| Poliovirus infection remodels intracellular membranes, creating vesicle structures that are closely associated with viral RNA replication proteins. Two such viral proteins, 2BC and 3A, can produce similar vesicles when expressed together, but not individually. Poliovirus-induced vesicles share hallmarks of autophagosomal vesicles including delimiting double membranes surrounding a cytosolic lumen, acquisition of endosomal marker LAMP-1, and recruitment of LC3. Autophagy results in the covalent addition of a lipid onto LC3, which confers LC3 ability to associate with membranes and provides a biochemical marker for the induction of autophagy.;To determine whether the covalent modification of LC3 was responsible for its membrane association during poliovirus infection, we monitored the mobility of endogenous or GFP-fused LC3 by immuno-blot. I found that both poliovirus infection and viral protein 2BC expression were capable of inducing the autophagy-like modification of LC3. The re-localization of LC3, observed during poliovirus infection, was dependent on modification. Mutagenesis of 2BC found that the N-terminal 200 amino acids was sufficient to cause the accumulation of modified LC3, and a point mutation in the amphipathic helix of 2C, I122K, was able to abrogate this activity.;To understand the mechanism behind the modification of LC3 by poliovirus and 2BC, we attempted to use inhibitors of autophagosome degradation and began to look for protein-protein interactions. Bafilomycin A1, a V-ATPase proton pump inhibitor, treatment resulted in dramatic increases in LC3-II amounts under all conditions tested, suggesting a extremely rapid rate of autophagy in resting cells. The amount of LC3-II that accumulated appeared very similar to infection, suggesting that poliovirus may inhibit autophagosome maturation, resulting in an accumulation of LC3. 2BC immunoprecipitation isolated many cellular proteins that could mediate the alteration of autophagy observed. |
