Fig 4. Reverse genetic analysis of the roles played by contacting CP side chains in virion assembly.

(A) Wild-type and mutant EV-E genome concentrations following transfection, quantified by qPCR of region 6,981–7,081 nts. Orange bars show encapsidated viral RNA concentrations, black lines show total viral RNA concentrations. The dark orange bar labelled virus is EV-E virion infection (using virus concentration equivalent to transfections), Cells refers to cell only control, GNN is EV-E mutant with inactivated replicase. (B) Plaque assays of transfection products. Quantification of three times-passaged EV-E transfection products by plaque assays and qPCR. Following qPCR, the products were run on a gel to confirm the expected length. Lanes on gel were loaded in the order from qPCR graph; only WT showed a band which corresponds with only WT being above detectable limit on qPCR, and only WT producing plaques. (C) Western blot of WT and W38A plasmid transfection lysate probed with anti-VP3 EV-E, stained with Western Blue. (D) Light-scattering of WT (green) and W38A (red) concentrated lysate with poliovirus (PV) ECs as reference (grey, dashed line) fractionated on a TSK 6000 column in PBS. EM of WT and W38A peak fractions, scale bar represents 100 nm. (E) Protein standards run on the same column and conditions as (D) showing a plot of elution points (mL) versus known molecular weight (kDa); vertical dashed line represents the peak elution point of ECs.