The recombinant RSV MTase-CTD protein.

(A) Schematic representation of the domain organization of the RSV L protein. Blue, RNA-dependent RNA-polymerase (RdRp) domain; green, polyribonucleotidyl-transferase (PRNTase) domain; yellow, connector domain (CD); orange, methyltransferase (MTase) domain; red, C-terminal domain (CTD). Amino acid residue numbers indicate the functional domain boundaries. The conserved regions (CR) within the L proteins of non-segmented negative-strand RNA viruses are indicated. Arrows denote the position of active site residues required for function, and dots indicate conserved motifs with the starting amino acid residue number in gray. (B) The RSV MTase-CTD fragment was defined based on the alignment with the VSV L protein. Arrow indicates the K₁₈₃₁-D₁₉₃₆-K₁₉₇₃-E₂₀₀₄ catalytic tetrad, typical of 2’-O-MTases. Dots indicate conserved motifs with the starting amino acid residue number in gray: the GxGxGx SAM/SAH binding site motif in the MTase domain, and the K-K-G motif, reminiscent of eukaryotic GTases, in the CTD domain. (C) SDS-PAGE of the purified recombinant RSV MTase-CTD protein containing an N-terminal histidine tag (49.3 kDa). Molecular weights (in kilodaltons) of the ladder are shown on the left, and the MTase-CTD band is labeled on the right.