Enhanced Fusion Pore Expansion Mediated by the Trans-Acting Endodomain of the Reovirus FAST Proteins
Figure 5
The endodomain functions as a fusion enhancer independent of direct interactions with the fusogen.
(A) Top panel: Live cells co-transfected with p14G2A and the p14 endodomain (black lines) or empty vector (grey lines) were immunostained using anti-p14ecto antiserum and analysed by flow cytometry. Grey-filled histogram represents auto-fluorescence from mock-transfected cells. Bottom panel: Surface expression of p14G2A at 8 h and 24 h post-transfection in cells co-transfected with empty vector (Vec) or the p14 endodomain (End), as determined by flow cytometry and Overton subtraction relative to mock-transfected cells. Numbers indicate the mean fluorescence intensity increase above mock-transfected cells±S.D. from a representative experiment in triplicate. (B) Cells were co-transfected with the indicated expression plasmids (p14, authentic p14; V, empty vector; FE, FLAG-tagged endodomain; p53, authentic p53; Fp53, FLAG-tagged p53), and cell lysates were immunoprecipitated using anti-FLAG antibody. The immunoprecipitates (IP) and unfractionated cell lysates (L) were processed for Western blotting using anti-p14 (top panel) or anti-p53 (bottom panel) antibodies. The top arrow in the bottom panel indicates the migration of FLAG-tagged p53 and the bottom arrow untagged p53.