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

Michael T. Parker; Smita Gopinath; Corey E. Perez; Melissa M. Linehan; Jason M. Crawford; Akiko Iwasaki; Brett D. Lindenbach

First, we identified conditions to specifically co-immunoprecipitate cGAS and 208 mtDNA, a known DNA ligand (34). As shown in Figure 5A To more broadly assess cytosolic and hcGAS-bound DNAs, we developed deep 220 sequencing libraries from cytosolic extracts or after immunoprecipitation of WT 221 hcGAS-HA3x. The first one-third of the mitochondrial genome was specifically 222 enriched in cytosolic preps from both uninfected and VSV-GFP-infected MEFs 223 ( Figure 5F ). Similarly, mtDNA was also highly enriched after immunoprecipitation of 224 hcGAS-HA3x, although there was a bias for the latter three-quarters of the genome 225 ( Figure 5G ). Importantly, there was no obvious difference in mtDNA pulldown 226 between uninfected and infected cells. Collectively, these data indicate that VSV 227 does not induce cytosolic release of mtDNA to stimulate cGAS activation. Consistent 228 with this, VSV-GFP replicated equally well in LMTK cells and mtDNA-depleted LMTK 229 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/434027 doi: bioRxiv preprint  0 cells (41), which express cGAS and STING ( Figure 5H ). Collectively, these data 230 suggest that mtDNA is dispensable for cGAS-mediated restriction of an RNA virus. 231