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.

Cloning and expression. The sequence encoding CR-VI þ was PCR amplified using primers that added a C-terminal SGHHHHHH-tag to the translation product, from a synthetic hMPV L gene (hMPV isolate 00-1, GenBank: AF371337.2), codon optimized for expression in Spodoptera frugiperda (Sf) cells (Geneart). The amplicon was cloned into pOPIN-E for expression in HEK293T mammalian cells (following transfection using Lipofectamine 2000; Invitrogen), in BL21 Star bacteria (Invitrogen) and in Sf21 (insect cells) following co-transfection with (flashBACULTRA) baculovirus (Oxford Expression Systems) using Cellfectin II (Invitrogen) 25 . Mutants were generated by PCR, using primers carrying the mutation (Supplementary Fig. 7) . The CR-VI þ DNA was used as a template for mutagenesis, except for the K 1991 Q/K 1995 Q and K 1991 Q/K 1992 Q/K 1995 Q mutants, which were obtained using the K 1995 Q DNA. For each mutation, the forward primer was combined with a vector-specific reverse primer (5 0 -AGTGGTATTTG TGAGCCAGG-3 0 ), and, in a second PCR, the reverse primer was used together with a vector-specific forward primer (5 0 -CCTTTAATTCAACCCAACAC-3 0 ). The two PCR products thus generated were digested with either BspQI or BtsI (New England Biolabs), restriction enzymes that cut within the CR-VI þ -specific primer regions. The amplicons were then rejoined using T4 DNA ligase (New England Biolabs), and the ligation products were PCR amplified using the vector-specific primers for insertion in the pOPIN-E vector.