Activity of polymerase proteins of vaccine and wild-type measles virus strains in a minigenome replication assay

Abstract

The relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.

FIG. 1.

Expression of CAT reporter protein by MV L proteins in a plasmid-based minireplicon assay. MVAT7-infected CV-1 cells were transfected with pTM1-EdmBil N, pTM1-EdmBil P, pMV107(−)CAT, and the indicated L plasmids (A); pTM1-EdmBil N, pMV107(−)CAT, the indicated L plasmids, and either pTM1-EdmBil P or the homologous P plasmid (B); pMV107(−)CAT, the indicated L plasmids with the homologous P plasmids, and either pTM1-EdmBil N or the homologous N plasmid (C); pTM1-EdmBil N, pTM1-EdmBil P, the indicated L plasmids, and either puc26A42G or puc26U42U (D). For the negative controls, one of the four plasmids was replaced with pTM1 as follows: the L plasmid in panel A, the P plasmid in panel B, the N plasmid in panel C, and the minigenome plasmid in panel D. Omission of any one of the four plasmids reduced the background to the same low level. CAT concentrations in cytoplasmic extracts were measured 42 to 48 h after transfection. Each panel shows the results of one experiment that is representative of three. In all of the experiments, the differences between the activities of the L proteins of attenuated strains and those of wild-type viruses were statistically significant, with a P value of <0.0001.

FIG. 2.

Levels of transcription and replication by MV L proteins in the plasmid-based minireplicon assay. MVAT7-infected CV-1 cells were transfected with pTM1-EdmBil N, pTM1-EdmBil P, pMV107(−)CAT and the indicated L plasmids. Micrococcal nuclease-resistant genomic RNA was hybridized with a positive-sense probe (A). Poly(A)⁺-selected mRNA was hybridized with a negative-sense probe (B). For the negative controls, the L plasmid was replaced with pTM1. Each panel shows one of three representative experiments. Molecular mass markers are shown on the right; the positions of genomic RNA and mRNA are indicated on the left. For both panels, RNA was separated by electrophoresis in formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with cat gene-specific, DIG-labeled riboprobes. For visualization by autoradiography, duplicate samples are shown and RNA extracted from one-fifth of a 35-mm-diameter dish was loaded onto the gel. For quantitation of band intensity, the amounts of RNA loaded were adjusted for expected signal strength to prevent saturation of the stronger signals. The signal produced by the CAM-70 L protein was defined as 100%, and the quantitative data are derived from quadruplicate samples. The asterisk denotes a background band that appeared primarily in lanes with strong signals, possibly as a result of incomplete denaturation of the sample. sd, standard deviation; nt, nucleotides.

FIG. 3.

Expression of MV N and P proteins. MVAT7-infected CV-1 cells were transfected with the indicated plasmids. ³⁵S-labeled proteins were immunoprecipitated with N- or P-specific rabbit antisera and separated by SDS-12% polyacrylamide gel electrophoresis. Molecular mass markers are shown on the right; the positions of the N and P proteins are indicated on the left. Vector: cells transfected with pTM1 as a control. (A) Expression of N proteins; B, expression of P proteins. (B) A protein of approximately 90 kDa was precipitated nonspecifically in every lane.