Post-transcriptional regulation of gene expression at the level of translation is central to the spatial and temporal control of protein production in many biological contexts, including viral infection. The eukaryotic translation initiation factor (eIF) 4F, which bridges the ribosome to the 5'methylated cap of the mRNA and provides helicase activity to facilitate ribosome scanning, plays a pivotal role in regulating cap-dependent translation initiation and is targeted by diverse viruses. Indeed, with their absolute dependence on their host translation system and the relative ease with which they can be genetically manipulated, viral systems have proven invaluable in the discovery and dissection of basic biological processes and have defined many translational control paradigms that operate in both infected and uninfected cells. However, much remains to be learned about the mechanisms involved in regulating translation initiation, in particular how selective and localized mRNA translation is controlled. Vaccinia Virus (VacV) is a member of the poxvirus family of large, double stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells within membrane- bound structures termed replication compartments or viral factories. In this proposal we aim to exploit the compartmentalized replication of VacV, whose mRNAs are capped and polyadenylated similar to their host counterparts, to study mechanisms of translation factor activation and redistribution, and their effects on viral and host protein synthesis. Recently, we have shown that VacV activates PI3K-Akt-mTOR signaling to inactivate translational repressor proteins and enhance the formation of host eIF4F complexes. This is accompanied by a redistribution of core components of eIF4F to discrete regions within viral factories where synthesis of viral proteins is thought to occur. Our preliminary data has identified a viral protein, I3 that binds the eIF4F scaffold protein, eIF4G, and both of these factors colocalize within the same regions of viral factories. Although VacV induces global suppression of host protein synthesis, our preliminary polysome profiling shows that translation of certain host mRNAs not only persists but, in select cases, is increased. We propose to characterize the role of I3 in regulating both eIF4F distribution and localized protein synthesis. Furthermore, we will determine the role of the cap-binding eIF4F subunit, eIF4E in the selective synthesis of specific host proteins and test the functional importance of this to infection. Finally, we will explore how upstream PI3K-Akt-mTOR signaling functions during the compartmentalized replication of VacV, complementing our studies of eIF4F redistribution to provide a comprehensive analysis of the role of this cap- binding complex in selective host and viral mRNA translation during poxvirus infection. Overall, these aims will provide important insights into the replication of poxviruses, which killed an estimated 300-500 million people in the 20th century, with the potential to uncover novel therapeutic targets, and will also contribute to our broader understanding of general mechanisms of both localized and selective mRNA translational control.
With their absolute dependence on host ribosomes, viruses have evolved a diverse array of strategies to gain control of their host's protein synthesis machinery and thwart host defenses aimed at crippling the synthesis of viral proteins. In this proposal we aim to determine how the poorly understood process of localized translation occurs during poxvirus infection and its role in regulating both viral and host protein synthesis. While advancing our understanding of how these deadly agents that killed hundreds of millions exploit host functions to replicate, with the potential to develop novel therapeutics, these studies will also provide valuable insights into fundamental mechanisms of translational control of gene expression.
| Meade, Nathan; Furey, Colleen; Li, Hua et al. (2018) Poxviruses Evade Cytosolic Sensing through Disruption of an mTORC1-mTORC2 Regulatory Circuit. Cell 174:1143-1157.e17 |
| Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele et al. (2017) Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase. Nature 546:651-655 |
| Jan, Eric; Mohr, Ian; Walsh, Derek (2016) A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells. Annu Rev Virol 3:283-307 |
| Walsh, Derek; Mohr, Ian (2014) Coupling 40S ribosome recruitment to modification of a cap-binding initiation factor by eIF3 subunit e. Genes Dev 28:835-40 |
