FIG 7.

Sla5045 can direct the initiation of RNA synthesis by the MNV RdRp in vitro. (A) Recombinant RdRps from MNV and the human GII.4 norovirus used in this study. The RdRps was expressed and purified as described in Materials and Methods. The gel images were from SDS-PAGE and Coomassie blue staining. (B) Schematics of the synthetic MNV proscript, MNVPs, and the GII.4 RNA proscript, GII.4Ps, used for the in vitro RNA synthesis assays. The initiation nucleotide used to initiate MNV subgenomic RNA synthesis is in bold and denoted with an arrow. A proscript containing a mutated initiation site named IM is used to control for specificity. (C) Denaturing PAGE analysis of in vitro RNA synthesis assays using the MNV NS7 with either MNVPs or the initiation mutant (IM). The positions of primer extension (PE) and de novo initiation products are highlighted. The lengths of the RNAs were assigned by comparison to RNAs of 8 to 51 nt that were labeled at the 5′ terminus with ³²P. (D) Virus genotype-specific RdRp-proscript interaction results in a higher level of RNA-dependent RNA synthesis. The results are representative of six independent experiments.