X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

MTase activities. In vitro, CR-VI þ most effectively binds and methylates synthetic RNAs containing the conserved start sequence of hMPV transcripts, preferring a substrate length of nine nucleotides (Fig. 2) . The methylation occurs at the GN7 and N 1 -2 0 O positions (Figs 2 and 3a), with 2 0 O-methylation preceding N7-methylation (Fig. 3a) , an uncommon order of events also occurring in VSV 8 . CR-VI þ , moreover, efficiently methylates uncapped RNAs with 5 0 -phosphate groups (especially pppRNA), primarily at the 2 0 O atom of N1 since almost no 3 H-methyl . Substrate specificity was determined as above, but using various synthetic RNA substrates, and allowing the reactions to proceed for 16 h. Substrates were compared with GpppGGGACAAGU (red and blue panels) and pppGGGACAAGU (green panel), for which the degree of methylation was set at 100% (#, marked bars). The red-shaded panel compares the degree of methylation of nine-nucleotide-long hMPV start sequences with different 5 0 -ends and methylation states (the lighter bars represent uncapped RNAs). The results indicate efficient methylation of RNAs that already carried a (cold) methyl group, either at their N7-guanine or 2 0 O-ribose position (confirming the occurrence of 2 0 O-and N7-methylation, respectively), and of uncapped RNAs (especially pppRNA). The pppRNA substrate is predominantly methylated at N 1 , as substrates that were methylated beforehand at this nucleotide do not undergo substantial additional methylation (green panel). CR-VI þ prefers the hMPV start sequence over the short GpppACCCC sequence, and over RNA-start sequences of Dengue virus and SARS coronavirus, irrespective of their lengths (blue panel). A nine-nucleotide hMPV substrate, however, is much preferred over one with only five nucleotides, indicating that additional interactions take place between the protein and nucleotides 6-9. Consistently a 10-times lower K D (dissociation constant) characterizes the interaction of CR-VI þ with the 9-mer, compared with that with the 5-mer. The K D s, measured in triplicate using a dot-blot assay and listed at the right of the diagram ( ± s.d.'s), also show that capped and uncapped hMPV sequences are bound with comparable affinities, and that 2 0 O-methylated substrates are preferred over unmethylated ones.