Characterization Of The Role Of The Poxvirus Extracellular Virion Specific Glycoproteins A33 A34 And B5 For Infectivity And Pathogenesis PDF Download

The Poxviridae family contains a considerable number of large, double-stranded DNA viruses. Vaccinia virus (VACV), the prototypical poxvirus, is a member of the Orthopoxvirus genus and is the virus that was used as the live-attenuated vaccine against smallpox. During infection, two antigenically distinct, infectious forms of the virus are produced, including intracellular mature virus (IMV) and extracellular virus (EV). EV contain an additional membrane envelope compared to IMV and are critical for cell-to-cell spread and dissemination during infection. Proteins A33, A34, A56, B5, F13, K2, and E2 are specific to the EV membrane. Of these, A33, A34, A56 and B5 are glycoproteins. With the exception of A56, deletion of any one of these proteins results in a small-plaque phenotype, representative of their important role in the production of infectious EV. The overall goal of this thesis study was to further elucidate the function of the glycoproteins (A33, A34, and B5) and their interactions for infectivity and pathogenesis. Our investigations led to the discovery of a number of previously unrecognized interactions. Furthermore, characterization of these interactions revealed an interdependency of the glycoproteins for their trafficking to the site of wrapping and for subsequent incorporation. Additionally, our data indicates that F13 and the glycoproteins serve as a matrix-like complex to facilitate envelopment of IMV to form EV. Further investigations revealed that A33 contains a unique structural mechanism that controls its interactions with A34 and B5, and these results indicated that the interactions between the glycoproteins are transient, and this transience is critical for the production of fully infectious EV. During our investigations, we developed several novel techniques, including flow virometry and fluorescence-activated virion sorting to analyze individual EV. Using these assays, we show a direct relationship between glycoprotein incorporation and infectivity. Altogether, this work reveals that a complex cascade of protein interactions exists to facilitate efficient morphogenesis and intracellular envelopment, and these interactions are tightly regulated to ensure optimal EV protein content and the production of fully infectious EV. This work has provided valuable insights regarding the roles of the glycoproteins for morphogenesis, intracellular envelopment, and infectivity. Taken together, the data presented here will be critical for the development of safer and more effective orthopoxvirus vaccines, poxvirus oncolytics, and viral vaccine vectors.