FIG 5.

Inhibition of MeV infection by an shRNA targeting the P mRNA (si2) is not alleviated by the expression of an shRNA-resistant P protein in trans. (A) Schematic representation of the MeV genome expressing GFP upstream of the N gene (top scheme). Inhibition of the expression of F (filled symbols, continuous line), gfp (open symbols, dotted line) after infection (MOI = 1) of parental 293T (∅, diamonds), or 293T cells constitutively expressing a shRNA against P (si2, triangles) or GFP (si1, squares) mRNA was assessed. The inset shows the expression of N and P proteins. Protein expression was determined by flow cytometry (curves) and Western blot at 96 hpi (inset). For cytometry analysis of F expression, cell-cell fusion was prevented by adding fusion inhibitor peptide z-fFG after MeV infection. (B) Kinetics of virus production after infection (MOI = 1) of parental (diamonds), si1 (squares), and si2 (triangles) cells and protein contents of purified virions collected at 96 hpi as determined by Western blotting (inset). (C) Ongoing RNA silencing does not affect infection by MeV or MeV expressing luciferase as the reporter gene, nor does MeV infection inhibit an ongoing RNA silencing. Cells constitutively expressing a miRNA-based shRNA targeting the luciferase gene or endogenous TNPO3 gene were infected with MeV or MeV-luc virus (MOI = 1) for 1 day. TNOP3 and virus N protein expression were determined by Western blotting. (D) Inability of P protein transiently expressed from a shRNA resistant transcript (rP) to restore MeV growth in si2 cells (MOI 4). The inset presents the protein expression, as determined by Western blotting, showing resistance and sensitivity to si2 shRNA of the rP and P constructs, respectively. In the absence of urea, P migrates as a doublet due to its phosphorylation heterogeneity.