Functional GFP expression in rhMPV/ΔM2-2/GFP6 poly A by A nucleotide insertion. A) rhMPV and rhMPV/ΔM2-2 viruses containing the native GFP ORF or GFPpolyA sequences, harboring an engineered poly A tract that silenced the translation of GFP, formed comparable plaques in Vero cells. B) Western blots indicated F expression was comparable between viruses. C) A duplicate Western blot was probed with antibody directed to actin to serve as a loading control. D) GFP was detected by Western blot in viruses that contained native GFP ORFs. E) Fluorescence was robustly detected in viruses that contained native GFP ORFs, was readily detectable in some fluorescent foci in rhMPV/ΔM2-2/GFPpolyA, and was not detected in rhMPV/GFPpolyA. F) Nucleotide insertion of one A restored function of GFPpolyA ORF. Nucleotide insertion of 3 As would not restore functional GFPpolyA, but indicated heterogeneity at this polyA locus.