A Time-Resolved Molecular Map of the Macrophage Response to VSV Infection

Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase-substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.