| The avian coronavirus infectious bronchitis virus (IBV) expresses two small nonstructural proteins, 3a and 3b, during infection. These proteins are highly conserved among different IBV strains, suggesting they are important for infection. The goal of this thesis was to uncover the functions of these proteins. The initial characterization of IBV 3a by cell biological and biochemical methods demonstrated that a pool of IBV 3a localized to the cytoplasm while another pool associated with smooth endoplasmic reticulum (ER) membranes. The short length of IBV 3a appeared to preclude efficient association with signal recognition particle (SRP), causing inefficient co-translational insertion of IBV 3a into membranes. Thus, IBV may limit IBV 3a levels at smooth ER membranes by a novel mechanism. Additional cell biological, genetic, and functional assays excluded several smooth ER localizations for IBV 3a, demonstrated that IBV 3a was not essential for replication in cell culture, and showed that a virus lacking IBV 3a had no observable fitness defects in cell culture. However, results did indicate that IBV 3a alters the transcriptional activity of nuclear factor (NF) kappaB, suggesting that IBV 3a may influence the levels of cell-survival or immunomodulatory proteins that are important for host infection.; IBV 3b localized to the nucleus in mammalian cells when using a vaccinia virus expression system, suggesting that IBV 3b and the severe acute respiratory sydrome (SARS) coronavirus 3b protein (also a nuclear localized protein) might be functional homologs. However, IBV 3b localized to the cytoplasm with apparent nuclear exclusion in chicken cells. IBV 3b was undetectable via microscopy of IBV-infected or transiently transfected mammalian cells because of a greatly reduced half-life. This rapid turnover in mammalian cells was proteasome-dependent while turnover in avian cells was proteasome-independent. These results highlighted the importance of using cells derived from the natural host when studying coronavirus non-structural proteins. These results also indicated that IBV 3b and SARS 3b are unlikely to be functional homologs. |
