The Induction Of Autophagy By Coxsackievirus B3 Infection And Its Molecular Mechanism

Coxsackievirus B3 (CVB3) is the main microbial pathogens that can cause a variety of diseases such as human viral myocarditis (VMC), and the infections are widespread in the crowd. In recent years, the incidence and occident with acute viral myocarditis in our country is going upward, and the disease becomes one of the main reasons of unexplained sudden death in young people, which is a serious threat to human health. At present, the pathogenic mechanisms of viral myocarditis induced by CVB3 are not fully clear. So it is urgent for us to pay more attention to the basic research of this virus.Autophagy, also called self-eating, is a catabolic process which is evolutionarily conserved and intracellular substances turnover in eukaryotes. It normally maintains steady state or homeostasis of the intracellular environment. However, dysregulation of autophagy may contribute to the development of multiple diseases, and this intracellular conserved process has been exploited by some microbial pathogens to benefit their replication, leading to the occurrence of the disease.This study aims to investigate the relationship between CVB3 and autophagy and the molecular mechanism of CVB3-induced autophagy. First of all, we treated 293A cells with different infection doses and time points, then detected the expression changes of LC3-I and LC3-II through Western blot. At the same time, the formation of autophagy-like vesicles was observed by LC3 labeling green fluorescent protein (GFP-LC3) with confocal microscopy. Then we detected the p62 protein expression change by Western blot. We also used stable cell lines (mCherry-GFP-LC3) observing the change of green fluorescence (GFP-LC3)、red fluorescence (mCherry-LC3) and yellow fluorescence with confocal microscopy.The localization of autolysosomes indicated by lysosomal marker such as LAMP1、LysoTracker was observed under confocal microscopy. Finally, we identified the relationship between CVB3, autophagosomes andlysosomes through Western blot, Real-time PCR, flow cytometry and siRNA silencing technology.In vitro cell experiments we found that CVB3-induced a dose-dependent and time-dependent intracellular autophagy. Confocal microscopy results further proved CVB3 can induce autophagy. At the same time we found that the autophagy-like vesicles maintained a stable red fluorescence when the cells were infected with CVB3. Moreover, this autophagy-like vesicles collocated with autolysosomes (yellow punctate distributions) indicated by lysosomes, suggesting that CVB3 infection induced an intracellular complete autophagic process. Inhibition of autophagy reduced the the viral replication.Taking together, we conclude that CVB3 can induce an intracellular complete autophagy, and inhibition of autophagy significantly reduced viral replication. It suggests that autophagy may play an important role in the proliferation, maturation and pathogenicity of virus. Our study may reveal the possible mechanism to provide clues and ideas on treatments of viral myocarditis and other diseases.