Regulation of Vaccinia Virus Replication: a Story of Viral Mimicry and a Novel Antagonistic Relationship Between Vaccinia Kinase and Pseudokinase

Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. 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. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. 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, and orchestrate yet-to-be-discovered nuclear events during infection. Advisor: Matthew S. Wieb

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

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and vi- ral 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 com- mon pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vac- cinia 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 repli- cation in the absence of B1, unlike other DNA viruses. Employing loss-of-function analy- sis, 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. More- over, 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 replica- tion 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-depen- dent manner

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

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

Vaccinia Virus Arrests and Shifts the Cell Cycle

Modulation of the host cell cycle is a common strategy used by viruses to create a proreplicative environment. To facilitate viral genome replication, vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. However, the cellular factors and viral effectors that mediate these changes remain unknown. Here, we set out to investigate the effect of VACV infection on cell proliferation and host cell cycle progression. Using a subset of VACV mutants, we characterise the stage of infection required for inhibition of cell proliferation and define the viral effectors required to dysregulate the host cell cycle. Consistent with previous studies, we show that VACV inhibits and subsequently shifts the host cell cycle. We demonstrate that these two phenomena are independent of one another, with viral early genes being responsible for cell cycle inhibition, and post-replicative viral gene(s) responsible for the cell cycle shift. Extending previous findings, we show that the viral kinase F10 is required to activate the DNA damage checkpoint and that the viral B1 kinase and/or B12 pseudokinase mediate degradation of checkpoint effectors p53 and p21 during infection. We conclude that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins. (209/200.

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

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

Regulation of Vaccinia Virus Replication: a Story of Viral Mimicry and a Novel Antagonistic Relationship Between Vaccinia Kinase and Pseudokinase

Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. 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. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. 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, and orchestrate yet-to-be-discovered nuclear events during infection. Advisor: Matthew S. Wieb

Regulation of Vaccinia Virus Replication: A Story of Viral Mimicry and a Novel Antagonistic Relationship between Vaccinia Kinase and Pseudokinase

Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. 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. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. 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, and orchestrate yet-to-be-discovered nuclear events during infection

Regulation of Vaccinia Virus Replication: A Story of Viral Mimicry and a Novel Antagonistic Relationship between Vaccinia Kinase and Pseudokinase

Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. 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. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. 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, and orchestrate yet-to-be-discovered nuclear events during infection

Antagonism of Protein Kinase R by Large DNA Viruses

Decades of research on vaccinia virus (VACV) have provided a wealth of insights and tools that have proven to be invaluable in a broad range of studies of molecular virology and pathogenesis. Among the challenges that viruses face are intrinsic host cellular defenses, such as the protein kinase R pathway, which shuts off protein synthesis in response to the dsRNA that accumulates during replication of many viruses. Activation of PKR results in phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), inhibition of protein synthesis, and limited viral replication. VACV encodes two well-characterized antagonists, E3L and K3L, that can block the PKR pathway and thus enable the virus to replicate efficiently. The use of VACV with a deletion of the dominant factor, E3L, enabled the initial identification of PKR antagonists encoded by human cytomegalovirus (HCMV), a prevalent and medically important virus. Understanding the molecular mechanisms of E3L and K3L function facilitated the dissection of the domains, species-specificity, and evolutionary potential of PKR antagonists encoded by human and nonhuman CMVs. While remaining cognizant of the substantial differences in the molecular virology and replication strategies of VACV and CMVs, this review illustrates how VACV can provide a valuable guide for the study of other experimentally less tractable viruses

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

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and vi- ral 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 com- mon pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vac- cinia 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 repli- cation in the absence of B1, unlike other DNA viruses. Employing loss-of-function analy- sis, 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. More- over, 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 replica- tion 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-depen- dent manner