FIG. 5.

Effect of expression of individual viral proteins on the accumulation of saponin-resistant GFP-LC3-I and GFP-LC3-II proteins. COS-7 cells were transfected with identical total amounts of DNA plasmids, in mixtures that contained GFP-LC3-expressing plasmids and increasing amounts of DNA that expressed 2BC (A), 2B (B), 2C (C), or 3A (D). The balance of the DNA samples comprised the VSV-G-expressing vector into which the poliovirus sequences were cloned. Cells were incubated for 48 h, collected by centrifugation, and subjected to extraction with 0.5% saponin. Saponin-resistant proteins were displayed by electrophoresis in 10% polyacrylamide gels, except as indicated, and probed with three different antibodies for each panel: anti-LC3, to detect GFP-LC3-I and GFP-LC3-II; anti-VSV-G, to normalize for transfection efficiency and toxicity; and anti-2B, anti-2C, or anti-3A, to monitor the expression of the viral protein of interest. (A) Effect of increasing amounts of poliovirus 2BC protein expression. (B) Effect of 2B protein expression. The gel used to resolve the 2B protein was 13.5% acrylamide. (C) Effect of 2C protein expression. (D) Effect of 3A protein expression, compared to the amount of GFP-LC3-I and GFP-LC3-II that was saponin resistant following poliovirus infection as for Fig. 1. The lower portion of each panel shows the amount of saponin-resistant GFP-LC3-I and GFP-LC3-II detected divided by the amount of VSV-G observed and normalized to the control transfection in which no poliovirus protein was expressed. Some toxicity was observed for the greatest amounts of DNA transfected.