Scanning mutagenesis identifies critical residues in the rinderpest virus genome promoter

Short regions at the 3' and 5' ends of the genome of Rinderpest virus (RPV) contain signals that regulate transcription of the viral genome, known as the genome promoter and the (complement to the) antigenome promoter, respec tively. An RPV minigenome construct carrying the CAT coding sequence was us ed as a reporter to investigate residues in the 3'-terminal region of the g enome important for these functions. Single-base scanning mutagenesis showe d that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader ha d an extremely inhibitory effect on transcription and/or encapsidation of t he minigenome, with CAT expression reduced to 0-10% of control values. Chan ges in any of the other first 22 nucleotides reduced the efficiency of the minigenome to 20-80% of the wild-type control, with the exception of nucleo tides 16, 17 and 20, where mutations did not affect CAT expression signific antly. Mutagenesis in blocks identified critical residues in positions 23-2 6, but changes to leader residues 27-48 had no major effect on CAT expressi on. A region of about 16 nucleotides (49-65) located around the start of th e nucleocapsid gene, including the intergenic triplet CTT, was identified a s essential for minigenome function. Mutations further into the nucleocapsi d gene (nt 66-89) had a moderate effect (CAT activity 20-60% of control), w hile at least one critical residue was found in positions 93-96. The import ance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G 79 was found to be optimal, though not critical, whil e a purine was required at 85 and 91. Although G 17 is conserved in morbill iviruses, all bases were equally effective at this position.