The measles virus RNA-dependent RNA polymerase consists of two virus-encoded subunits, the phosphoprotein (P) and the large (L) protein. The P mRNA also codes for a C protein in the +1 reading frame relative to P. The activities of the measles P and C proteins from the vaccine strain, EdB, a wild-type CM strain, and an SSPE P4 strain were investigated using a CAT reporter minigenome assay. CAT is synthesized following replication and transcription of a DI-CAT minigenome supported by individual P, L, and N plasmids expressed in a mammalian expression system. As measured by CAT activity, CMP1 and P4P1 stimulate transcription and replication four- to six- and six- to eightfold, respectively, better than EdP. There are 10 and 16 amino acid changes in the P protein and three and four changes in C in CMP1 and P4P1, respectively, relative to EdP. By constructing chimeric P genes we showed that mutations throughout P4P1 were required for enhanced polymerase activity, while 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 CMP1S 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 proteins give a differential inhibition that accounts for most of the differences in RNA synthesis by EdP and CMP1.
Copyright 2001 Academic Press.