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 comparisons will provide insights into both core principles and distinct strategies that govern how adenoviruses exploit cellular ubiquitin to dismantle host defenses and facilitate viral pathogenesis.
Viruses produce proteins that reprogram the infected cell in order to promote viral progeny production by exploiting cellular resources and counteracting antiviral host defenses. One cellular pathway harnessed by viruses is ubiquitination, whereby the small cellular protein ubiquitin is attached to other proteins in a process that can regulate functions by directing degradation or altering cellular localization and protein interactions. In this project will use mouse adenovirus infection as a model to identify ubiquitination targets during infection and to define molecular mechanisms that control virus infections.
