Matthew S. Wiebe
Date of this Version
Olson, Annabel T. Regulation of vaccinia virus replication: A story of viral mimicry and a novel antagonistic relationship between vaccinia kinase and pseudokinase. Doctoral Dissertation. University of Nebraska-Lincoln. April 2019.
Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways.
We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent.
We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. Interestingly, our characterization of the adapted viruses reveals that mutations correlating with a loss of function of the vaccinia B12 pseudokinase provide a striking fitness enhancement to this virus. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. Together, our studies of B1 and B12 present novel evidence that a paralogous kinase-pseudokinase pair can exhibit a unique epistatic relationship in a virus, and orchestrate yet-to-be-discovered nuclear events during infection.
Advisor: Matthew S. Wiebe