FIG. 7.
De novo initiation of RNA synthesis on sapovirus anti-subgenomic RNA. (A) RNA synthesis was examined in the presence of a synthetic anti-subgenomic RNA (Anti-sG RNA) used as a template and in the presence wild-type sapovirus 3Dpol (3Dpol) or an active site 3Dpol mutant (m3Dpol, YGD343GD344G), as indicated. Reaction products were analyzed on nondenaturing agarose gel and visualized by UV transillumination after ethidium bromide staining. In the reaction using m3Dpol instead of wild-type 3Dpol, the residual band observed corresponds to the synthetic anti-subgenomic RNA template used in the reaction. T7, synthetic anti-subgenomic RNA generated by T7-mediated in vitro transcription; M, RNA molecular mass marker; +, present; −, absent. (B) Strand separation analysis of the reaction product of in vitro RNA synthesis by sapovirus 3Dpol. The reaction product was generated from RNA synthesis by sapovirus 3Dpol from a synthetic anti-subgenomic RNA (Anti-sG RNA) used as a template, as indicated. Reaction products were visualized on denaturing formaldehyde-agarose gels. T7, synthetic anti-subgenomic RNA generated by T7-mediated in vitro transcription; M, RNA molecular mass marker. (C) The sapovirus 3Dpol synthesis product (3Dpol-RNA) was generated from synthetic anti-subgenomic RNA (T7) used as a template. The RNA synthesis product was then incubated in the presence (+) or absence (−) of S1 nuclease (S1), in the presence of low-salt (50 mM NaCl) or high-salt (250 mM NaCl) concentrations, or after heating (95°C, 5 min) and rapid chilling on ice for 5 min, as indicated. Reactions products were analyzed on nondenaturing agarose gels and visualized by autoradiography. 3Dpol-RNA, sapovirus 3Dpol synthesis product using anti-subgenomic RNA as a template. T7, synthetic anti-subgenomic RNA generated by T7-mediated in vitro transcription.