Fig 2. In vitro polymerase activity of TaV rORF1 and TaV_pol.

Representative autoradiograms of in vitro TaV RdRP activity assays analyzed in 7% acrylamide TBE gels. (A) Left panel, first lane, ssRNA template labeled with α-³²P GTP; second and third lines, RdRP activities of TaV rORF1 and TaV_pol, respectively, using the previously unlabelled 311-nts long ssRNA template corresponding to the TaV 3’-UTR. Right panel, polymerization activity of TaV_pol performed in: i) absence of template (first lane), ii) presence of the TaV 3’-UTR template (second lane), iii) presence of the TaV 3’-UTR hybridized to a short oligonucleotide primer complementary to an internal template sequence (third lane) and, iv) presence of a non-related viral template (the 3’-UTR of the SJNNV nodavirus; fourth lane). (B) Polymerization activity of TaV_pol on short ssRNA templates. Oligonucleotides of 6-, 12- and 16-nts in length (left) derive from the TaV 3’-UTR and the 13- and 25-nts in length (right) contain TaV unrelated sequences. The negative control (-) was performed in absence of RNA. (C) Different ions (Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺) and concentrations ranging from 1 to 25 mM were employed in polymerization experiments performed under otherwise optimal conditions (see Methods and S1 Fig). A negative control (-) was performed using a reaction mixture lacking metal ions.