Analysis of the protein expression and virus recovery from the T7-IRES genomic clones. (A) Comparison of the in vitro translation products derived from the FL MNV genome constructs and from clones pCINterm, pCINTPase, and pCIPol encoding sequences of NS1-2, NS3 and NS7 proteins (Sosnovtsev et al., 2006). The plasmids were translated in the TnT system (Promega) in the presence of [35S]-methionine. The synthesized radiolabeled proteins were resolved in a 4–20% gradient Tris-Gly polyacrylamide gel and visualized by autoradiography. Translational products corresponding to MNV nonstructural proteins NS1-2, NS3, NS4-5-6-7, and NS7 are indicated with arrows. (B) Plaque formation assay of the recovered MNV and FCV viruses. The BHK-T7 cells were transfected with T7-IRES-FL MNV or FCV clones as described in text, and production of infectious virus particles was assayed on RAW264.7 and CRFK cells, respectively.