Receptor Determinants And Entry Pathways Of Coronavirus And Influenza Virus
Avian coronavirus infectious bronchitis virus (IBV) and influenza virus are two respiratory viruses that have great impact on veterinary and human health globally. The two respiratory viruses described in this thesis share many commonalities on the molecular mechanisms of entry in host cells. Following cell surface attachment via receptor binding, these enveloped viruses need to be internalized into subcellular compartments to initiate fusion and uncoating. Although often studied in certain prototypical cell types, many details of attachment and entry pathways are overlooked in more in vivo-relevant cell types. Despite many speculations on its receptor usage, the authentic IBV receptor has not been discovered although the attachment factor sialic acid is documented. When I expressed DC-SIGN and L-SIGN, the C-type lectins, in mammalian cells, I discovered that IBV are able to infect these non-permissive cells that are usually refractory to IBV infection. In addition, the infection in DC-SIGN-expressing cells is independent on the level of sialic acid on the cell surface. When I examed whether sialic acid also plays a role in IBV infection of chicken peripheral blood derived monocytes (chPBMCs), cells that potentially harbor the authentic proteinaceous receptor for IBV, I unexpectedly found that the established attachment factor sialic acid does not play a critical role in the IBV infection in chPBMC. To further evaluate the entry pathways utilized by IBV in chPBMCs, I examined the IBV infection in the presence chemical inhibitors used to disrupt endocytic components. Using immunofluorescence microscopy, I identified that caveolae-dependent endocytosis and macropinocytosis pathways were used by IBV entry in chPBMCs. In a similar chemical and molecular approach, in the presence of endocytic inhibitors and dominant- negative proteins implicated in the endocytic pathways, I evaluated the influenza virus entry in polarized epithelial cells-- Madin-Darby canine kidney (MDCK) cells, a model for the cells at primary sites of influenza infection in vivo. My study showed that in polarized MDCK II cells, influenza virus has a differential utilization for CME pathway and requires Eps15 protein for entry In all, these studies of coronavirus and influenza virus entry provide a clearer understanding of the molecular mechanisms involved in enveloped virus entry into host cells. The results of this study will enrich our knowledge of enveloped virus pathogenesis.
Infectious bronchitis virus; Influenza; entry pathways
Whittaker, Gary R
Jin, Moonsoo; Parker, John Stuart Leslie
Ph.D. of Microbiology
Doctor of Philosophy
dissertation or thesis