Figure 4

PFV Bet does not counteract feA3Z2b. (A) HEK293T cells were cotransfected with 4 μg of pCF-BBtr, 0.8 μg pcDNA or pfeA3Z2b, and 6 μg of plasmid expressing wt FFV or PFV Bet. Viral titers were determined two d.p.t. In the presence of feA3Z2b, FFV-BBtr titers decreased about 3 logs. Unlike FFV Bet, which completely rescues the viral titer, PFV Bet did not counteract feA3Z2b activity. The line above the graph indicates the presence of feA3Z2b. For co-IP experiments, 293T cells were cotransfected with 6 μg of plasmid expressing feA3Z2b-HA and 12 μg of FFV Bet (B) or PFV Bet (C). Two d.p.t., cell lysates (CL) were subjected to co-IP with monoclonal mouse anti-HA IgG or beads only (mock). Precipitated FFV Bet but not PFV Bet proteins were detected by immunoblotting using monoclonal V5 tag-specific antibody and polyclonal hyperimmune sera against PFV Bet, respectively. feA3Z2b-HA, along with a lower molecular weight band that also appeared in mock samples, was detected in all samples using anti-HA IgG.