Investigating the Regulation of the DNA Sensor cGAS Following Viral Infection

Abstract

Pathogenic DNA sensing is a crucial component of the immune system. One key protein functioning within the mammalian immune response is cyclic GMP-AMP synthase (cGAS), which produces 2’3’-cGAMP that ultimately leads to the production of antiviral cytokines and the induction of immune responses. Despite the extensive characterization of the pathway through which cGAS induces these cytokines, little is known about how cGAS functionality is controlled upon infection of the cell. Here, I identify novel cGAS post-translational modification (PTM) sites as well as confirm already discovered sites through immunoaffinity purification combined with tandem mass spectrometry. I analyze levels of PTMs in uninfected cells and following infection with HSV-1 wild type or d106 strain, and further probe the importance of these PTM sites by constructing stable cGAS point mutation cell lines. Additionally, I investigate the ability of these mutant cell lines to induce the antiviral immune response through qPCR analysis of cytokine levels, as well as their influence on the induction of apoptosis. Furthermore, I compare the protein interactome for mutant cGAS with the wildtype counterpart following HSV-1 infection. In concert with this exploration into cGAS functionality, I also examine the role of the nuclear population of cGAS by constructing a FLAG-NLS-cGAS and its ability to induce immune signaling. These findings expand the understanding of how cGAS can be dynamically controlled within the cell by PTM sites as well as identify specific sites of interest and their effect on cytokine induction and apoptosis.