Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response

We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-kappaB is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response.     

Role of cyclophilin B in activation of interferon regulatory factor-3

IRF-3 is a member of the interferon regulatory factors (IRFs) and plays a principal role in the induction of interferon-beta (IFN-beta) by virus infection. Virus infection results in the phosphorylation of IRF-3 by IkappaB kinase epsilon and TANK-binding kinase 1, leading to its dimerization and association with the coactivators CREB-binding protein/p300. The IRF-3 holocomplex translocates to the nucleus, where it induces IFN-beta. In the present study, we examined the molecular mechanism of IRF-3 activation. Using bacterial two-hybrid screening, we isolated molecules that interact with IRF-3. One of these was cyclophilin B, a member of the immunophilins with a cis-trans peptidyl-prolyl isomerase activity. A GST pull-down assay suggested that one of the autoinhibition domains of IRF-3 and the peptidyl-prolyl isomerase domain of cyclophilin B are required for the binding. A knockdown of cyclophilin B expression by RNA interference resulted in the suppression of virus-induced IRF-3 phosphorylation, leading to the inhibition of the subsequent dimerization, association with CREB-binding protein, binding to the target DNA element, and induction of IFN-beta. These findings indicate that cyclophilin B plays a critical role in IRF-3 activation.     

Hsp90 regulates activation of interferon regulatory factor 3 and TBK-1 stabilization in Sendai virus-infected cells

Interferon regulatory factor 3 (IRF3) plays a crucial role in mediating cellular responses to virus intrusion. The protein kinase TBK1 is a key regulator inducing phosphorylation of IRF3. The regulatory mechanisms during IRF3 activation remain poorly characterized. In the present study, we have identified by yeast two-hybrid approach a specific interaction between IRF3 and chaperone heat-shock protein of 90 kDa (Hsp90). The C-terminal truncation mutant of Hsp90 is a strong dominant-negative inhibitor of IRF3 activation. Knockdown of endogenous Hsp90 by RNA interference attenuates IRF3 activation and its target gene expressions. Alternatively, Hsp90-specific inhibitor geldanamycin (GA) dramatically reduces expression of IRF3-regulated interferon-stimulated genes and abolishes the cytoplasm-to-nucleus translocation and DNA binding activity of IRF3 in Sendai virus-infected cells. Significantly, virus-induced IRF3 phosphorylation is blocked by GA, whereas GA does not affect the protein level of IRF3. In addition, TBK1 is found to be a client protein of Hsp90 in vivo. Treatment of 293 cells with GA interferes with the interaction of TBK1 and Hsp90, resulting in TBK1 destabilization and its subsequent proteasome-mediated degradation. Besides maintaining stability of TBK1, Hsp90 also forms a novel complex with TBK1 and IRF3, which brings TBK1 and IRF3 dynamically into proximity and facilitates signal transduction from TBK1 to IRF3. Our study uncovers an essential role of Hsp90 in the virus-induced activation of IRF3.     

Identification of novel alternative splicing variants of interferon regulatory factor 3

Interferon regulatory factor 3 (IRF-3) plays a crucial role in host defense against viral and microbial infection as well as in cell growth regulation. IRF-3a is the only structurally and functionally characterized IRF-3 splicing variant and has been established to antagonize IRF-3 activity. Here, five novel splicing variants of IRF-3, referred to as IRF-3b, -3c, -3d, -3e, and -3f, were identified and shown to be generated by deletion of exons 2, 3, or 6 or some combination thereof. RT-PCR examination revealed that these novel splicing variants were more frequently expressed in human liver, esophagus, and cervical tumor tissues than in their normal counterparts. Additionally, electrophoretic mobility shift assay and subcellular localization showed only IRF-3 and IRF-3e were capable of binding the PRDI/III element of interferon-beta (IFNβ) promoter in vitro and underwent cytoplasm-to-nucleus translocation following Poly(I:C) stimulation. Coimmunoprecipitation assay revealed that only IRF-3c (3f) of novel splicing variants associated with IRF-3 in vivo. Further luciferase assay showed IRF-3c (3f) and IRF-3e failed to transactivate PRDI/III-containing promoter but appeared to inhibit transactivation potential of IRF-3 to varying degrees. Taken together, our findings suggest novel splicing variants may function as negative modulators of IRF-3 and may be correlated with pathogenesis of human tumors.