Innate Immune Priming by cGAS as a Preparatory Countermeasure Against RNA Virus Infection

The detection of nucleic acids by pattern recognition receptors is an ancient and conserved component of the innate immune system. Notably, RNA virus genomes are sensed by mammalian cytosolic RIG-I-like receptors, thereby activating interferon-stimulated gene (ISG) expression to restrict viral replication. However, recent evidence indicates that the cGAS-STING DNA sensing pathway also protects against RNA viruses. So far, the mechanisms responsible for DNA sensing of RNA viruses, which replicate without known DNA intermediates, remain unclear. By using cGAS gene knockout and reconstitution in human and mouse cell cultures, we discovered that DNA sensing and cGAMP synthase activities are required for cGAS-mediated restriction of vesicular stomatitis virus and Sindbis virus. The level of cGAMP produced in response to RNA virus infection was below the threshold of detection, suggesting that only transient and/or low levels of cGAMP are produced during RNA virus infections. To clarify the DNA ligands that activate cGAS activity, we confirmed that cGAS binds mitochondrial DNA in the cytosol of both uninfected and infected cells; however, the amount of cGAS-associated mitochondrial DNA did not change in response to virus infection. Rather, a variety of pre-existing cytosolic DNAs, including mitochondrial DNA and endogenous cDNAs, may serve as stimuli for basal cGAS activation. Importantly, cGAS knockout and reconstitution experiments demonstrated that cGAS drives low-level ISG expression at steady state. We propose that cGAS-STING restricts RNA viruses by promoting a preparatory immune activation state within cells, likely primed by endogenous cellular DNA ligands.