The Influence Of Hepatitis B Virus Integration On Metabolic Function And Its Underlying Mechanisms

Objective:The primary objective of this study is to elucidate the effects of Hepatitis B virus(HBV)integration on metabolic function,as well as to unravel the underlying mechanisms contributing to HBV integration events.Methods:1.Sequencing Analysis:This study employs a combined analysis of metabolomics and transcriptomics to elucidate the influence of HBV integration on energy metabolism.A cohort of 49 hepatocellular carcinoma(HCC)patients undergoing surgery at the Oriental Hepatobiliary Surgery Hospital was recruited,along with corresponding adjacent noncancerous tissue samples.HBV capture sequencing and transcriptome sequencing techniques were utilized to investigate the quantity and proportion of HBV integration,as well as the expression levels of APOBEC family genes and endonuclease genes.2.Cellular Experiments:This study utilizes confocal microscopy imaging techniques to explore the subcellular localization relationships between HBV,APOBEC3B,and mitochondria.It assesses the effects of HBV integration on APOBEC3B expression,mitochondrial copy numbers,as well as cellular apoptosis and proliferation.3.Statistical methods:The Shapiro-Wilk test was used for normality testing in two-sample comparisons.If the data exhibited a normal distribution and the variances were homogeneous,independent sample t-tests were performed;if the variances were not homogeneous,twosample t-tests with unequal variances were used.When data failed to follow a normal distribution,the Mann-Whitney test was applied for analysis.Metabo Analyst 5.0 was utilized to perform metabolite pathway analysis on differential metabolites identified by organ-like samples.Multiple comparisons were corrected using the Bonferroni method.Correlation analysis utilized the Spearman test to calculate the correlation coefficient for non-normally distributed data.A two-tailed P<0.05 was considered statistically significant.Result:1.The impact of mitochondrial-HBV integration on folate and coenzyme A metabolic pathways.Compared to organelles lacking HBV integration,those with integration display significant increases in metabolites and metabolic enzymes within the folate cycle and its metabolic pathways,while coenzyme A levels remain unchanged.2.The impact of mitochondrial-HBV integration on arginine metabolic pathways.Mitochondrial-HBV integration significantly increases metabolites and metabolic enzymes within the ornithine cycle and its metabolic pathways,while urea levels remain unchanged.3.The impact of mitochondrial-HBV integration on nucleotide biosynthesis pathways.Within the purine biosynthesis pathway,cytidine diphosphate,deoxyguanosine,adenosine diphosphate,adenosine,and adenine significantly decrease in organelles with HBV integration,while inosine nucleoside significantly increases.Within the pyrimidine biosynthesis pathway,thymidine,thymidine monophosphate,cytidine,and uridine diphosphate significantly decrease in organelles with HBV integration.Correspondingly,the expression levels of the corresponding metabolic enzymes also significantly decrease.4.The impact of mitochondrial-HBV integration on glutathione metabolism pathway.While glutamate increase in organelles with HBV integration,the oxidative and reduction forms of glutathione decrease significantly due to upregulation of metabolic enzymes in the bypass.The ratio of reduced to oxidized glutathione between organelles with and without integration remains unchanged.5.An association exists between APOBEC3B expression and endonuclease.The overall correlation coefficient between high expression of endonuclease genes in cancer-adjacent tissue and the APOBEC gene family is 0.755(P<0.001).Notably,among the APOBEC gene family,APOBEC3B exhibits the strongest correlation with each endonuclease.6.The expression of APOBEC3B is increased in cells with integration.Specifically,cells with integrated HBV show higher levels of APOBEC3B expression.Both self-measured samples and the analysis of The Cancer Genome Atlas Program data indicate that APOBEC3B is highly expressed in HCC tumor tissue(P<0.001).Moreover,high APOBEC3B expression is associated with shortened patient survival time(HR=1.5,P=0.031).7.The expression of APOBEC3B and ANG is positively correlated with HBV expression and integration in cells.In cell experiments,the expression levels of APOBEC3B and ANG exhibit a consistent trend with changes in HBV expression and mitochondrial HBV integration.Furthermore,there is a positive correlation between the expression levels of APOBEC3B and ANG in cells(r=0.707,95%CI: 0.397-0.873,P<0.001).8.HBV integration promotes apoptosis,inhibits proliferation,and increases mitochondrial copy number in HCC cells.Results of cell experiments indicate that cells with HBV integration exhibit increased apoptosis and inhibited proliferation(P<0.05).Additionally,the mitochondrial copy number in cells with HBV integration increases(P<0.05).9.HBV,APOBEC3B,and mitochondria co-localize in the cytoplasm of cells.Confocal microscopy imaging shows that HBV,APOBEC3B,and mitochondria are all diffusely distributed in the cytoplasm and overlap in certain regions.10.The impact of mitochondrial-HBV integration on mitochondrial function.The impact of mitochondrial-HBV integration on mitochondrial function is mainly manifested in differences in metabolic processes such as cellular respiration and aerobic respiration,which are mainly encoded by genes located in the mitochondrial matrix and inner membrane.Consequently,genes related to mitochondria are enriched in metabolic pathways such as central carbon metabolism,amino acid metabolism,and biosynthesis of co-factors.Conclusion:Mitochondrial-HBV integration can alter aerobic respiration and induce oxidative stress in tumor cells.HBV infection may induce HBV integration by upregulating the expression of APOBEC3B and activating nucleases,such as ANG,through inflammatory factors.