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

intermediates, it has been reported that VSV-specific cDNAs can arise in infected 233 cells, presumably through reverse transcriptase (RT) activity encoded by 234 endogenous retroelement(s) (42). We therefore investigated whether such viral 235 cDNAs arose during VSV-GFP infections in our laboratory. Indeed, VSV N 236 gene-specific cDNAs were generated in infected cells, although in extremely low 237 abundance, ~1 copy/10 4 cells ( Figure 5I ). The cDNA origin of the N gene template 238 was confirmed by nuclease treatment ( Figure 5J ), by its sensitivity to tenofovir, an 239 RT inhibitor that had no effect on VSV replication ( Figure S3A ), and by its enhanced 240 expression in cells devoid of TREX1 nuclease ( Figure S3B ). We also identified 241 virus-specific cDNAs in cells infected with yellow fever virus (YFV), a positive-strand 242 RNA virus ( Figure S3C ), suggesting that cDNA formation is a general feature of RNA 243 virus infections. Finally, to determine whether cDNA formation was specific to 244 virus-infected cells or to viral transcripts, we examined whether cDNA forms of an 245 abundant housekeeping gene, GAPDH, arose in uninfected cells. Indeed, 246 splice-dependent GAPDH cDNAs were identified in low abundance by qPCR ( Figure 247 5K). Importantly, VSV or retroelement cDNAs were not detected in deep sequencing 248 analyses of whole cytosol or cGAS-HA immunoprecipitations, likely due to their low 249