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.

As a GDP is transferred onto a PRNTase-bound pRNA intermediate during cap synthesis in VSV 21 , the presence of NTPase activity in CR-VI þ would suggest that this domain is involved in cap addition. It is unclear from the structure of VSV L how capping, cap methylation and RNA synthesis are coordinated 14 , but an involvement of CR-VI þ in cap addition is consistent with the dynamic nature of the multi-domain polymerases of RNA viruses in general, exemplified by flu where the C-terminal two-thirds of PB2 has been shown to be extremely mobile 22, 23 . The presentation of uncapped (but CR-V-linked) pRNA to CR-VI þ would also explain why hMPV L does not require a high-affinity cap-binding site. The integrity of both the PRNTase and K-K-G motifs, moreover, has been shown to be essential for viable mRNA synthesis in human parainfluenzavirus 2 (hPIV-2) 5 , in line with capping involving a concerted action of CR-V and CR-VI þ . Assuming the transcript is presented to CR-VI þ by the PRNTase, 2 0 O-methylation may actually occur before the cap is added (as a cap is not required for the reaction; Fig. 2b ), which would also be consistent with 2 0 O-methylation preceding N7-methylation. The possibility that 2 0 O-methylation precedes cap addition was also suggested by the ability of virions of the rhabdovirus spring viremia of carp virus to synthesize uncapped, N1-methylated oligonucleotides 24 . We did not observe significant methylation of free GTP by CR-VI þ (levels were o5% of that of GpppGGGAC AAGU methylation), suggesting GN7-methylation takes place after G is added to the RNA. In addition 2 0 O-methylated substrates (GpppG m GGACAAGU and pppG m GGACAAGU) show an B2-fold higher affinity for CR-VI þ , compared with their unmethylated counterparts (Fig. 2b) , which presumably reflects an RNA repositioning mechanism allowing the GN7 atom access to the SAM methyl donor in the second methylation step.