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, respectively. An RPV minigenome construct carrying the CAT coding sequence was used as a reporter to investigate residues in the 3'-terminal region of the genome important for these functions. Single-base scanning mutagenesis showed that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader had an extremely inhibitory effect on transcription and/or encapsidation of the minigenome, with CAT expression reduced to 0-10% of control values. Changes 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 nucleotides 16, 17 and 20, where mutations did not affect CAT expression significantly. Mutagenesis in blocks identified critical residues in positions 23-26, but changes to leader residues 27-48 had no major effect on CAT expression. A region of about 16 nucleotides (49-65) located around the start of the nucleocapsid gene, including the intergenic triplet CTT, was identified as essential for minigenome function. Mutations further into the nucleocapsid gene (nt 66-89) had a moderate effect (CAT activity 20-60% of control), while at least one critical residue was found in positions 93-96. The importance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G79 was found to be optimal, though not critical, while a purine was required at 85 and 91. Although G97 is conserved in morbilliviruses, all bases were equally effective at this position.