RNAseq analysis of PARP9 bound RNA during reovirus infection

The innate immune sensor retinoic acid-inducible gene-I (RIG-I) like receptors (RLRs) including RIG-I and melanoma differentiation-associated protein 5 (MDA5) detect double-stranded RNA (dsRNA) derived from RNA viruses. RIG-I preferably recognizes dsRNA short poly I:C from 0.2 kb to 1kb, while MDA preferably recognizes dsRNA short poly I:C from 1.5 kb to 8kb. Here we we report that the poly(ADP-ribose) polymerase 9 (PARP9), a member of PARP family enzymes, is highly induced in human primary dendritic cells (DCs) by interferon (IFN) alpha and serves as an unique size dependent RNA sensor that signals via phosphoinositide 3-kinase (PI3K)/AKT3 metabolic pathway to produce type I IFN post RNA virus infection. To identify the physiological RNA species that are recognized by PARP9 during reovirus infection, we purified the RNAs that co-immunoprecipitated with anti-PARP9 antibody in wild-type (WT) and PARP9 knockout (KO) mouse bone marrow-derived dendritic cells (BMDC) infected with reovirus at multiplicity of infection (MOI) of 200. As controls, RNA species bound to PARP9 in uninfected (Mock) WT and PARP9 KO BMDC were also purified. PARP9 bound RNA extracted from Mock and reovirus-infected WT and PARP9 KO BMDC were analyzed by RNAseq, and the resulting sequences were mapped to both mammalian orthoreovirus 3 Dearing strain T3D and mouse genome (mm10). This analysis revealed that physiological dsRNA species recognized by PARP9 during reovirus infection were 8bp to 1187 bp of S4 gene (Reo1187), 11bp to 1198 bp of S3 gene (Reo1198), 16bp to 1320 bp of S2 gene (Reo1320), and 9bp to 1410 bp of S1 gene (Reo1410).