The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2␣ phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

IntroductionThe application of molecular technologies to identify proteins differentially expressed by transformed cells is providing large numbers of candidate antigens that can be potentially targeted to selectively eliminate tumor cells by cancer immunotherapy. 1,2 Efforts to vaccinate patients to such antigens have yielded some provocative results, but only a small subset of patients have demonstrated therapeutic responses, likely reflecting the many in vivo obstacles to generating potent responses to these proteins, particularly in patients with an established malignancy. 3 An alternative approach of isolating and expanding reactive T cells ex vivo followed by adoptive transfer into the patient circumvents many of these in vivo obstacles. Although this adoptive therapy approach has demonstrated significant clinical promise, 4 generating the large numbers of T cells required for adoptive therapy of cancer patients, particularly within the time constraints posed by progressive tumors, is often not feasible. Molecular technologies have now provided a means to more broadly capture the therapeutic potential of this treatment strategy. Genes encoding the ␣ and ␤ chains of a T-cell receptor (TCR) can be isolated from a T cell reactive with the antigen of interest and restricted to a defined HLA allele, inserted into a shuttle expression vector, and then introduced into large numbers of T cells of individual patients sharing the restricting allele and the targeted protein. 5 This approach is already being pursued clinically, 6 and the goal is to establish a library of such defined TCR genes that could provide reagents for treating a diverse set of patients and diseases. Multiple virus-and tumor-reactive TCR genes have already been successfully isolated and re-expressed in T cells, including TCR genes with specificity for HLA*0201 (HLA-A2)-restricted epitopes from melanoma antigens 7-9 and HLA-A2-and HLA*2402-restricted WT1-derived epitopes. 10,11 The avidity of a T cell for its target reflects many factors, including the affinity of the TCR for its cognate antigen 12 and the level of TCR expression. [13][14][15] One difficulty with the TCR-transfer approach is that the TCR-transduced T cells are often of lower avidity than the parental T cell from which the TCR was derived due to failure to achieve wild-type levels of TCR expression, which likely contributed to the limited efficacy observed in the recently reported clinical trial pursuing this strategy. 6 Thus, the TCR chains introduced into T cells need to be initially selected for appropriate affinity 10,16 and inserted into vectors that can achieve and maintain high-level expression. 17 However, even if these criteria are met, the introduced exogenous ␣ and ␤ chains can potentially assemble as pairs not only with each other but also with the endogenous TCR ␣ and ␤ chains, thereby reducing the number of appropriately matched exogenous ␣␤TCR pairs at the cell surface and decreasing the achievable T-cell avidity. Such mismatched pairing poses a second substantive p...

Background Burnout is characterized by physical and emotional exhaustion from long-term exposure to emotionally demanding work. Burnout affects interpersonal skills, job performance, career satisfaction, and psychological health. However, little is known about the burden of burnout among healthcare providers in sub-Saharan Africa. Methods Relevant articles were identified through a systematic review of PubMed, Web of Science (Thomson Reuters), and PsycINFO (EBSCO). Studies were selected for inclusion if they examined a quantitative measure of burnout among healthcare providers in sub-Saharan Africa. Results A total of 65 articles met our inclusion criteria for this systematic review. Previous studies have examined burnout in sub-Saharan Africa among physicians (N = 12 articles), nurses (N = 26), combined populations of healthcare providers (N = 18), midwives (N = 2), and medical or nursing students (N = 7). The majority of studies assessed burnout using the Maslach Burnout Inventory. The highest levels of burnout were reported among nurses, although all healthcare providers showed high burnout. Burnout among healthcare providers is associated with their work environments, interpersonal and professional conflicts, emotional distress, and low social support. Conclusions Available studies on this topic are limited by several methodological challenges. More rigorously designed epidemiologic studies of burnout among healthcare providers are warranted. Health infrastructure improvements will eventually be essential, though difficult to achieve, in under-resourced settings. Programs aimed at raising awareness and coping with burnout symptoms through stress management and resilience enhancement trainings are also needed.