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

As shown in Fig. 4A , we designed a reverse transcription primer specific for the viral genome, as well as primers for semiquantitative PCR in the region surrounding SvsRNA-1 (shown in red): one reverse primer (R) and three forward primers, upstream (F U ) or downstream of the putative modification (F D1 and F D2 ). After retrotranscription with low and high dNTP concentrations, we performed multiplex PCR using primers F U /F D1 (in the SvsRNA-1 region) or primers F D1 /F D2 (in the control region) and then compared the signal intensity ratios of PCR products amplified with different numbers of PCR cycles (Fig. 4B ). Our data indicated that only the F U /F D1 ratio changed depending on the RT conditions (Fig. 4B, top panel) , suggesting that at a low dNTP concentration, the reverse transcriptase paused in the SvsRNA-1 region, most probably due to the presence of a modification on the viral genome in the region of interest.