To successfully withstand a pathogen attack, organisms must first recognize the presence of an invader and then mount an immune response. C. elegans lack adaptive immunity and have no known professional immune cells, meaning that they rely exclusively on epithelial innate immunity for pathogen defense. The goal of this project is to better understand the interactions between viruses and host innate immune signaling. The Orsay virus, a natural pathogen of C. elegans, is a positive single-stranded RNA virus with an unusually small genome containing only four proteins (Felix et al., 2011). Orsay viral infection induces an innate immune response in C. elegans known as the Intracellular Pathogen Response (IPR) (Bakowski et al., 2014; Reddy et al., 2017, 2019). The IPR is a transcriptional innate immune response that provides defense against stress and intracellular pathogens (Reddy et al., 2017). The activity of the RNA-dependent RNA polymerase encoded by the Orsay virus RNA1 genome segment is known to trigger induction of the IPR through a mechanism dependent on the DRH-1 receptor, and expression of the RNA-dependent RNA polymerase alone is sufficient to turn on the IPR in the absence of infection (Sowa et al., 2020). However, the individual effects of the other three Orsay viral proteins on IPR induction have not yet been investigated. In this project, we will assess the effects of the Orsay viral capsid, delta, and capsid-delta fusion proteins on induction of the IPR. Thus far, molecular cloning has been used to construct plasmids for heat-shock-inducible overexpression of the capsid, delta, and capsid-delta fusion proteins. These constructs will be used to create transgenic lines overexpressing each of the three viral proteins. To visualize IPR activation levels in these strains, we will use the
pals-5p::GFP IPR transcriptional reporter strain, in which the promoter for
pals-5 (a gene which is part of the IPR) drives the expression of GFP when the IPR is triggered (Bakowski et al., 2014). By assessing the degree of IPR activation in the viral protein overexpression strains vs non-transgenic siblings, under different stress conditions we will determine whether the Orsay viral capsid, delta, or capsid-delta fusion proteins affect IPR signaling.