Figure 2.

Deletion of the miRNA cassette neither affects BFV Gag and Pol expression and processing nor BFV particle release and composition in transfected and MDBK-co-cultured HEK293T cells. Each two sub-clones of plasmids encoding high titer pCMV-BFV-MDBK 24 (wt BFV) and pCMV-BFV-MDBK 24-ΔmiRNA (ΔmiR BFV) and the pcDNA2.1 control plasmid were transfected into HEK293T cells together with an EGFP expression plasmid using the PEI method. Two days later, MDBK cells were added and three days later, cells were harvested for protein analyses. Cell culture supernatants were used to enrich BFV particles by sucrose cushion sedimentation. Each 15 µg of cell lysates and regular aliquots of enriched particles were subjected to immunoblotting (as given above the panels) using a cross-reactive rabbit anti PFV IN antiserum [39] (top panels) and a serum pool from BFV-infected cattle (middle panels). The BFV-specific proteins detected are labeled between the panels. A directly conjugated antibody against β-actin served as the loading control (bottom panels).