The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12

Abstract

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). 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 virus protein 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, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus’s 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 coprecipitation of B12 by B1 and VRK2. 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 analyses 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 coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner. IMPORTANCE 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 virus 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 virus B12 protein 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.

Dysregulation of Cellular VRK1, BAF, and Innate Immune Signaling by the Vaccinia Virus B12 Pseudokinase

“…While the critical role of protein pseudokinases in governing molecular signal transduction is becoming increasingly apparent, the contribution of pseudokinases during viral infection remains poorly understood. The VACV B12R gene encodes a pseudokinase capable of acting as a potent inhibitor of VACV DNA replication in the absence of VACV B1 regulation ( 6 , 11 , 48 , 56 ). Previous studies indicate that B12 impacts the poxviral life cycle via association with VRK1, a cellular kinase with which B12 copurifies and may redirect to the nucleus during infection ( 11 ).…”

“…pHAGE-HYG-MCS ( 97 ), pHAGE-HYG-3×Flag-BAF (the plasmid was generously provided by Paula Traktman), pHAGE-HYG-3×FLAGB1 ( 56 ), and pHAGE-HYG-3×FLAGVRK1 ( 11 ) were described previously. pHAGE-HYG-3×FLAGVRK1-ΔNLS, pHAGE-HYG-3×FLAGVRK1-ΔBasic, pHAGE-HYG-3×FLAGVRK1-ΔNLSΔBasic, pHAGE-HYG-3×FlagVRK1 1-369, and pHAGE-HYG-3×FLAGVRK1 1-331, pHAGE-HYG-meGFP, and pHAGE-HYG-meGFPhVRK1 were produced through overlap PCR and cloning.…”

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62

“…VACV WR WT, WR E EGFP ( Chomczynski and Mackey, 1995 ; Stiefel et al, 2012 ), WR L EGFP ( Chomczynski and Mackey, 1995 ; Schmidt et al, 2013 ; Stiefel et al, 2012 ), and WR mCherry-A4 ( Mercer and Helenius, 2008 ; Schmidt et al, 2013 ) were previously published. WR ΔB1mutB12 ( Olson et al, 2019 ; Rico et al, 2019 ) was a kind gift from the Wiebe lab.…”

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62

“…VACV WR WT, WR E EGFP (Chomczynski & Mackey, 1995;Stiefel et al, 2012), WR L EGFP (Chomczynski & Mackey, 1995;Schmidt et al, 2013;Stiefel et al, 2012), WR mCherry-A4 (Mercer & Helenius, 2008;Schmidt et al, 2013) were previously published. WR ΔB1mutB12 Rico et al, 2019) was a kind gift from the Wiebe lab.…”