Host Control of Viral Protein Function: Phosphorylation in the Papillomavirus E2

Indiana University Indianapolis, Indianapolis IN

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Abstract

DESCRIPTION (provided by applicant): We used linear ion trap mass spectrometry to identify previously unreported post-translational modifications in the E2 protein of bovine papillomavirus type 1, a model for human papillomavirus studies. Our analysis revealed a novel phosphorylation site in the E2 transactivating domain at tyrosine 102. E2 is a master regulatory protein for all papillomaviruses; it is necessary for transcriptional activation and repression, vial genome replication, and equal partitioning of genomes to dividing host cells. E2 is most active and most highly expressed upon terminal differentiation of its keratinocyte host, leading to viral genome amplification and eventual packaging of new virions; however, the cellular-viral crosstalk that initiates this upregulation of E2 are not well understood. Our preliminary data indicate that tyrosine 102 is a crucial residue in determining global E2 activation and that a poin mutation approximating phosphorylation is defective for both transcriptional and replicative functions. We propose that phosphorylation occurs at Y102 early in the viral life cycle before the host cell undergoes terminal differentiation and that this modification is relieved to allow full activity of E2 once differentiation occurs. Aim 1 will test our hypothesis that phosphorylation of E2 at Y102 occurs during viral maintenance in dividing keratinocytes but is diminished upon host-cell differentiation. We will accomplish this aim by monitoring phosphorylation of stably expressed E2 directly in proliferating versus differentiated cells by immunoprecipitation and Western analysis with p-Tyr antibody. Southern blot and qPCR will be used to assess viral genome copy number in cells stably transfected with WT or Y102 mutant genomes, allowing us to correlate simulated E2 phosphorylation state, cell differentiation state, and capacity of E2 to drive genome amplification. Since our preliminary data and previous reports in the field demonstrate a role for cell cycle- regulatory kinases and phosphatases in controlling E2 activity, we anticipate that the enzymes responsible for Y102 phosphorylation will participate in host cell cycle regulation as well. Specifically, our second aim will test the hypothesis that E2 phosphorylation is controlled by a kinase inhibited during host-cell differentiation. Potential E2-interacting tyrosine kinases identified by predictive software will be verified by co- immunoprecipitation and in vitro kinase assay with 32P labeling; differences in expression of the kinase(s) of interest in basal (proliferating) versus superficial (differentiating) keratinocyte epithelia will be assessed by immunohistochemistry. RNA levels of viral proteins and de novo viral DNA synthesis will also be examined to confirm differentiation in the epithelium, cementing the relationship between this cell state and kinase expression. These experiments will allow us to form a more complete picture of spatiotemporal regulation of E2 in a model of the natural host environment.