Increased type I IFN induction by viral replicase Triggers the Shutdown of Mutant SFV Replication.

<p>(A–D) MEFs were infected at 37°C with SFV4-Rluc and SFV4-Rluc-RDR at an MOI of 1. At the indicated times, cell lysates were prepared for the Renilla luciferase reporter assay (A), western blot assay (C and D), and IFN-β secreted into the cell culture supernatants (B) was measured by ELISA. RLU, relative light units; ND, not detectable; h.p.i., hr post-infection; *, non-specific cellular protein, recognized by nsP4 antibody. (E–H) MEFs were infected at 37°C (E and F) or 28°C (G and H) with the mutant SFV4-Rluc-RDR reporter virus at various MOI values prior to the performance of functional assays, as described in (A–D). (I) Scheme for capped SFV4-Rluc-RDR viral RNA genome replication and transcription driven by the viral replicase. RRR→RDR, nsP2 NLS mutation; SG, subgenomic promoter; n, variable number of residues; (+), positive-strand RNA; (−), negative-strand RNA. (J) MEFs were infected at 37°C or 28°C with the SFV4-Rluc-RDR virus at an MOI of 0.1 or an MOI of 1. Total RNA was extracted and analyzed by northern blotting with a digoxigenin-UTP labeled RNA-probe complementary to the viral positive (upper panel) or negative (lower panel) RNA strands. Equal amounts of total RNA were loaded into each lane. The data are representative of two (A, B, and J) or three (E–H) independent experiments. Immunoblots (C and D) are representative examples of two independent experiments.