SUMMARY As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell.

Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1͞2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1͞2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

The BTK (Bruton's tyrosine kinase) inhibitor ibrutinib is associated with an increased risk of bleeding. A previous study reported defects in collagen- and adenosine diphosphate (ADP)-dependent platelet responses when ibrutinib was added ex vivo to patient samples. Whereas the collagen defect is expected given the central role of BTK in glycoprotein VI signaling, the ADP defect lacks a mechanistic explanation. In order to determine the real-life consequences of BTK platelet blockade, we performed light transmission aggregometry in 23 patients receiving ibrutinib treatment. All patients had reductions in collagen-mediated platelet aggregation, with a significant association between the degree of inhibition and the occurrence of clinical bleeding or bruising (P=0.044). This collagen defect was reversible on drug cessation. In contrast to the previous ex vivo report, we found no in vivo ADP defects in subjects receiving standard doses of ibrutinib. These results establish platelet light transmission aggregometry as a method for gauging, at least qualitatively, the severity of platelet impairment in patients receiving ibrutinib treatment.

Members of the protein kinase C (PKC) family are expressed in many different cell types, where they are known to regulate a wide variety of cellular processes that impact on cell growth and differentiation, cytoskeletal remodelling and gene expression in the response to diverse stimuli. The broad tissue distribution and redundancy of in vitro function have often hampered the identification of definitive roles for each PKC family member. However, recent in vivo studies of PKC isoenzyme-selective knockout and transgenic mice have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies utilizing PKC isoenzyme-specific cDNA (wild-type, constitutively active and dominant-negative), antisense oligonucleotides (ASO), RNA interference (RNAi), and pharmacological inhibitors, indicate that PKC-regulated signalling pathways play a significant role in many aspects of immune responses, from development, differentiation, activation and survival of lymphocytes to macrophage activation. The importance of PKCs in cellular immune responses suggests that improved understanding of the molecular events that govern their actions could point to new avenues for development of treatments for immune disorders.

Chronic infection with hepatitis C virus (HCV) is an emerging global epidemic. The development of effective HCV antiviral therapeutics continues to be a daunting challenge owing to the absence of adequate animal models and tissue-culture systems for analysis and propagation of the virus. Despite these obstacles, inhibitors of the replicative elements of HCV, immune modulators and non-specific hepatoprotective agents are being pursued and exciting progress has been made. Successful therapeutic intervention of HCV will probably require combination approaches and new approaches, including host drug discovery targets.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) phosphoprotein has been intensely studied due to its ability to subvert the host interferon-induced antiviral response. However, more recent studies suggest that it may also play an important regulatory role in HCV RNA replication as well as modulate host intracellular signaling pathways. Phosphorylation of NS5A appears to be a highly regulated process and several cellular protein kinases responsible for NS5A phosphorylation have been identified in vitro. Studies utilizing the HCV replicon cell culture system have suggested a provocative role for the differential phosphorylation of NS5A in the regulation of viral RNA replication through its association with the viral replication complex, including several host cell factors. Importantly, recent in vivo data linking loss of NS5A hyperphosphorylation to non-productive HCV replication in the chimpanzee model have provided high validation for targeting the cellular kinases involved, particularly the kinases responsible for NS5A phosphorylation, for antiviral therapeutic intervention. Understanding the process of NS5A phosphorylation and the definite identification of the culprit cellular protein kinase(s) will shed light on the mechanisms of HCV RNA replication and/or pathogenesis.

The protein kinase Cθ (PKCθ) serine/threonine kinase has been implicated in signaling of T cell activation, proliferation, and cytokine production. However, the in vivo consequences of ablation of PKCθ on T cell function in inflammatory autoimmune disease have not been thoroughly examined. In this study we used PKCθ-deficient mice to investigate the potential involvement of PKCθ in the development of experimental autoimmune encephalomyelitis, a prototypic T cell-mediated autoimmune disease model of the CNS. We found that PKCθ−/− mice immunized with the myelin oligodendrocyte glycoprotein (MOG) peptide MOG35–55 were completely resistant to the development of clinical experimental autoimmune encephalomyelitis compared with wild-type control mice. Flow cytometric and histopathological analysis of the CNS revealed profound reduction of both T cell and macrophage infiltration and demyelination. Ex vivo MOG35–55 stimulation of splenic T lymphocytes from immunized PKCθ−/− mice revealed significantly reduced production of the Th1 cytokine IFN-γ as well as the T cell effector cytokine IL-17 despite comparable levels of IL-2 and IL-4 and similar cell proliferative responses. Furthermore, IL-17 expression was dramatically reduced in the CNS of PKCθ−/− mice compared with wild-type mice during the disease course. In addition, PKCθ−/− T cells failed to up-regulate LFA-1 expression in response to TCR activation, and LFA-1 expression was also significantly reduced in the spleens of MOG35–55-immunized PKCθ−/− mice as well as in in vitro-stimulated CD4+ T cells compared with wild-type mice. These results underscore the importance of PKCθ in the regulation of multiple T cell functions necessary for the development of autoimmune disease.

Objective. Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor ␣ (TNF␣) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNF␣ and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. Methods. Cultures of human fibroblast-like synoviocytes (FLS