Mitochondrial HIGD1A Inhibits Hepatitis B Virus Transcription And Replication Through The Cellular PNKD-NFκB-NR2F1 Nexus

BackgroundChronic hepatitis B virus(HBV)infection remains a severe health problem worldwide.Chronic HBV infection can be classified into five phases,in which Immune tolerant(IT)and Inactive carrier(IC)phase have similar inactivated immune status,yet the HBV viral load is hugely different,little was known about the mechanism.Objective and ValueOur team aims to investigate the inhibitory effect of human hepatocyte membrane protein HIGD1A on HBV replication in human hepatoma cell lines and HBV replication mouse model,the mechanisms under which will be revealed simultaneously.The results suggest that HIGD1A may be a new target to suppressing HBV replication.MethodsPotential host genes inhibiting HBV replication will be uncovered by analyzing the liver gene expression profiles in total 83 patients with chronic HBV infection including those in immune tolerance and Inactive carrier phases through transcriptome microarray analysis;Human and mouse HIGD1A expression plasmids together with si-HIGD1A and sh-HIGD1A are constructed to investigate the effect of HIGD1A on HBV transcription,replication and antigen production in Huh7,HepG2,HepG2.2.15,HepG2-NTCP12 cells and in HBV replication mouse model;Tetracyclin induction experiment and Luciferase reporter assay are performed in HepAD38 cells and Huh7,HepG2 cells respectively to elucidate the timepiont HIGD1A acting on HBV life cycle and effect of HIGD1A on different HBV promoters;Different regions of HIGD1A gene are deleted to investigate its effect on the regulation HBV replication;Subcellular location and expression of HIGD1A will be confirmed by Western Blot and Confocal microscopy;Key signaling molecules are examined in Huh7,HepG2,HepG2.2.15 cells through enhancing or weakening HIGD1A expression and blocking PNKD or NFκB.ReslultsCompared with those immune tolerance patients with high HBV load,the liver HIGD1A level was significantly increased in immune control patients with low HBV load(p=0.0011)and negatively correlated with serum HBV DNA level(p=0.0017,r=-0.379);HIGD1A overexpression could inhibit HBV transcription and replication,inhibit the secretion of viral particles,HBsAg and HBeAg with a dose-dependent manner;while silence of HIGD1A promotes HBV replication and gene expression.In the HBV replication mouse model,establishing by hydrodynamic injection of pAAV-HBV1.2 into the tail veins,the effects of HIGD1A over-expression or silence on HBV replication and gene expression are consistent with in vitro experiment.Mechanismly,Mechanistically,HIGD1A is located on the mitochondrial inner membrane and activates NFκB signaling pathway through binding to PNKD,which further enhanced the expression of a transcription factor NR2F1 to inhibit HBV transcription and replication.Consistently,knockdown of PNKD or NR2F1 and blockage of NFκB signaling pathway abolished the inhibitory effect of HIGD1A on HBV replication.In addition,studies have found that the expression of HIGD1A promotes the proliferation of liver cancer cells and inhibits autophagy,possibly associated with its antiviral effects.ConclusionsHIGD1A exploits the PNKD-NFκB-NR2F1 nexus to act as a host restriction factor of HBV infection.Our study thus shed new lights on the regulation of HBV by hypoxia-related genes and related antiviral strategies.