Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

ARF GTPases and their ubiquitous role in intracellular trafficking beyond the Golgi

Molecular switches of the ADP-ribosylation factor (ARF) GTPase family coordinate intracellular trafficking at all sorting stations along the secretory pathway, from the ER-Golgi-intermediate compartment (ERGIC) to the plasma membrane (PM). Their GDP-GTP switch is essential to trigger numerous processes including membrane deformation, cargo sorting and recruitment of downstream coat proteins and effectors such as lipid modifying enzymes. While ARFs (in particular ARF1) had mainly been studied in the context of coat protein recruitment at the Golgi, COPI/clathrin independent roles have emerged in the last decade. Here we review the roles of human ARF1-5 GTPases in cellular trafficking with a particular emphasis on their roles in post-Golgi secretory trafficking and in sorting in the endo-lysosomal system