Fig. 7

Polymorphisms of puma and bobcat A3Z3 and their association with PLV Vif sensitivity. a Feline A3Z3 polymorphisms. Additional A3Z3 sequences from five pumas, one cheetah and one bobcat were determined and the phylogenetic tree was reconstructed using the ML method. The A3Z3 sequences used in the experiments above (summarized in Fig. 1c) are indicated with open circles and designated hap I. Additional A3Z3 haplotypes detected in the puma and bobcat are indicated with filled circles and designated hap II. The nucleotide/amino acid substitutions detected in puma and bobcat A3Z3 hap II are indicated in parentheses. Note that the new cheetah A3Z3 sequence was identical to the previously determined sequence (Fig. 1c). The scale bar indicates 0.002 nucleotide substitutions per site. b–f Antiviral activity of the puma and bobcat A3Z3 hap II proteins and their sensitivity to PLV Vif. b, c Different amounts of HA-tagged expression plasmids for A3Z3 hap I and II of puma or bobcat (0, 100, 200 and 400 ng) and the three plasmids used to produce the vif-deficient FIV-based reporter virus (FIV plasmids; pFP93 [200 ng], pTiger-luc [150 ng] and pMD.G [50 ng]) were co-transfected into HEK293T cells. b Western blotting. Representative results are shown. c FIV reporter assay. FIV infectivity is shown as the percentage of the value of “no A3Z3”. Note that “hap I” is identical to those used in the other experiments shown in Figs. 1, 2, 4, 5 and 6. d–g HA-tagged expression plasmids for puma A3Z3 hap II (d, e) or bobcat A3Z3 hap II (f, g) (200 ng) and the three plasmids used to produce the vif-deficient FIV-based reporter virus (FIV plasmids; pFP93 [200 ng], pTiger-luc [150 ng] and pMD.G [50 ng]) were co-transfected with or without His-tagged PLV Vif expression plasmids (400 ng) into HEK293T cells. d, f Western blotting. Representative results are shown. e, g FIV reporter assay. FIV infectivity is shown as the percentage of the value of “no A3Z3”. In b, asterisks indicate significant differences (P < 0.05 by Student’s t test) between hap I and hap II. In e and g, asterisks indicate significant differences (P < 0.05 by Student’s t test) versus “no Vif”. The assays were independently performed in triplicate. Data represent averages with SDs