New Ops: Mechanisms of early vaccinia viral morphogenesis (trans-RCE proj)

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Members of the poxvirus family have been investigated for their applications as vaccines and expression vectors, and more recently intently studied because of their use as potential biological weapons. Vaccinia virus, the prototypic member, evolves through multiple forms in which membrane morphogenesis plays a key role. Even though viruses generally usurp host factors for its use, it remains unclear whether key host transport factors that are well known to act in membrane morphogenesis of host cellular compartment participate in early vaccinia membrane morphogenesis, a process critical in acquiring viral infectivity. We have now identified a role for coatomer, a host protein complex well characterized to form Coat Protein I (COPI) transport vesicles. However, insights into how it participates in early viral morphogenesis reveals that the virus bypasses key regulatory mechanisms that form host COPI vesicles, but instead interacts with two proteins, the viral K7 and the host KDEL receptor (KDELR) through critical di-Iysine residues on these interacting proteins. Thus, to gain further insight into how coatomer and its two interacting proteins contribute to viral replication, we propose two major aims. In one aim headed by Victor Hsu with proposed funding from NERCE, perturbation of coatomer will be examined for its effect in potentially accumulating particular viral forms. Moreover, the KDELR will be examined with respect to its distribution on viral forms and also whether its ligands have a role in viral replication. As another aim headed by Stuart Isaacs with proposed funding from MARCE, the viral K7 protein will be examine for effects upon its deletion and also when a mutant form that cannot interact with coatomer is expressed. This collaborative effort is responsive in multiple ways to the request for application under the "New Opportunities" initiative by the two respective Regional Centers of Excellence. First, we will be gaining insights into a Category A pathogen, for which we will not only elucidate novel mechanisms by which a host protein functions dUring viral replication, but also identify potential key target(s) for the future design of intervention against the most abundant infectious form of the virus. Second, funding of this proposal will allow one of the main investigators (Victor Hsu) to collaborate with a poxvirus expert (Stuart Isaacs), and thus, applying outside expertise in mechanisms of vesicular transport to critical issues in poxvirus biology. As viruses generally commandeer host mechanisms for their interaction with the host rather than inventing completely novel ones, the complementary expertise of the collaboration will likely enhance the elucidation of how vaccinia virus interacts with its host. Third, potential funding of this proposal will likely set the stage for an eventual more comprehensive application in the future, such as through the NIH R01 mechanism.