Infectious bronchitis virus attaches to lipid rafts and enters cells via clathrin mediated endocytosis

Abstract

Due to its economic importance to in poultry industry, the biology and pathogenesis of infectious bronchitis virus (IBV) have been investigated extensively. However, the molecular mechanisms involved in IBV entry are not well characterized. In this study, systematic approaches were used to dissect IBV entry process in various susceptible cells. First, we observed that lipid rafts were involved in IBV attachment. Second, low pH in intracyplasmic vesicles was required for virus entry. By using the specific clathrin mediated endocytosis (CME) inhibitor or knock down of clathrin heavy chain (CHC), we demonstrated that IBV mainly utilized the CME for its entry. Furthermore, GTPase dynamin1 was involved in virus containing vesicle scission and internalization. Surprisingly, CME adaptor Eps15 had no effect on IBV internalization. Third, the penetration of IBV into cells led to active cytoskeleton rearrangement. After internalization, virus particles moved along with the classical endosome/lysosome track, as evidenced by co-localization of R18 labeled IBV with vehicle markers Rab5/Rab7/LAMP1 along with the infection time course. Functional inactivation of Rab5 and Rab7 significantly inhibited IBV infection. VCP, a protein helps early endosome maturation, was involved virus trafficking. Finally, by using the dual R18/DiOC labeled IBV, we observed that membrane fusion with late endosome/lysosome membranes was induced between 2-3 h.p.i.. Taken together, our findings demonstrate that IBV virions attach to lipid rafts and are internalized into cells via CME, move along with early/late endosomes-lysosomes, finally fuse with late endosome-lysosome membranes, release virus genome into cytoplasm. This study provides comprehensive images of IBV attachment-internalization-trafficking-fusion steps.\n\nIMPORTANCEIBV, the avian coronavirus isolated in 1937, infects chicken and causes economic loss in poultry industry. It has been reported that the entry of IBV requires low pH. However, the molecular mechanisms underlying IBV internalization and trafficking remain to be clarified. Therefore, we employed multiple chemical and molecular approaches to dissect the entry mechanisms of IBV in susceptible cells. Our results showed IBV entry was significantly inhibited when clathrin-mediated endocytosis (CME) was blocked by chemical inhibitor or depletion of clathrin protein. Moreover, by using R18-labeled IBV, we found that IBV particles attached to lipid rafts, led to actin rearrangement, and moved along with the entire endosomal system. R18/DiOC labeling method showed that IBV fused with late endosomes or lysosomes. This is the first report to describe the entire entry process of IBV, allowing for a better understanding of the infection process of group III avian coronavirus.