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Fusion seems unlikely given the absence of any virus-encoded proteins in the quasi-envelope (McKnight et al

Fusion seems unlikely given the absence of any virus-encoded proteins in the quasi-envelope (McKnight et al., 2017), whereas the selective targeting of eHAV to lysosomes suggests that the quasi-envelope, despite SL251188 being stable at pH 5.0, might be degraded by cholesterol transporter proteins and hydrolytic enzymes expressed within late endosomes and lysosomes (Feng et al., 2013; Kolter and Sandhoff, 2010). 2B. Abstract Many non-enveloped viruses, including hepatitis A computer virus (HAV), are released non-lytically from infected cells as infectious, quasi-enveloped virions cloaked in host membranes. Quasi-enveloped HAV (eHAV) mediates stealthy cell-to-cell spread within the liver, whereas stable naked virions shed in feces are optimized for environmental transmission. eHAV lacks virus-encoded surface proteins, and how it enters cells is unknown. We show both virion types enter by clathrin- and dynamin-dependent endocytosis, facilitated by integrin 1, and traffic through early and late endosomes. Uncoating of naked virions occurs in late endosomes, whereas eHAV undergoes ALIX-dependent trafficking to SL251188 SL251188 lysosomes where the quasi-envelope is usually enzymatically degraded and uncoating ensues coincident with breaching of endolysosomal membranes. Neither virion requires PLA2G16, a phospholipase essential for access of other picornaviruses. Thus naked and quasi-enveloped virions enter via comparable endocytic pathways, but uncoat in different compartments and release their genomes to the cytosol in a manner mechanistically unique from other also reduced both eHAV and HAV uptake and spread in H1-HeLa cells (Physique 1F,G). Consistent with these results, pre-treating Huh-7.5 cells with an RGD peptide made up of an integrin 1-binding motif reduced uptake of both virion types by about 50% (Determine 1H). On the other hand, pre-treating cells with antibodies that activate integrin 1 by binding to and stabilizing specific 1 conformations (Su et al., 2016) increased viral uptake compared to an inert integrin 1 antibody (K-20), SL251188 and revealed differences in the conversation of integrin 1 with eHAV versus HAV (Physique 1H). The activating antibody TS2/16, which binds an open conformation of 1 1 (Su et al., 2016), enhanced eHAV but not HAV access, whereas 8E3 and HUTS-4, which bind extended and open headpiece 1 conformations, respectively, had the opposite effect, enhancing naked HAV but not quasi-enveloped eHAV access. These data hint at Rabbit Polyclonal to RAB18 differences in the ligands, yet to be recognized, that are bound by integrin 1 during eHAV and HAV access. In contrast to the impact of integrin 1 depletion, depletion experiments failed to confirm a requirement for any specific integrin in the uptake of either virion (Physique 1D, Physique 1figure product 2B). While RNAi-mediated depletion of integrin 1 caused a modest but statistically significant decrease in HAV uptake in Huh-7.5 cells, this was not confirmed in H1-HeLa cells with CRISPR/Cas9 knockout of (Determine 1D, Determine 1figure supplement 3). Confocal microscopic imaging also suggested eHAV was associated with integrin 1, both at the surface of Huh-7.5 cells at 4C and during virion internalization at 37?C (Physique 1I), but not with either 5 or V integrins (Physique 1E, Physique 1figure product 4). Collectively, these results demonstrate that HAV and eHAV are dependent on unique integrin 1 interactions for uptake by clathrin- and dynamin-mediated endocytosis, but leave unanswered the role of integrins. Distinct intracellular trafficking routes for naked and quasi-enveloped HAV Several GTPases are well-known for their SL251188 role in the sorting of cargo through functionally unique endosomes, with Rab5A and Rab7a involved in trafficking through early and late endosomes, respectively (Mellman, 1996; Mercer et al., 2010). Confocal microscopy of infected Huh-7.5 cells revealed transient co-localization of the capsid antigen in both naked and quasi-enveloped virions with Rab5A+ and Rab7a+ compartments around~1C2 hpi (Determine 2A). In contrast, neither type of virion was associated with Rab11A+ recycling endosomes. RNAi-mediated depletion of Rab5A or Rab7a, but not Rab11A, resulted in a significant reduction in the accumulation of intracellular HAV RNA (Physique 2B, Physique 2figure product 1). Thus, both types of HAV virions traffic through early and late endosomes shortly after uptake into the cell through clathrin-mediated endocytosis. Open in a separate window Physique 2. Distinct endocytic sorting of naked and quasi-enveloped HAV.(A) Confocal micrographs of Huh-7.5 cells immunolabeled with anti-HAV capsid (K24F2) and anti-Rab5A, Rab7a, or Rab11A at two hpi. Level bar, 10 m. (B) Effect of siRNA-mediated depletion of Rab GTPases on HAV and eHAV access.