| Coronaviruses acquire their envelopes by budding into cis-Golgi network membranes. This thesis examines the mechanisms by which avian infectious bronchitis virus (IBV), collects its envelope proteins, spike (S), matrix (M), and envelope (E), in the cis-Golgi network for assembly. The small E protein is of low abundance in the virion envelope, and plays an important, but undefined, role in virus budding. This work seeks to provide further understanding of the role of the IBV E protein in virus assembly. Chapter 2 details the cell biological properties of IBV E, which is an integral membrane protein with its carboxyl-terminus in the cytoplasm. Indirect immunofluorescence showed that IBV E is localized to the Golgi region. IBV E was found to colocalize with IBV M, a cis-Golgi protein, in cotransfected cells. Chapter 3 describes an immunoelectron microscopic study of the localization of IBV E in transfected cells when it is expressed alone and with IBV M. IBV E was found to be localized throughout the Golgi stack. When coexpressed with IBV M, the localization of IBV E was restricted to a compartment adjacent to the Golgi stack, which is likely to be the cis-Golgi network budding site. Chapter 4 discusses the mechanism by which the IBV E protein is localized to the Golgi complex. Analysis of cells expressing an amino-terminal truncation of IBV E indicated that the cytoplasmic tail of IBV E is sufficient for localization to the Golgi complex. Studies in brefeldin A-treated cells suggested that the cytoplasmic tail of IBV E mediates its association with Golgi scaffold proteins. Chapter 5 examines the interactions between the IBV E and M proteins that facilitate virus assembly. Wild type and mutant E and M proteins were analyzed using in vivo chemical crosslinking and virus-like particle (VLP) formation assays. These studies showed that the cytoplasmic tails of IBV E and M are involved in their interaction. In summary, our results suggest that the cytoplasmic tail of IBV E interacts with both Golgi scaffold proteins and IBV M, which may reflect dual functions for IBV E in virus assembly. |
