A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase
Fig 5
Mutations perturbing the JEV-mode interface interactions impaired DENV2 RdRP initiation.
A) Left: A diagram of construct T30/P2 used in all polymerase assays and related NTP-driven reactions to generate products with different lengths. Middle: reaction flow chart of the P2-driven EC formation (IC2 to EC9) and the subsequent single-nucleotide extension (EC9 to EC10). Right: the EC9 was in a form of precipitate and was able to extend to EC10 upon CTP addition under high-salt condition. B-C) The EC9 formation comparison with the WT NS5 for the JEV-mode (B) and DENV3-mode (C) mutants. The relative intensity of the 9-mer was used to estimate the polymerase activities (the WT value for each time point series was set to 100). D) Comparison of the WT and two representative mutants (R3 for the JEV-mode; M_67A/68A for the DENV3-mode) in the multiple-turnover P3 formation and in the single-turnover P9 formation assays. The pppGGA was synthesized when GTP and ATP were provided as the only NTP substrates using the P2-free T30 template and was used as a migration marker. Note that pppGGA migrated at similar position as pGG, but faster than pGGA since it contains two extra phosphate groups at the 5′ end. A chemically synthesized 9-mer loaded with an equal molar amount to T30 was used as a quantitation standard (STD, lanes 62 and 81). The average intensity of the STD bands was set to 1. Based on previously reported evaluation, the intensity-molar amount starts to deviate from a linear relationship when the relative intensity approaches 4–5 under similar experimental settings [12]. Therefore, the intensity reported in lanes 64–65 and 76–77 are underestimated. The P9 product migrated faster than the chemically synthesized STD RNA due to its 5′-phosphate inherited from the pGG dinucleotide.