Identification of RNase L-Dependent, 3'-End-Modified, Viral Small RNAs in Sindbis Virus-Infected Mammalian Cells

Girardi, Erika; Chane-Woon-Ming, Béatrice; Messmer, Mélanie; Kaukinen, Pasi; Pfeffer, Sébastien

We therefore focused on the characterization of the positivestrand-derived small RNAs. One hypothesis regarding their identity could be that some of these SvsRNAs are in fact virus-encoded miRNAs. Using bioinformatics tools such as miRDeep2 (45, 46) and miRanalyzer2 (47, 48) , we excluded the possibility that putative miRNA precursor structures were present in the viral genome. Nonetheless, we tested whether the miRNA machinery was required for the production of viral small RNAs by knocking down Drosha, Dicer, and Ago2. The downregulation of these factors affected neither SINV replication nor accumulation of the three most abundant identified SvsRNAs, indicating that their production must involve another RNase. We discovered that the RNase L endonuclease is implicated in SvsRNA genesis. Indeed, its downregulation causes a dramatic reduction in the accumulation of Sindbis viral small RNAs detected by Northern blot analysis; this is accompanied by an upregulation of the viral genomic RNA. In order to extend this observation, we also generated small RNA libraries from HEK293 cells treated with siRNAs against RNase L and infected with SINV. We found that, under these conditions, the global population of SINV sRNAs was downregulated. RNase L is an endoribonuclease that is present in the cell in the form of an inactive monomer. Upon type I interferon induction, and more specifically 2=-5= oligoadenylate synthetase activation (6) , it can dimerize and then act upon its viral RNA targets. RNase L is known to play an antiviral role against viruses such as West Nile virus (49), hepatitis C virus (50) , and SINV (51) . We also looked at the involvement of exoribonucleases in the production of SvsRNAs and found that, indeed, the accumulation of these small RNAs is counteracted by the cellular 5=-3= exonuclease Xrn1. This suggests that SvsRNAs represent stable viral products of cellular decay enzymes. We therefore hypothesize that RNase L is the enzyme primarily involved in the generation of small RNAs and that the further processing operated by other uncharacterized enzymes could give rise to the final 20-to 30-nt-long viral RNAs. It also remains possible that some of the fragments we detected are direct products of RNase L but are further modified by kinases and dephosphorylases, which would allow their cloning.