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The Vaccinia B1 and Cellular VRK2 Kinases Promote Vaccinia Replication Factory Formation through Phosphorylation Dependent Inhibition of Vaccinia B12.

Research paper by Amber B AB Rico, Zhigang Z Wang, Annabel T AT Olson, Alexandria C AC Linville, Brianna L BL Bullard, Eric A EA Weaver, Clinton C Jones, Matthew S MS Wiebe

Indexed on: 30 Jul '19Published on: 26 Jul '19Published in: Journal of virology



Abstract

Comparative examination of viral and host homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called VRKs (vaccinia related kinases). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1 as compared to other DNA viruses. Employing loss of function analysis, we demonstrate that vaccinia dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining B1 and VRK2 co-precipitation of B12. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating, but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analysis of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling co-regulation and support a model in which these enzymes modulate B12 in a phosphorylation dependent manner.Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious, mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia B12 via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog, VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase. Copyright © 2019 American Society for Microbiology.