PROJECT SUMMARY Viruses exert an extensive network of dynamic interactions with host components to promote infection by dismantling cellular intrinsic and innate defenses. A central arm of viral takeover of cellular processes relies on viral exploitation of the cellular ubiquitin system to induce degradation of host factors. However, there is a gap in our understanding of the molecular mechanisms by which ubiquitin is harnessed by viral proteins. Here we propose a cross-species comparison of the human and mouse adenovirus systems to explore how viruses subvert host defenses via ubiquitin. Human adenovirus 5 (HAd5) is a prominent nuclear-replicating DNA virus that redirects cellular Cullin E3 ubiquitin ligase activity via complex formation with two viral early proteins (E1B55K and E4orf6). We recently developed a proteomics approach to define host proteins ubiquitinated when the HAd5 E1B55K/E4orf6 complex is expressed. By combining our ubiquitome analysis with whole cell proteomics, we were able to define which substrates are ubiquitinated and which are subsequently degraded as a result of the E1B55K/E4orf6 complex. The strict species-specificity of adenovirus infection limits our ability to study HAd in its natural host, but mouse adenovirus type 1 (MAV-1) provides an alternative tractable system. Based on genetic similarities, MAV-1 is thought to encode orthologs (mE1B55K and mE4orf6) to the HAd5 complex, and these proteins are presumed to redirect cellular ubiquitin in a parallel fashion. We have applied our proteomics pipeline to MAV-1 infected cells, and used global ubiquitin-profiling to identify proteins modified and degraded by the virus. Distinct from HAd5, we discovered that MAV-1 uniquely facilitates degradation of several canonical and non-canonical proteins involved in nucleic acid sensing and antiviral interferon signaling, including PKR and STING. Contrary to the prevailing dogma of how the HAd5 E1B55K/E4orf6 complex employs the E1B55K component to select ubiquitination substrates, we surprisingly discovered that mE4orf6 is sufficient to reduce abundance of the antiviral RNA sensor PKR in a proteasome- and Cullin- dependent manner, independent of mE1B55K. These findings collectively suggest divergence in the composition, mechanisms of assembly, and substrate selectivity between the HAd5 and MAV-1 directed E3 ligases. An overarching implication is that the MAV-1 and HAd5 complex exploit ubiquitin in different ways to counteract intrinsic and innate immune responses. In Aim 1 we will leverage a multidisciplinary, quantitative proteomics approach to systematically define the endogenous cellular ubiquitin substrates and associated pathways targeted during MAV-1 infection. We will also determine the functional consequences of substrate ubiquitination during infection. In Aim 2 we will establish the composition, and mechanisms of substrate selection for the MAV-1 directed E3 ligase complex and compare to HAd5. Results of our cross-species...