Hepatitis B virus production is enhanced through early autophagy, but degraded through autophagosome-lysosome fusion

 

Background and aims: Autophagy is a catabolic process that mediates the removal of long-lived proteins and damaged organelles through a lysosomal degradative pathway. Recently, several studies reported that celluar autophagy may have complex interconnections with hepatitis B virus (HBV) replication and envelopment. However, the definite mechanisms of different stages of the autophagic process affecting HBV production are unclear.

Methods: In the present study, we investigated whether and how HBV production was modulated through early and late autophagy. Two proteins Rab5 and -7 related to the formation of early autophagic bodies and autophagosomes were chosen to study their roles in HBV replication.

Results: HBV replication, HBsAg secretion, and intracellular HBsAg expression were significantly decreased by Rab5 silencing. The intracellular level of HBsAg was strongly decreased about 40% by Rab5 silencing. These results were consistent with previous findings that blocking initiation of autophagy by silencing the components of ULK1 complex or using 3-MA significantly reduced HBV replication and HBsAg formation.

In constrast, silencing Rab7 significantly enhanced HBV replication and HBsAg production. The level of intracellular HBsAg increased about 33% by Rab7 silencing. Interestingly, Rab7 silencing led to the promotion of autophagic flux without reduced autophagic protein degradation. In reverse, Rab7 overexpression significantly decreased the level of HBV replication and HBsAg production. Considering the function of Rab7 in autophagosome formation, our results suggest that the delivery of HBV capsids and HBsAg to the lysosome may facilitate their degradation. Thus, enhanced HBV replication and HBsAg production by Rab7 silencing was a result of blocking the fusion of autophogosome with lysosome. This hypothesis could be confirmed by silencing or overexpression of the tethering genes SNAP29 and VAMP8, which are involved in mediating autophagosome-lysosome fusion, showing the same effect on HBV replication of HBsAg. In the same line, the blockage of lysosome function by chloroquine increased HBV replication and HBsAg formation. Confocal microscopic analysis directly demonstrated the accumulation of HBsAg in autophagosomes in the presence of chloroquine.

Conclusion: Our findings provide new evidence indicating that HBV replication and HBsAg production requires early autophagic peocess but will be degraded to a significant part in autophagosome compartment.