Abstract

Poxviruses employ sophisticated, but incompletely understood, signaling pathways that engage cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. For example, the vaccinia B1 protein kinase plays a vital role in inactivating the cellular antiviral factor BAF, and likely orchestrates other pathways as well. In this study, we utilized experimental evolution of a B1 deletion virus to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. After several passages of the ΔB1 virus we observed a robust increase in viral titer of the adapted virus. 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. In support of predictions that reductive evolution is a driver of poxvirus adaptation, this is clear experimental evidence that gene loss can be of significant benefit. Next, we present multiple lines of evidence demonstrating that expression of full length B12 leads to a fitness reduction in viruses with a defect in B1, but has no apparent impact on wild-type virus or other mutant poxviruses. From these data we infer that B12 possesses a potent inhibitory activity that can be masked by the presence of the B1 kinase. Further investigation of B12 attributes revealed that it primarily localizes to the nucleus, a characteristic only rarely found among poxviral proteins. Surprisingly, BAF phosphorylation is reduced under conditions in which B12 is present in infected cells without B1, indicating that B12 may function in part by enhancing antiviral activity of BAF. 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, perhaps serving to enhance B1 conservation during poxvirus evolution and to orchestrate yet-to-be-discovered nuclear events during infection.

Highlights

  • Protein kinases regulate the function of a large fraction of cellular proteins, governing numerous molecular processes [1,2,3]

  • Vaccinia virus is the archetype member of the Poxviridae family. This virus encodes ~200 genes that contribute to viral propagation in the cytoplasm and coordinate restriction of the host antiviral response through mechanisms that are incompletely understood

  • Fitness gains observed following adaption of the ΔB1 virus correlate with an indel mutation within the B12R gene

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Summary

Introduction

Protein kinases regulate the function of a large fraction of cellular proteins, governing numerous molecular processes [1,2,3]. A group of eukaryotic kinases have homology (~40% amino acid identity) to the vaccinia B1 protein [7,8,9,10] These proteins are named vaccinia related kinases (VRKs) and have been found to share at least one common substrate with B1, demonstrating that the B1/VRK enzymes represent an intersection of viral and host signaling pathways. Much of what we know regarding the function of B1 is based on studies of temperature-sensitive mutant viruses with point mutations in the B1 locus [4, 5, 11,12,13,14,15,16,17] as well as a recently described B1-knockout virus [18] Progeny of these B1-deficient viruses are markedly reduced in number during infection, due to profound defects in viral DNA replication [4, 5, 15, 18]

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