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Title: The effect of APOBEC3 deaminases on HBV replication and the role of HBx in the inhibition of RNA interference and in the development of hepatocellular carcinoma
Author: Mohammed, Essam Mohammed Ahmed
Awarding Body: Imperial College London (University of London)
Current Institution: Imperial College London
Date of Award: 2007
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Abstract:
Hepatitis B is one of the world's major infectious diseases. Some 350 million people are chronic carriers of the virus (HBV), a significant minority go on to develop cirrhosis or cancer of the liver and over 1 million die annually from HBV liver disease. An effective vaccine has been available for nearly 20 years, however, vaccination cannot be used to treat established infections. The replication strategy of this virus has been described in detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. The main objective of this thesis is to investigate the interaction ofHBV, in particular the HBx protein, with recently identified anti-viral pathways (RNA interference and APOBEC3 deaminases) and the relationship of HBV proteins to viral pathogenesis. The DNA editing enzyme APOBEC3G has been shown to cause lethal G to A mutations in replicating retroviruses during reverse transcription. HBV replication involves a reverse transcription step and recent evidence indicates that APOBEC3G can also interfere with HBV replication. I hav(fr.shown by RT-PCR that HepG2 and Huh7, liver cell lines, express very little APOBEC3G mRNA. Consequently, to investigate the effect ofAPOBEC3 deaminases on HBV, using transient and long term HBV replication models, I have constructed recombinant adenoviruses that express APOBEC3 enzymes. The expression of APOBEC3 deaminases by these recombinant adenoviruses has been shown, by in situ immunostaining, in HepG2, Huh7 and HepG2.2.15 (a HBV producing cell line) cells ,with a transduction efficiency of about 95%. I have shown that APOBEC3G can inhibit HBV replication by up to 90% and it causes extensive G to A mutations in the HBV genome. Other members of the APOBEC3 family -APOBEC3B, APOBEC3C and APOBEC3F- can also inhibit HBV replication to different levels. Using purified APOBEC3G' protein and DNA oligonucleotides corresponding to HBV precore/core sequences I have shown, in a cell free assay that a G at position 1896 can be converted to an A This corresponds to the G to A mutation which is observed in patients who are chronically infected with HBV but have become HBeAg negative. The results of these experiments suggest that APOBEC3G might be responsible for the in vivo precore 1896 G to A mutation. lhave also shown that the expression ofAPOBEC3G in hepatoma cell lines resulted in a 25-60 % increase in the intracellular HBsAg, which is the direct result of the inhibition ofHBV virion maturation. Other experiments indicate that HBx has some inhibitory effects on RNA interference. Finally as part of a collaborative study with Dr. Betty Slagle, Baylor College' of Medicine, Texas, USA, a double-transgenic HCV/HBx mouse, which is predisposed to liver disease (steatosis and hepatocellular carcinoma), has been shown to express HBx and HCV NS3 proteins by immunohistochemistry indicating that the co-expression of these proteins could have a synergistic effect leading to an increase in liver pathology.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.485554  DOI: Not available
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