The Molecular Mechanism And Inter-genotype Disparity Of Cell Apoptosis Induced By Hepatitis B Virus X Protein

Hepatitis B virus?HBV?can replicate in the infected hepatocytes constantly infections,is an important pathogen that will bring serious harm to human health.HBV infection can promote acute or chronic hepatitis and cirrhosis,which is closely related to the initiation and development of hepatocellular carcinoma?HCC?.Past studies showed that,Hepatitis B virus X protein had been thought as an important protein during the pathogenesis of HBV infection.HBx is a multifunctional protein,which is involved in transactivation of several genes expression,change multiple cellular signaling pathways,promoting cell proliferation and influence the cell cycles,etc.However,to our knowledge,investigators have been busy in focusing on HBx-mediated activation of several cellular signaling pathways in an indirect manner.In this study,we have demonstrated that HBx can interact with Bcl-2 and Bcl-xl through its BH3-like motif in HepG2 cells,then induce cytosolic calcium elevation and cell death.When two mutations?G124L and I127A?are introduced into wildtype HBx,the BH3-like motif of HBx is totally destroyed.HBx mutant with G124L and 1127A mutations fail to bind Bcl-2 and Bcl-xl so as to reduce cell apoptosis through decreasing cytosolic calcium.Otherwise,HBx can interfere the interaction of Bcl-2 and IP3R by competing with Bcl-2 to activate the cellular Ca²⁺signaling pathway.We hypothesize that HBx may promote cell apoptosis through Ca²⁺/Bcl-2 pathway during HBV pathogenesis.Given the facts that the heterogeneity of HBx amino acids across all HBV genotypes,we explore the disparity of four HBx subtypes on regulating cell apoptosis.We establish four doxycycline regulated HBx stable cell lines with genotype A?B?C and D using a method of lentivirus transduction.All HBx subtypes can significantly inhibit the proliferation of hepG2 cells.Several cellular apoptotic markers,such as full length or cleaved caspase-3 and full-length caspase-8,are induced in these four HBx-expressing stable cells.Furthermore,RNA sequencing is employed to analyze the difference of gene expression between HepG2 cells with or without HBx expression.13 apoptosis related genes are found with differentiated expression analysis.Among of them,2 pro-apoptotic genes are upregulated and 9 anti-apoptotic genes are downregulated,which suggests that multiple genes are involved in HBx mediated cell apoptosis.In summary,we try to explore the target genes and molecular mechanism of HBx protein during HBV pathogenesis.We find HBx protein targets the Ca²⁺/Bcl-2 signaling pathway to induce cell apoptosis;we also explore the underlying differences of different HBx subtypes on inducing cell apoptosis.These observations will provide us novel evidences to study the mechanisms of HBV pathogenesis.Therefore,the BH3-like motif of HBx or other related genes could be designed as potent and effective targets for the prevention and management of HBV infection.