A poxvirus pseudokinase represses viral DNA replication via a pathway antagonized by its paralog kinase

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.

Emerging concepts in pseudoenzyme classification, evolution, and signaling

“…Outside of these notable pseudoenzyme families, relatively few examples of competitor and/or "decoy" pseudoenzymes have been identified to date. However, new data confirm their expansion within plant, fungal, and pathogen proteomes (24-27), including, for example, a new viral pseudoenzyme that provides the pathogen with an advantage by directly competing with host cell defense mechanisms (28).…”

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

“…The precise function of vaccinia virus B12 during infection has remained enigmatic to date. Specifically, previous studies of a B12 deletion mutant, lacking 83% of the B12R gene, revealed that this B12 deletion virus replicated efficiently in cultured cells and in vivo (26); however, when B12 was deleted in the context of the vaccinia virus B1 deletion mutant ΔB1mutB12, B12 was revealed to be repressive to vaccinia virus replication (6). Considering this evidence that B12 acts as a repressor of vaccinia virus replication upon loss of B1, we sought to determine whether the requirement of vaccinia virus for either vaccinia virus B1 or the cellular kinase VRK2 is functionally linked to vaccinia virus B12.…”

“…The vaccinia virus B1 kinase is expressed early during infection and promotes multiple facets of the vaccinia virus life cycle (6)(7)(8)(9)(10)(11). Genetic and biochemical investigations of the B1 kinase using temperature-sensitive viruses (7,9,(12)(13)(14) and a B1 deletion mutant virus (6,15) have provided insights into the essential roles played by B1 during vaccinia virus infection.…”

“…The vaccinia virus B1 kinase is expressed early during infection and promotes multiple facets of the vaccinia virus life cycle (6)(7)(8)(9)(10)(11). Genetic and biochemical investigations of the B1 kinase using temperature-sensitive viruses (7,9,(12)(13)(14) and a B1 deletion mutant virus (6,15) have provided insights into the essential roles played by B1 during vaccinia virus infection. Importantly, the severity of the phenotype resulting from B1 mutant virus infections in those studies was cell type dependent and was limited at the stages of DNA replication (13)(14)(15)(16)(17), intermediate transcription (7,11), or morphogenesis (9), signifying a function for B1 in promoting viral replication at each of these steps.…”

“…The nature of this additional BAF-independent pathway by which B1 and VRK2 promote vaccinia virus replication was informed by recent findings regarding adaptive evolution experiments with ΔB1 vaccinia virus (6). When ΔB1 vaccinia virus was subjected to iterative passaging, an adapted virus variant emerged that is referred to herein as ΔB1mutB12.…”