Genetic and biochemical evidence for pseudoreversion through upregulation of mRNA4.

<p>(A) Endpoint titres after multicycle replication in MDCK cells of viruses with the indicated mutations to segment 7. Values are plotted are the mean + SEM of between 2 and 18 independent rescues. NR; not rescuable in 2 or more attempts. Viruses were also visually classified into normal (black bar) and small (white bar) plaque phenotypes. (B, C) Segment 7 mRNA accumulation. Total RNA isolated from cells infected with the indicated viruses at 6 h p.i. was analysed by RT-primer extension and urea-PAGE using primers specific for segment 7 mRNAs, vRNA or (as a loading control), cellular 5S rRNA. (C) The amounts of mRNAs 1–4 were quantified by phosphorimager and plotted as the mean ± SD of 3 experiments (D) Segment 7 polypeptide accumulation was monitored by western blot analysis of lysates from 293T cells transfected with reverse genetics plasmids for the indicated viruses at 72 h post transfection with the indicated antisera.