, another ISG. By using mutants of ADAR1, we show that this activity is linked to the ability of the protein to bind PKR. In astrocytes that do not replicate HIV efficiently due to an enhanced PKR response, ADAR1 partially restores viral expression. Surprisingly, PACT binds to and inhibits PKR activity. All three dsRBPs, TRBP, ADAR1 and PACT prevent PKR activation and the phosphorylation of its downstream target, eIF2alpha. Together, our results highlight the key function of PKR in innate immunity and its multiple-level of regulation during HIV-1 replication.L'infection d'une cellule par un virus induit un état antiviral, caractérisé par l'expression de l'interféron (IFN) et de plusieurs gènes induits par l'IFN. Le traitement par l'IFN est efficace pour inhiber la réplication du virus de l'immunodéficience humaine (VIH) dans des cellules infectées en culture, mais ne montre aucun effet bénéfique chez les patients infectés par ce même virus. Cette disparité qui existe entre les résultats de recherche in vivo et in vitro n'est toujours pas résolue. PKR, une protéine kinase induite par les IFNs, est activée par phosphorylation et joue un rôle central dans le mécanisme antiviral de l'IFN. Nos résultats démontrent que PKR est activée de manière transitoire suite à l'infection de cellules lymphocytaires Jurkat ou de lymphocytes/monocytes primaires du sang périphérique par le VIH. Par contre, la kinase n'est plus activée durant et après le pic d'infection, lorsque la réplication du virus est intense. Par immunoprécipitation, nous avons démontré que PKR forme un complexe ribonucleoprotéique avec plusieurs protéines cellulaires qui lient l'ARN double-brin, soit la protéine liant l'ARN TAR, TRBP, l'adénosine déaminase ADAR, ainsi que la protéine activatrice de PKR, PACT, pendant la réplication virale. La surexpression de PKR est suffisante pour inhiber la production du VIH dans les cellules HEK 293T. Cette inhibition est supprimée par l'expression d'ADAR1, une des protéines induite par les IFNs. Par différentes mutations dans la séquence protéique d'ADAR1, nous avons démontré que cette activité d'ADAR est liée à sa capacité de lier PKR. Dans les astrocytes, qui ne répliquent pas le VIH efficacement en raison d'une activation accrue de PKR, ADAR1 rétablit partiellement l'expression virale. Étonnamment, PACT se lie à PKR et inhibe son activité dans les cellules infectées par le VIH. Testées en parallèle, TRBP, ADAR1 et PACT empêchent l'activation de PKR ainsi que la pho

Improvement of functioning in patients with schizophrenia: real-world effectiveness of aripiprazole once-monthly (REACT study)

Abstract Background Functional impairment affects many patients with schizophrenia. Treatment with the long-acting injectable antipsychotic aripiprazole once-monthly (AOM) may help improve functioning. Objectives To explore changes in functioning in patients with schizophrenia who received AOM treatment in observational studies. Methods Here we report functional outcomes in the form of Global Assessment of Functioning (GAF) scores in a pooled analysis of data from two observational studies from Canada (NCT02131415) and Germany (vfa non-interventional studies registry 15960N). Data from 396 patients were analyzed. Results At baseline, the mean GAF score was 47.7 (SD 13.4). During 6 months of treatment with AOM, the mean GAF score increased to 59.4 (SD 15.8). Subgroups stratified by patient age (≤35 years/>35 years), sex, disease duration (≤5 years/>5 years) and disease severity at baseline had all significantly improved their GAF at month 6. 51.5% of the patients showed a GAF score increase of at least 10 points, which was regarded as clinically meaningful, and were considered responders. Conclusions These data show that treatment with AOM may help improve patient functioning in a routine treatment setting. Trial registration NCT02131415 (May 6, 2014), vfa non-interventional studies registry 15960N

ADAR1 Interacts with PKR during Human Immunodeficiency Virus Infection of Lymphocytes and Contributes to Viral Replication▿

The interferon-induced protein kinase RNA activated (PKR) is activated after virus infection. This activation is transient during the human immunodeficiency virus type 1 (HIV-1) infection of lymphocytes, and the protein is not activated at the peak of infection. We observed that interferon-induced adenosine deaminase acting on RNA 1-p150 (ADAR1-p150) and ADAR1-p110 expression increases while the virus replicates actively. Furthermore, both forms of ADAR1 show enhanced interactions with PKR at the peak of HIV infection, suggesting a role for this protein in the regulation of PKR activation. We observed that ADAR1-p150, as previously shown for the TAR RNA binding protein (TRBP), reverses the PKR inhibition of HIV expression and production in HEK 293T cells. This activity requires the Z-DNA binding motif and the three double-stranded RNA binding domains but not the catalytic domain. In astrocytic cells, ADAR1-p150 increased HIV expression and production to an extent similar to that of TRBP. Small interfering RNAs against ADAR1-p150 moderately decreased HIV production. These results indicate that two interferon-induced proteins, ADAR1 and PKR, have antagonistic functions on HIV production. They suggest that ADAR1 and TRBP belong to a multiprotein complex that inhibits PKR during the HIV infection of lymphocytes

The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

Background: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. Results: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. Conclusion: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.</p