Hepatitis C Virus Suppresses Autophagosome Degradation by Arl8b-mediated Lysosomal Positioning

HCV infection increases steady-state autophagosome numbers but the mechanism of this effect is poorly understood. Flux assays suggest that an increase in autophagosomes results from a block in the fusion of autophagosomes with lysosomes. The aim of this study was to determine how HCV prevents autophagosome degradation. Autophagy induction and flux were measured by a flux assay involving western blotting for LC3-II after addition of the lysosomal inhibitor, bafilomycin A1. Flux through the autophagy pathway is greatly diminished compared to uninfected cells. To determine if HCV suppressed autophagosome-lysosome fusion events, control and HCV-infected cells were transfected with tandem RFP-GFP-LC3, where the quenching of the GFP fluorescence indicates autophagolysosome formation. In HCV-infected cells, there was no loss of GFP fluorescence from puncta indicating lack of fusion between autophagosomes and lysosomes even under conditions that activate autophagy. We then assessed the activity and maturation of lysosomal hydrolases from HCV-infected cells and determined that HCV did not decrease global lysosomal proteolytic activity. We next performed fusion assays with isolated subcellular organelles to assess whether lack of fusion was intrinsic to autophagosomes and lysosomes themselves. Vesicles isolated from HCV-infected cells fused with each other normally in vitro suggesting that the cellular fusion defect resulted from trafficking rather than inability of vesicles to fuse. To test this hypothesis, we studied organelle positioning focusing on Arl8b, an Arf-like GTPase that specifically localizes to lysosomes. In control cells, Arl8b was primarily found in a perinuclear localization and co-localized with LC3-positive autophagosomes. In JFH-1-infected cells, Arl8b localization was more diffuse and peripheral and there was a complete failure of Arl8b to co-localize with LC3, even after autophagy induction. Since Arl8b has the ability to link lysosomes to the outwardly-directed motor protein kinesin, we examined the effect of HCV on Arl8b itself. After HCV infection, mRNA and protein levels of Arl8b were both elevated three-fold. Lentiviral-mediated knockdown of Arl8b in infected cells restored autophagic flux to levels seen in control cells. In conclusion, HCV suppresses autophagic flux and increases the steady-state levels of autophagosomes by increasing the expression of Arl8b. This GTPase links lysosomes to kinesin, repositions them toward the cell periphery and suppresses their ability to fuse with autophagosomes. These trafficking changes may promote the HCV lifecycle

An Analysis of the Factors that Influence Vaccination Rates

Gemstone Team IPOVDue to the current rise of the vaccine hesitancy movement, there has been an increase in vaccine-preventable disease outbreaks (Mnookin, 2011; Reich, 2016). Parental rationalizations for opting out of vaccinations vary; however, some of the more commonly cited rationalizations include concerns for their child’s safety, distrust of medical professionals, and protection of civil liberties and individual decision-making processes (Glanz et al., 2013). Team IPOV has added to the literature body by examining how parents’ levels of knowledge about diseases, vaccine beliefs, and trust in institutions, medical professionals, and vaccines influence their levels of vaccine hesitancy, while adding the additional scope of the varicella and influenza vaccines and diseases. A hierarchical linear regression test revealed that trust exhibits the highest marginal impact on vaccine acceptance, followed by beliefs, and then, knowledge. Thus, while all three factors provided significant predictive insight into parents’ levels of vaccination hesitancy, parents’ trust in the varicella and influenza vaccines appear to possess the most significant impact over parents’ levels of vaccine hesitancy. Consequently, in considering future methods of alleviating vaccine hesitancy and increasing herd immunity, it is important to consider the ways in which trust can be built for the varicella and influenza vaccines.Gemstone Team IPO