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.

A potential role of CR-VI þ in cap addition. K-K-G residue K 1995 corresponds to the Gp binding lysine in the signature motif of eukaryotic GTases, but here is in a loop instead of an a-helix ARTICLE Mg þ þ , resulted in radioactive protein bands on denaturing SDS gels, the level of radioactivity was low, and was not diminished by acid treatment before SDS-polyacrylamide gel electrophoresis (SDS-PAGE), implying it is not due to phosphoamide bond formation. A second, strong argument against a GTase-based capping mechanism in hMPV (and in favour of a PRNTase-based one) is the fact that in the closely related RSV the cap is formed by Gpp ligation to pRNA 20 . We observed that CR-VI þ also displays NTPase activity, converting GTP into GDP and ATP into ADP (Fig. 7) . GTPase activity, which is required for PRNTase-based capping, was previously reported in Mononegavirales L (ref. 21 ), but as yet could not be linked to a specific domain within the protein. The reaction observed with CR-VI þ , however, is quite slow, possibly because other parts of L, or other co-factors, are needed for full activity. In line with this, we were not able to identify key activesite residues. Using the mutants listed in Fig. 3b , the greatest reductions in GTPase activity were obtained with E 1697 C (corresponding to the flexible residue at the bottom of SAM P; Fig. 4d ) and S 1669 A (part of SUB P, Fig. 4b) to 54 (±20) and 57 ( ± 16) % of the wild-type activity, respectively (n ¼ 3). The deletion of the dipeptide G 1645 K 1646 from the long N-terminal loop resulted in a somewhat more pronounced B70% reduction ( Supplementary Fig. 6 ). The NTPase activity was confirmed using crystallized CR-VI þ (Fig. 7 ).