Hsp90 Inhibitors Suppress HBV Replication By Degradation Of Hbc

Hepatitis B virus(HBV)infection is a major risk factor for chronic hepatitis,cirrhosis,and even liver cancer.Heat shock protein 90(Hsp90)is an important molecular charperone in cells,which assists in the correct folding,successful assembly,functional stability and degradation of abnormal proteins.Studies have shown that Hsp90 inhibitors prevent the substrate protein from being properly folded by Hsp90,which may promote Hsp90 to recruit E3 ubiquitin ligase to degrade the substrate protein of Hsp90.Hsp90 has become a hotspot in development of cancer drug,because of its ability to block cell growth,proliferation and signal transmission.Studies have shown that Hsp90 inhibitors can block the interaction of HBV polymerase with the pregenomic RNA(pgRNA),reduce the formation of ribonucleic protein(RNP)complex,which in turn inhibit viral RNA packaging and DNA replication.However,this study only demonstrated the effect of Hsp90 inhibitor on the replication of HBV in vitro,and the relevant studies in vivo were not reported.In addition,previous studies have shown that HBV core protein(HBc)is also a substrate protein of Hsp90,and HBc plays an important role in virus replication cycle and pathogenesis.It is not clear whether and how Hsp90 inhibitors act on HBc and affect HBV replication.Therefore,this study intends to explore the mechanism how hsp90 inhibitor affects HBc,and further confirm its effect in HBV transgenic mice.Firstly,Huh7-HBc stable cell line was constructed,and the expression of HBc was verified by Western Blot.Huh7-HBc was treated with Hsp90 inhibitor(17AAG),and the expression of HBc protein was down-regulated by Western Blot.By constrast,17 AAG had no effect on HBc mRNA level through real-time qPCR detection.In addition,17 AAG was demonstrated to reduce HBc protein without relying on HBx or HBpol.Then,the half-life of HBc protein was shortened by treatment of Huh7-HBc with a cycloheximide.Besides,in order to detect the effect of 17 AAG on the replication capacity of HBV,the HepG2.2.15 cells were treated with 17 AAG.HBc,capsid and HBV DNA level were detected by Western Blot,1% agarose gel electrophoresis and real-time qPCR in HepG2.2.15 cells,respectively,demonstrating that 17 AAG inhibited HBV packaging and replication at the cellular level.Finally,HBV transgenic mice were treated with 17 AAG.The HBV DNA and HBsAg levels in serum of transgenic mice were reduced by quantitative detection.Combined results of gel electrophoresis and immunohistochemical detection,17 AAG could reduce HBc level in the liver of transgenic mice and inhibit the formation of nucleocapsid,while 17 AAG had no significant effect on the histological morphology of liver.17 AAG was detected by Southern Blot to inhibit the synthesis of HBV replication intermediates in the liver of transgenic mice.By doing these,17 AAG was found to inhibit the packaging and replication of HBV in vivo.To sum up,the research results showed that Hsp90 inhibitor 17 AAG made the balance tends to the degradation of HBc,thereby could inhibit the formation of HBV capsid and the packaging and replication of HBV.The inhibitory effect of Hsp90 inhibitor on HBV replication was demonstrated for the first time in HBV transgenic mice.This will provide new ideas for the treatment of HBV infection.