The role of autophagy in enterovirus-71 infection and the therapeutic effect of autophagy inhibition by Saikosaponin D

Abstract

Enterovirus-71 (EV71) is a positive single-stranded RNA virus from the Picornaviridae family. Autophagy is a conserved degradation process that helps cells to maintain homeostasis. Previous studies reported that EV71 infection induced autophagy in vitro and in vivo, but the underlying mechanism for the autophagy induction, as well as the role of autophagy in EV71 reproduction, remain unclear. The way in which cellular organelles or components participate in the EV71 life cycle is also unknown. Herein, I studied the life cycle of EV71 infection and investigated the role of autophagy during EV71 replication. I found that EV71 infection induced autophagy in the early stage, independent of mammalian target of rapamycin (mTOR) in HeLa cells. EV71 replication occurred in both autophagosomes and endosomes, but not lysosomes. Autophagic induction in the early stage promoted EV71 replication and virions reproduction. Moreover, autophagic inhibition in the late stage increased the accumulation of EV71 double-stranded RNA (dsRNA), which is the replication intermediate, due to the accumulation of autophagosomes, and reduced EV71 virions reproduction. These data suggest that induced accumulation of autophagosomes, either by autophagic induction in the early stage or autophagic inhibition in the late stage, increased the accumulation of EV71 replication intermediates and that the autophagosome maturation process is required for EV71 virions reproduction. Furthermore, EV71 infection triggered Golgi fragmentation, which could be further regulated by autophagy modulators. I also found that early endosomes and endoplasmic reticulum (ER) were involved in the EV71 infection process. On the other hand, EV71 infection did not affect the general endosomal-lysosomal degradation pathway. These findings suggest that autophagy modulation may be a possible and novel method to reduce EV71 infection, since autophagy played an important role in EV71 replication and virions reproduction. EV71 infection causes Hand Foot and Mouth Disease (HFMD); currently, there is no specific drug against EV71 for the treatment of HFMD. Many traditional Chinese medicines function as autophagy modulators, and some active compounds isolated from Chinese medical herbs have shown anti-virus effects. I investigated the way in which Saikosaponin D (SsD), one active component from Bupleurum falcatum, modulated autophagy, and whether it may be useful against EV71 infection. I found that SsD reversibly blocked fusion of autophagosomes with lysosomes in HeLa cells, and slightly inhibited the endosomal-lysosomal degradation pathway. SsD-induced lysosomal alkalinization may contribute to its inhibition effect on autophagy. Ras-related protein 5A (RAB5A) is a small GTPase involved in lysosomal fusion: knockdown of RAB5A or expression of the dominant negative form of RAB5A (RAB5A-DN) inhibited SsD-induced autophagosome accumulation, suggesting that RAB5A was necessary for autophagy arrest by SsD. SsD strongly inhibited EV71-induced cell death and it inhibited EV71 reproduction, possibly by blockage of fusion between autophagosomes and lysosomes. This is consistent with the suggestion that inhibition of autophagy by SsD likely reduced EV71 infection. Therefore, my study suggests that SsD is a specific and potent inhibitor of autophagy in the late stage and that it may be useful as a potential therapeutic drug for EV71 infection.