Reconstitution of RSV RNA synthesis <i>in vitro</i>.

<p>(A) Sequence of the 25 nt TrC RNA used in the <i>in vitro</i> assay and the expected complementary Tr sense product. The first A residue the RdRp would encounter at position +14 of the template and the corresponding U residue in the product are underlined. The 3′ nt of the template is marked +1, reflecting the numbering system used throughout the paper, and the +3 initiation site identified in this study is indicated by a black dot. (B) Page Blue stained gel showing isolated wt L/P (lane 1) and mutant L_N812A/P (lane 2) complexes. The bands correlating to the expected migration patterns for L (250 kDa) and P (27 kDa) are indicated. Mass spectrometry of a representative gel showed that the bands indicated with asterisks and dots contain L and P specific polypeptides, respectively. (C) Hsp70 and/or HSC70 co-purifies with RSV L/P complexes. Total insect cell lysates (lane 1) and isolated wt L/P complexes (lane 2) were subjected to SDS-PAGE and Western blot analysis, and probed with an anti-Hsp70/HSC70 antibody. (D) Substitution of asparagine 812 to alanine in the GDNQ motif in L abolishes RSV RNA synthesis in a minigenome assay. Northern blot analysis of RSV transcription and replication products (CAT mRNAs 1/2 and anti-minigenome, respectively) generated from a dicistronic CAT minigenome in cells transfected with plasmids expressing minigenome RNA together with N, P, M2-1 and either wt L or mutant (L_N812A), as indicated. (E) RNA products synthesized by the RSV RdRp. Isolated wt (lanes 2 and 4) or mutant (L_N812A) (lane 3) RdRp was incubated with 0.2 µM template RNA consisting of either TrC 1–25 (lanes 2 and 3) or its complement Tr 1–25 (lane 4), with 200 µM of each NTP in the presence of [α-³²P]ATP. The labeled products were separated by denaturing gel electrophoresis and visualized by autoradiography. Lane 1 shows the molecular weight ladder, representing nts 1–25 of the anticipated Tr product.