Recombinant MeVs with editing site-proximal variants are V-defective.

(A) Genomes of the recombinant MeVs with single P gene substitutions. The numbers on the left of each genome indicate the position of the nucleotide changes relative to the G-insertion site, and the corresponding amino acid sequences are indicated above the P gene. (B) Growth curves of the recombinant viruses on Vero-hSLAM cells. Colors correspond to panel A. Values are shown as the mean of three replicates ± SD. (C) Analysis of RNA editing by the recombinant viruses after infection of HeLa-hSLAM cells 48 h post infection at MOI 0.1. Vertical lines within the chromatograms indicate the G-insertion site. Asterisks above nucleotides indicate the position of the variant nucleotides. Secondary peaks downstream of the G-insertion site reflect the efficiency of RNA editing. (D) Proteins expressed by the recombinant viruses. Westerns blots were performed using lysates from the same infections as in (C), with sera against the N, P, and V proteins. Actin was used as a loading control. Size markers are indicated on the left of each panel. (E) Quantitation of protein expression levels. Values shown as mean of n = 3, ± SD. Calculations were normalized to the p1 ratio of P or V to N. Asterisks indicate statistically significant differences compared to p1 (**, P<0.01, Student’s t-test).