Translation is a critical stage at which the abundance, timing, and localization of gene expression is regulated. In the case of infection, precise control of translational machinery and processes can determine whether an effective antiviral program is initiated or whether these programs are impeded by the invading pathogen. Although required for both virus replication and host defense, mechanisms by which translation is regulated early after virus infection are not fully understood. Moreover, the importance of translational regulation in limiting viral replication remains to be elucidated. Understanding functions for translational regulation during virus infection and innate immune responses will provide insights into virus-induced disease and uncover novel mechanisms of antiviral immunity. The main goal of this proposal is to investigate functions of early regulation of translation in host susceptibility to virus infection.
In Specific Aim 1, I will use ribosome profiling and pulse-labeled mass spectrometry to identify and quantify translationally regulated mRNAs in immune stimulated cells. Cells deficient for specific translationally regulated mRNAs will be used to determine the importance of these mRNAs in viral replication and antiviral immunity.
In Specific Aim 2, I will utilize confocal microscopy of pulse-labeled cells to define subcellular sites of early translational events that follow virus infection and immune activation. Translation of specific antiviral mRNAs also will be examined by selective tagging and visualized by live-cell microscopy.
In Specific Aim 3, I will study ribosomal stalk proteins, which were recently identified as pro-viral host factors in a CRISPR knockout screen, and their roles during virus infection. Expression and localization of the stalk proteins will be determined following immune stimulation, and translation and viral replication will be assessed in stalk protein-deficient cells. Stalk protein-deficient cells will be reconstituted with genetically altered constructs to define sequences that contribute to stalk protein function. Together, these studies will elucidate functions for translational regulation in host susceptibility to virus infection and will enhance our understanding of host-pathogen interactions. Knowledge gained from this proposed research may illuminate new approaches for therapeutically targeting broad families of viruses.
Translational regulation shapes gene expression to control a multitude of cellular responses, but mechanisms by which translation is regulated during virus infection are not understood. The proposed research will determine the function of host and viral translational regulation in host susceptibility to virus infection. These studies will enhance an understanding of antiviral immunity and viral pathogenesis and reveal novel strategies for the development of antiviral therapeutics.
