Host Cellular Metabolic Responses To HBV Replication

Hepatitis B virus (HBV) infection threatens people’s health. It is estimated that approximately350million people are chronically infected by HBV all over the world. Persistent HBV infection can induce fatty liver and hepatofibrosis, and finally develop into hepatocellular carcinoma. Presently, the pathological mechanisms behind HBV-induced diseases are not totally clear, and there is no available medicine, which can cure HBV chronic infection completely. HBV replication is a complicated interaction between the virus and the host cell. Therefore, to investigate the metabolic responses to HBV replication at the cellular level is very important to understand the pathogenesis and the strategy of treatment.In this thesis, an HBV expressing cell line HepG2.2.15and its parental cell line HepG2were used. We aimed to develop nuclear magnetic resonance (NMR)-based metabonomics method with multivariate data analysis for cultured cells, and then to investigate the metabolic responses of the host cell to HBV replication using metabonomics, proteomics and validate the results by a range of molecular biological assays.Here, we established an efficacious method of preparing samples of cultured cells for NMR-based metabonomics analysis. We also improved the data normalization methods for statistical analysis, including cell wet weight normalization for cell extract and normalization for medium extract with adjusted area under the cell growth curve.Metabonomics results showed that HBV replication induced significant alterations in metabolic profiles of host cells. These alterations were characterized by the elevations in the levels of metabolites involved in the central carbon metabolism, increased levels of UDP-N-acetyl glucosamine (UDP-GlcNAc) and UDP-N-acetyl galactosamine (UDP-GalNAc) produced in hexosamine biosynthesis pathway, increased levels of metabolites participating in the anabolism of nucleotides, as well as decreases in metabolites involved in the catabolism. In addition, decreased levels of choline and GSH, and increased levels of phosphocholine were also found in HepG2.2.15cells. GC-MS results showed that HBV replication induced obvious changes of the composition of fatty acids in host cells.The results from proteomics and references confirmed the metabolic alterations further. Taking these results together, we concluded that HBV could modulate the metabolism of host cells by regulating the expressions of corresponding enzymes. HBV replication induced the promotion of glycolysis, the TCA cycle, the pentose phosphate pathway and the biosynthesis of nucleotides. HBV replication also resulted in alterations of fatty acid composition, the accumulation of total fatty acids, as well as deficiency of the essential fatty acids. These metabolic changes could provide biomass and energy for the replication of HBV.Through the target RT-PCR analysis based on the metabonomics results, we showed that HBV replication caused up-regulated hexosamine biosynthesis via GFAT1transcriptional activation and phosphatidylcholine biosynthesis via transcriptional regulation of CHKA, PHOSPHO1and PCYT1A. HBV replication also consumed GSH, giving rise to oxidative stress. These metabolic alterations were closely associated with inflammation, fatty liver and carcinogenesis. Moreover, the inhibition of the activity of GFAT1could repress the replication and expression of HBV; down-regulation of CHKA transcription could inhibit HBV replication as well.In summary, this thesis provides a relatively systematic research on cellular metabolic responses of host cells to HBV replication using the methods of metabonomics in combination with proteomics and molecular biological techniques. The results provide further understandings of the pathogenesis of HBV-induced diseases and shed light on new drug targets for treating HBV infection.