The coding sequence of wt, truncated, and/or substituted L and/or P proteins from MeV (Moraten strain) was subcloned in frame either downstream or upstream of the Gaussia luciferase glu1 and glu2 domains under a cytomegalovirus promoter in the pCi vector (2) and under the T7 promoter in the pEMC vector (30). Some P variants were subcloned into the duplicated P2 gene position in the biG-biS–modified p(+)MVNSe vector (Fig. 2A) (2). The constructs for the eukaryotic expression of the P variants were generated by polymerase chain reaction (PCR) using dedicated oligonucleotides, and the PCR products were introduced into pCi, pEMC, and p(+)MVNSe by recombination using the InFusion Kit (Clontech). Whenever possible, new restriction sites with silent mutations were also introduced in the oligonucleotides to facilitate the screening of Escherichia coli–transformed clones. Plasmids used in the minigenome assay have been already described (12).

The bacterial constructs encoding variants of the P(304-375) MD with a C-terminal hexahistidine tag were subcloned in the pDEST14 vector (Invitrogen) by Gateway recombination (Invitrogen) of PCR products amplified from the corresponding pEMC constructs. All insert sequences were verified and validated by sequencing.

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