Investigating the Role of Vaccinia Virus Protein A34 in Viral Morphogenesis and Its Interaction with B5 and A33

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2010.Vaccinia virus, the most studied member of Poxviridae family, is well known as the live vaccine used in the global eradication of smallpox. This large complex DNA virus, which replicates in the cytoplasm of cells, produces three forms of virus; intracellular mature virus (IMV) which accounts for a large majority of the progeny virions, intracellular enveloped virus (IEV), and extracellular virus (EV). EV is further defined into two groups. Virions that remain on the cell surface are cell-associated enveloped virus (CEV) and those released from the cell surface are extracellular enveloped virus (EEV). CEV are responsible for cell-to-cell spread and EEV are responsible for long-range dissemination of the virus. A34, A33, and B5 are proteins found specifically on the enveloped form of the virus (IEV, CEV and EEV) and A36 is found exclusively on IEV. Deletion of any one of these genes attenuates the virus and the mutants display reduced plaque size on cell monolayer cultures. While interactions between A33-A36, A33-B5, A34-A36, and A34-B5 have been reported, only the A33-A36 interaction has been shown to have a specific function. In addition, while there have been a fair number of studies on A33, A36 and B5, less information is known about A34. The overall goal of this research project was to determine the role of A34 in viral morphogenesis and how it interacts with other enveloped viral proteins. A recombinant virus that expresses B5-GFP in place of B5 and had the A34 gene deleted (vB5R-GFP/ΔA34R) was created to look at viral morphogenesis in the absence of A34. Fluorescent microscopy and subcellular fractionation of cells infected with vB5R-GFP/ΔA34R revealed that in the absence of A34, B5-GFP was mislocalized in the endoplasmic reticulum and failed to traffic to the site of wrapping compared to cells infected with a normal B5-GFP expressing virus (vB5R-GFP). Trans-complementation assays in cells infected with vB5R-GFP/ΔA34 showed that the luminal domain of A34 was responsible for the proper trafficking of B5-GFP and correlated with co-immunoprecipitation studies in which an interaction between A34 and B5-GFP was only detected when the luminal domain of A34 was present. Next EEV from vB5R-GFP/ΔA34 infected cells were analyzed and found to contain greatly diminished levels of B5-GFP compared to the levels found in EEV from vB5R-GFP infected cells. These results show that during infection, A34 is required for the proper targeting of B5-GFP to the site of wrapping and incorporation into enveloped virions. In addition, we show that A34 and B5 interact through their luminal domains. The next objective was to further define the region within the luminal domain of A34 required for interaction. Several mutants of A34 were created and co-expressed with B5 to test for interaction by co-immunoprecipitation. Results from the luminal domain deletion mutants and truncation mutants revealed that A34 residues 1-80 were sufficient for interaction and residues 1-120 were required for full interaction. When small A34 regions were tested for interaction, we found two separate regions that were able to interact with B5. We hypothesized that A34 localized to the site of wrapping through targeting signals in its transmembrane or cytoplasmic domain and that expressing soluble A34 or an interacting A34 region during normal infection would act as a dominant negative displacing endogenous A34 and mislocalizing B5. Immunofluorescence studies of cells expressing the small A34 regions during infection showed that only full length soluble A34, which contains both regions, mislocalized B5. This set of data demonstrates that there are two sites of interaction between A34 and B5. A33 has also been reported to require A34 for proper localization and incorporation into enveloped virions and this is thought to be through A33’s interaction with B5. Co-expression of A34 and A33 followed by co-immunoprecipitation revealed that the two proteins interact through their luminal domains. Using recombinant epitope-tagged A34 viruses we were also able to detect this interaction during normal infection. This body of work shows that the interaction between A34 and B5 is required for the proper localization of B5 and its efficient incorporation into enveloped virions. We also show that A34 and B5 have two sites of interaction and that A34 interacts with A33. Taken together we show that A34’s interactions with other enveloped virion proteins are important for viral morphogenesis by determining the composition of the enveloped virion membrane