Polypharmacology-based kinome screen identifies new regulators of KSHV reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identify specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch.

Polypharmacology-based kinome screen identifies new regulators of KSHV reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu ), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch. Author Summary: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

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.

Vaccinia Virus Arrests and Shifts the Cell Cycle.

Modulation of the host cell cycle is a common strategy used by viruses to create a pro-replicative 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 B12 Pseudokinase Represses Viral Replication via Interaction with the Cellular Kinase VRK1 and Activation of the Antiviral Effector BAF.

The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulates 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 phosphoinactivate 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 virus infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms that poxviruses use to hijack homologous cellular signaling pathways during infection.IMPORTANCE Viruses from diverse families encode 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. Here, 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.

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.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.

Generation of Vaccinia Virus Gene Deletion Mutants Using Complementing Cell Lines.

This protocol describes how to couple two techniques, the generation of complementing cells lines and production of viral deletion mutants, to rapidly construct novel tools for poxvirus analysis. Specifically, the production and utilization of a complementing cell line expressing a poxvirus gene of interest are critical for the generation of poxvirus mutants in which essential genes are disrupted. Complementing cells are also valuable for the characterization of vaccinia genes in the absence of infection. Here, we detail the process of isolating vaccinia virus deletion mutants. Deletion mutant generation involves homologous recombination between replicating viral DNA and transfected DNA followed by selection and screening on a complementing cell line that provides the deleted gene in trans. Finally, deletion is confirmed by polymerase chain reaction, sequencing, and functional assays if available.

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.

Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2.

The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (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 both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.