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.

defined, nor is it clear if CR-VI acts on its own or in conjunction with other domains, whether it mediates both N7-and 2 0 O-methylation and which of these methylations would take place first. Research into the activities of L and the mechanisms underpinning them has historically been hampered by a complete, order-wide absence of high-resolution structures. To redress this, we set out to study L domains of hMPV, a paramyxovirus of the Pneumovirinae subfamily closely related to respiratory syncytial virus (RSV). Like RSV, hMPV is highly contagious and causes respiratory tract disease 7 . As a part of this study, we expressed CR-VI þ , a 406-residue fragment comprising CR-VI and the adjoining ' þ domain', the variable region carrying the K-K-G-motif, investigated its MTase activity and solved its crystal structure. Besides sequentially methylating the 2 0 O and N7 atoms of small capped RNAs, CR-VI þ also 2 0 O methylates uncapped substrates and displays nucleotide triphosphatase (NTPase) activity. Both the CR-VI domain, which assumes a fairly standard MTase fold, and the K-K-G motif of the (mainly helical) þ domain are required for the MTase reactions. Combined, the data provide new insights into the modification of the 5 0 -ends of transcripts emerging from the polymerase domain of L. This structural information on a mononegavirus L protein, and the new insights in the capping mechanism it provides, should spur the development of novel antiviral drugs against this important group of highly pathogenic viruses.