This is an application for renewal of a grant to study picornavirus genome replication. All positive-strand RNA viruses hijack and/or remodel host membranes to create an organelle that serves as the site of genome replication. Poliovirus has served as an important model system for elucidation of viral factors, host factors and corresponding interactions required for biogenesis of the replication organelle. Historically, our laboratory has been interested in the process of genome replication and elucidation of the roles of P3-encoded proteins in this process. During the previous funding period, we made the unexpected observation that the PV protease and RNA-binding protein, 3CD, contributes to formation of the replication organelle and is needed for this organelle to efficiently transfer replicated genomes from the site of synthesis into capsids. Interestingly, the role of 3CD in formation of the replication organelle is concentration dependent and can be complemented in trans, suggesting an interaction of 3CD with a host factor. Recently, the Altan-Bonnet lab showed that phosphatidylinositol-4-phosphate (PI4P) increases in abundance during picornavirus infection and localizes to the replication organelle. How a lipid contributes to formation and/or function of the replication organelle remains to be elucidated. However, we have now shown that ectopic expression of 3CD is sufficient to alter the localization and perhaps even increase the abundance of PI4P lipids in cells. In addition, our biochemical, biophysical and computational experiments support the conclusion that PV 3CD is a phosphoinositide-binding protein. These observations, placed into the context of the pioneering work of Belov and Ehrenfeld showing that PV 3CD interacts with membranes and is sufficient for activation of Arf1, a small G protein involved in phosphoinositide metabolism and other transactions of/on membranes, leads to the central hypothesis driving the aims of this application. We propose that 3CD binding to membranes is governed, at least in part, by the interaction of this protein with PI4P and perhaps other phosphoinositides. The membranes hijacked for incorporation may therefore change as a function of time post-infection in a manner dependent on both the strength of the 3CD-phosphoinositide interaction and concentration of 3CD. With different phosphoinositides come different effectors that may change the composition, form and/or function of the replication organelle. Temporal alterations in function would include a transition from replication to encapsidation. Activation of Arf1 and/or its homologues on the different membranes may contribute to changes to the phoshphoinositide pools expressed on the replication organelle during infection, with the potential for negative consequences on pathways that rely on "normal" levels and localization of the these lipids. During the next funding period, we will test this model by pursuing the following specific aims: (1) Elucidation of the mechanism and function for induction of PI4P by 3CD;(2) Elucidation of the mechanism and function for phosphoinositide binding to 3CD;(3) Elucidation of the mechanism and function for induction of other phosphoinositides during PV infection.
Picornaviruses represent an existing and emerging threat to US public health. Achievement of the goals of the application will provide novel targets and mechanisms for development of inhibitors to treat infections by picornaviruses, especially those for which vaccines are not available.
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