Figure 4
3Dpol enzymes efficiently bind acidic membranes only in the presence of membrane-tethered 3B. (A) Recombinant 3Dpol enzymes from several +RNA viruses bind PI4P poorly in a liposome pulldown assay. 3Dpol from the Aichi virus (AiV), Polio virus (PV), Coxsackie virus B3 (CVB3) and Enterovirus 71 (EV71) show no binding to PI4P liposomes at 60 µM concentration. SidC was used as a positive control and 14-3-3ζ protein as a negative control. Liposomes were centrifuged and the pellets (denoted P) and supernatants (denoted S) were analyzed using SDS PAGE. For the full length gels see the SI Fig. 2. (B) GUV recruitment assay. GUVs of different composition were used. Upper panel – neutral membrane (5% PS), middle panel – PI4P acidic membrane (5% PS + 5% PI4P), lower panel – PS acidic membrane. Aichi 3Dpol fluorescence signal in blue (left), membrane in red (middle) and merged image (right). A typical result of three independent experiments. Scale bar = 20 µm. (C) Quantification of Aichi 3Dpol binding to membranes of different composition. Composition dependent membrane accumulation of CFP-3Dpol: excess of the fluorescence signal of CFP in the GUV pixels relative to the signal of unbound CFP-3Dpol. The error bars indicate a 95% confidence interval. (D) Cros-scorrelation curves of CFP – 3Dpol and LUVs. FCS curves representing temporal cross-correlation of the CFP-3Dpol (Aichi 3Dpol on the left panel and PV 3Dpol on the right panel) and ATTO647N-DOPE labelled LUVs at various membrane lipid compositions (neutral, enriched by PI4P, and enriched by PS; see SI Table 1). The curves show the cooperative effect of the 3B peptide on 3Dpol membrane recruitment. The concentration of the Aichi or PV 3B peptide (attached to the membrane surface by His-tag – 18:1 DGS-NTA(Ni) interaction) was 0 µM (black), 1.8 µM (red), and 2.7 µM (blue).
