Mutations in the measles virus C protein that up regulate viral RNA synthesis

The measles virus RNA-dependent RNA polymerase consists of two virus-encode d subunits, the phosphoprotein (P) and the large (L) protein. The P mRNA al so codes for a C protein in the +l reading frame relative to P,The activiti es of the measles P and C proteins from the vaccine strain, EdB, a wild-typ e CM strain, and an SSPE P4 strain were investigated using a CAT reporter m inigenome assay CAT is synthesized following replication and transcription of a DI-CAT minigenome supported by individual P, L, and N plasmids express ed in a mammalian expression system, As measured by CAT activity, CMP1 and P4P1 stimulate transcription and replication four- to six- and six- to eigh tfold, respectively, better than EdP, There are 10 and 16 amino acid change s in the P protein and three and four changes in C in CMP1 and P4P1, respec tively, relative to EdP. By constructing chimeric P genes we showed that mu tations throughout P4P1 were required for enhanced polymerase activity, whi le only mutations in the 5 ' -terminal portion, encompassing the C ORF, of the CMP1 gene mediated stimulation. Abrogation of C expression from the Ed and CM P genes resulted in an increase in RNA synthesis of twofold for CMP1 S and four- to fivefold for EdPS. With the addition of C protein expressed from a separate plasmid that contains only the C ORF, EdC reduces viral RNA synthesis more strongly than CMC, These data suggest that EdC and CMC prot eins give a differential inhibition that accounts for most of the differenc es in RNA synthesis by EdP and CMP1. (C) 2001 Academic Press.