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

The production of SvsRNAs does not rely on the miRNA biogenesis machinery. The identification of small RNAs from RNA viruses could be seen either as a result of a productive mechanism for the pathogen (as for virus-encoded miRNAs) or as the consequence of a host response to control the infection by degrading viral RNAs. SINV expresses four enzymes that play key functions during the viral replicative cycle. However, none of these enzymes is known to possess an endoribonuclease activity (22) that could enable the processing of small RNAs from a viral RNA precursor. Therefore, cellular enzymes have to be involved in the biogenesis of SINV sRNAs. In order to identify them, we decided to knock down cytoplasmic enzymes with a known RNase activity. We first focused on proteins important for the production of small RNAs, such as miRNAs. Hence, we transfected siRNAs against Drosha, Dicer, or Ago2 followed by an infection with SINV and an analysis of viral small RNA accumulation. Although Drosha is known to be mostly nuclear, we also took it into consideration because it was recently shown to relocalize to the cytoplasm upon SINV infection (21) . Although we were able to downregulate very efficiently Drosha, Dicer, and Ago2 (see Fig. S4A to C in the supplemental material), the knockdown of these factors neither affected the viral load nor the accumulation of SvsRNA-1, -2 and -3 ( Fig. S4D to G). We also examined whether we could detect an siRNA signature among all viral reads by looking for small RNAs that could perfectly base pair on 19 or 20 nt and present a 2-nt overhang in 3=. A very limited number of such duplexes could be found, but they were not more represented than larger duplexes presenting with 3= or 5= overhangs (data not shown). It has been reported that small RNAs, which are not generated by Dicer or Drosha cleavage, could nevertheless be assembled into Argonaute proteins (e.g., tRNA fragments [27] ). We therefore immunoprecipitated Argonaute proteins followed by Northern blot analysis to see whether SvsRNAs could be detected within the RISC. However, although we could detect the accumulation of the cellular miRNA miR-16, using both an antibody specific for Ago2 (Fig. S4H , left panel) and an antibody able to recognize all four Ago proteins (Fig. S4H , right panel), we were unable to detect SvsRNAs within immunoprecipitated RISC.