Adenovirus regulation of cellular RNA binding proteins

Children'S Hosp Of Philadelphia, Philadelphia PA

Investigators

Abstract

PROJECT SUMMARY This proposal addresses mechanisms used by adenovirus (AdV) to manipulate host RNA binding proteins (RBPs) in order to exploit cellular RNA processing pathways while overcoming host defenses. DNA viruses are reliant on cellular RNA processing machinery to generate viral transcripts and synthesize viral proteins. Productive viral infection is achieved by redirecting cellular RBPs through changes in post-translational modifications and subcellular localization. Although RNA viruses are known to co-opt or counteract RNA-protein complexes, there is much less knowledge about the fundamental mechanisms employed by DNA viruses to harness cellular pathways for efficient mRNA transcription, transport, and translation. Adenovirus presents a highly tractable model DNA virus which replicates in the cell nucleus and harnesses host RNA processing machinery. The transcriptionally complex pattern of gene expression from the AdV DNA viral genome provides a powerful system to study temporal regulation of RNA splicing and RNA fate. Our preliminary studies employed mass spectrometry proteomics and RNA Binding Region Identification (RBR-ID) to monitor post-translational modifications and RNA-protein interactions during infection. Integrating these unbiased proteome-wide approaches revealed a unique set of nuclear RBPs which are decreased for both arginine methylation and RNA- binding during infection with AdV serotype 5 (Ad5). We discovered that the multifunctional Ad5 late protein L4- 100K is methylated, binds cellular methylase Protein Arginine Methyltransferase 1 (PRMT1), and causes PRMT1 cytoplasmic relocalization. We propose that L4-100K redirects arginine methylation to alter RNA-binding capacity and function of diverse cellular RBPs. In Aim 1 we will define mechanisms by which modulation of RBP methylation promotes infection through changes to splicing and RNA processing. Insights into mechanisms for redirecting RBPs during AdV infection have also come from preliminary data using viral mutants that are defective for splicing (DE4 lacking early region E4) and protein translation (DVA lacking the viral associated RNA). We found that infection with these mutants induces signaling pathways that lead to assembly of two distinct ribonucleoprotein (RNP) complexes in cytoplasmic granules known to have antiviral activity against other viruses. The field has lacked a suitable viral system for selectively inducing granules, and here we demonstrate that AdV can be used to identify the RBP composition of distinct cytoplasmic granules and define their antiviral properties. In Aim 2 we will examine viral evasion of cytoplasmic granules and the impact of manipulating host responses. Together these studies will employ innovative technologies to define RNA binding and localization of host RBPs during infection, with systematic approaches of global proteomics and high-throughput imaging. Understanding changes in RNA-protein complexes mediated by RBP modification during infection will provide fundamental knowledge of virus-host interactions and will point to new antiviral therapeutic targets.