The Vaccinia Virus B12 Pseudokinase Represses Viral Replication via Interaction with the Cellular Kinase VRK1 and Activation of the Antiviral Effector BAF

Abstract

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulate a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phospho-inactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms poxviruses use to hijack homologous cellular signaling pathways during infection. IMPORTANCE Viruses from diverse families encode for both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Herein, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication.

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

“…Our previous study demonstrated that B12 coprecipitates VRK1 in VACV-infected or uninfected cells and influences events downstream of VRK1 ( 11 ). We sought to further investigate the properties of the B12/VRK1 complex, hypothesizing that B12 activity during VACV infection was linked to complex formation and regulation of VRK1.…”

“…Our previous study indicated that B12 can reduce VRK1-mediated phosphorylation of the DNA cross-bridging protein BAF ( 11 ). However, how this impacts the cellular properties of BAF remains incompletely 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 ). Positing not only that B12 regulates VRK1 but also that VRK1 may modulate B12, we pursued an interconnected series of structure/function studies of these two proteins alone and in combination.…”

“…In addition, B12-mediated inhibition of VACV DNA replication has been determined to be cell type specific and influenced by the levels of host VRK2 protein, arguing that both viral and cellular enzymes modulate how B12 impacts on infection ( 11 ). More recently, investigation into the mechanism of action for B12 revealed the formation of a complex containing B12 and the host VRK1 kinase ( 11 ).…”

“…During VACV infection, both VRK1 and VACV B1 kinase phosphorylate and inhibit the antiviral activity of the DNA binding protein BAF ( 11 , 36 ). Unphosphorylated BAF binds and cross-bridges DNA and thereby inhibits VACV DNA replication, but phosphorylation overcomes this host antiviral defense ( 11 , 36 – 38 ). Recent studies have demonstrated that, due to its high affinity for DNA, cytoplasmic BAF limits innate immune signaling leading to expression of type I interferon (IFN) downstream of viral and endogenous DNA sensing ( 39 , 40 ).…”