Sunday, December 22, 2024
12/22/2024
Viral manipulation of mitotic and antiviral signal transduction determines the outcome of infection, but remains poorly understood. To address this knowledge gap, our laboratory studies a family of protein kinases comprised of homologs widely expressed in poxviruses and in all multicellular eukaryotes. The long term goal of our research is to determine how poxviruses usurp and redirect signaling cascades governing mitotic and host defense effectors responsive to foreign DNA. Mammalian poxviruses express two proteins, B1 and B12, which are homologous to each other and to three eukaryotic protein kinases named vaccinia related kinases (VRKs). Comparative studies of B1 and VRK1 revealed that both enzymes directly modify the cellular protein BAF. Importantly, BAF acts as both a mitotic regulator and antiviral effector by binding and compacting dsDNA, a property that is inactivated via phosphorylation by B1 or VRK1. Our new data argue that B1 and cellular VRKs co-regulate other pathways as well, including an antiviral pathway activated by the B12 protein. Our data indicate that B12 directs strong repression of vaccinia DNA replication via partly unknown mechanisms also governed by B1. Intriguingly, B12 is a nuclear poxviral protein and a non-catalytic kinase or `pseudokinase', which are of key innovative importance for this proposal. Pseudokinases are members of the pseudoenzyme family, about which little is known in viruses. It is our central hypothesis that vaccinia B1 and B12 form a novel signaling axis that supplants and redirects cellular VRK pathways regulating BAF and other VRK substrates such as histones and the HUSH (Human Silencing Hub) complex. To test our hypothesis, we propose three aims. AIM 1) Determine how B1 and B12 remodel VRK1-responsive signaling during poxvirus infection. This Aim tests the hypothesis that B12 interacts with VRKs in the nucleus, thereby altering H2A, BAF, and HUSH regulation. Characterization of B12 interaction with BAF, B1, and cellular VRKs in vitro and in cultured cells will be achieved. AIM 2) Determine the molecular mechanisms governing B12 repression of poxvirus DNA replication. This Aim tests the hypothesis that the B12/VRK1 complex plays key roles in the mechanism of B12 signaling dysregulation. Structure/function analysis of B12 through targeted mutational analysis, novel loss of function screens, and investigation of B12 phosphoregulation are outlined in this Aim. AIM 3) Determine how viral/cellular pseudokinases mediate repression of the poxvirus lifecycle and converge with protein phosphatase signaling. This Aim will test the hypotheses that B1 and VRK2 kinases regulate B12 via direct phosphorylation while VRK3 and the phosphatase PP2A control dynamic regulation of B1/VRK1 substrates, leading to manipulation of downstream antiviral responses. The completion of this work will: fill gaps in our understanding of poxvirus manipulation of nuclear processes, yield broadly relevant insights to the field of kinase-pseudokinase biology, and provide needed information of how mitotic and antiviral signaling interweave.
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