Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients. Author summaryHepatitis B virus (HBV) infects more than 250 million people worldwide chronically. It is a major pathogen causing liver cirrhosis and hepatocellular carcinoma now. The HBV capsid protein (HBc) plays multiple roles in the viral life cycle, and many antivirals targeting HBc such as Heteroaryldihydropyrimidine compounds (HAPs) are under clinical trial recently. This study aimed to investigate how a HAP compound Bay41-4109 induces the degradation of HBc protein. Bay41-4109 induces aberrant non-capsid polymers, which form in complex with the chaperone-binding E3 ubiquitin ligase protein STUB1 and co-chaperone BAG3 and are transported to the perinuclear compartment. Subsequently, Bay41-4109-induced aberrant non-capsid polymers are removed by p62-mediated macroautophagy and lysosomal degradation. STUB1 overexpression accelerates Bay41-4109-induced degradation of HBc protein, and thus enhances the effect of Bay41-4109 on inhibiting secretion of HBeAg and HBV virions. When Bay41-4109 are enforced during HBV infection, de novo cccDNA formation were also negatively regulated by STUB1 overexpression. Altogether, this study provides novel mechanistic insights into developing more potent and safe HAP-based antiviral treatment.

Automated summary

This study investigates the mechanism of degradation of HBc protein induced by HAP compound Bay41-4109. Bay41-4109 induces the formation of aberrant non-capsid polymers, which are removed by p62-mediated macroautophagy and lysosomal degradation. Overexpression of STUB1 enhances the effect of Bay41-4109 on inhibiting secretion of HBeAg and HBV virions, and negatively regulates de novo cccDNA formation during HBV infection.