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

Total RNA was isolated from uninfected cells or cells infected with SINV at a multiplicity of infection (MOI) of 0.01 plaque forming units (PFU) per cell 16 h postinfection (hpi). Using these conditions, we could detect cells with a high viral load without observing any apparent cell death as assessed by propidium iodide staining (see Fig. S1 in the supplemental material). We also confirmed that until 24 hpi, cells were still actively proliferating (data not shown). High-throughput sequencing yielded 201,793 and 1,193,893 reads that mapped to the SINV genome in infected HEK293 and Vero cells, respectively (Fig. 1A) ; these two numbers of reads represented a percentage of 1.66 and 7.99%, respectively, of total mapped small RNAs in HEK293 and Vero cells. The greater fraction of viral reads in Vero cells is consistent with the fact that these cells are known to be more permissive to viral infections due to a defect in interferon production (26) . The distribution of the reads on the viral genomic (positive) and antigenomic (negative) strands is indicated in Fig. 1A . Interestingly, the vast majority of the reads (98 to 99%) originate from the genomic strand of the virus. Nonetheless, we identified limited sequencing reads that were derived from the negative strand, albeit at extremely low levels in both libraries (Fig. 1A) . Their genomic distribution showed a peak at the 3= end of the antigenome (see Fig. S2A in the supplemental material). This specific region corresponds to the viral promoter, necessary for the replication of the viral genome and is known to fold into a conserved 44-nt stemloop structure (25) . The length distribution of the antigenomic small RNA reads peaked at a size of 22 nt (Fig. S2B) , which corresponds to the 5= arm of the stem-loop structure (Fig. S2C ). Although we could validate the presence of the 44-nt-long RNA by Northern blot analysis, we were unable to detect the accumulation of the small 22-nt RNA as a discrete species (Fig. S2D) .