Figure 2From: Identification of the feline foamy virus Bet domain essential for APOBEC3 counteractionBet and Bel2ORF suppress feA3Z2b-mediated restriction. (A) Schematic representation of full-length Bet and N-terminal Bet deletion mutants. Dark and light gray boxes represent the different MEME motifs in Bet. Bet and Bel2ORF contain all six conserved motifs, while downstream N-terminal Bet deletion mutants do not contain the first conserved motif. Numbers indicate the first and the last amino acids of the deletion mutants. (B) HEK293T cells were cotransfected with 4 μg of pCF-BBtr, 0.8 μg pcfeA3Z2b-HA, and increasing amounts of wt Bet and Bet deletion mutant expression plasmids as indicated in the legend. pcDNA was used to compensate for different plasmid amounts. Viral titers were determined in triplicate using the FeFab titration assay and are presented as mean values of three measurements; error bars represent standard deviations. Labels below the columns indicate cotransfected clones. The line above the columns indicates the presence of feA3Z2b. The first column shows the viral titer in the absence of feA3Z2b, the second, in the presence of feA3Z2b. The other columns show the titer in the presence of feA3Z2b and the coresponding Bet clones, as indicated in the figure. 0.7 μg of Bet expression plasmid and 5.6 μg of Bel2ORF expression plasmid yielded similar levels of feA3Z2b counteraction. In both cases, the titer increased more than 1 log. The other N-terminal deletion mutants did not counteract feA3Z2b-mediated restriction. (C) 40 μg of protein of transfected HEK293T cells was used for immunoblotting. Bet proteins were detected with the FFV Bet- specific serum. Proper loading was confirmed by detecting β-actin (data not shown). (D) Densitometric analysis of the relative levels of Bet protein expression. Wt Bet at 5.6 μg of transfected DNA was set to 100%. The legend indicates the amount of expression plasmid used to obtain corresponding protein amounts.Back to article page