published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

Hepatitis B virus (HBV) is a significant global threat to human health due to its ability to cause chronic infections that can lead to hepatocellular carcinoma (HCC). While the process by which HBV increases the risk of HCC is unclear, evidence suggests that the hepatitis B X protein (HBx) may be a contributing factor. Cellular senescence is an important barrier to tumorigenesis, blocking the proliferation of cells that harbor excessive DNA damage or contain activated oncogenes. Autophagy is a non-proteasomal degradative pathway used by cells to recycle cytoplasmic contents under periods of nutrient starvation. This pathway is induced in response to a wide range of cellular stress factors, and has also been characterized as an effector mechanism for the establishment of cellular senescence. In this study, retroviral transduction of HepG2 cells with HBx resulted in the induction of cellular senescence and autophagy. The mechanism by which HBx can induce senescence is unclear. However, an increase in the accumulation of DNA damage was observed. HBx did not modulate the levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, or Mcl-1, which can inhibit autophagy through interactions with the autophagy regulator Beclin 1. As well, the activity and phosphorylation status of JNK/SAPK, an inducer of autophagy via Bcl-2 phosphorylation, was unchanged. These results suggest that senescence may act as a barrier to HBx-induced oncogenesis, and may offer some explanation as to why HBx does not function as a more potent oncogene. Also, we propose that HBx modulates autophagy through a mechanism other than Bcl-2 phosphorylation or expression over the time course of this study.

No description available.

Hepatitis B virus genotype G (HBV/G) is a unique genotype of HBV which contains a 36-nucleotide insertion in the Core gene as well as 2 mutations that lead to stop codons in the Pre-Core coding region. Chronic infection with HBV/G is not known to occur without a co-infecting HBV genotype, suggesting that it is defective on its own. This study aims to look at the replication capacity of HBV/G, HBV/A, and HBV/A/G recombinant strains circulating in Canada and to determine the relationship between co-infecting strains. Four full-length HBV genomes were isolated from 2 different patients and transiently transfected into the HepG2 human hepatoma cell line for phenotypic analysis of each strain. HBV/G, HBV/A and HBV/A/G recombinant strains were isolated from Patient 1, while a different HBV/A/G recombinant strain was isolated from Patient 2. HBV replication capacity was measured using a quantitative real time PCR assay. Markers of replication, such as secreted HBsAg and HBeAg, intracellular core particles and replicative DNA intermediates were measured by ELISA, Western blot and Southern blot, respectively. HBV/G demonstrated a higher replicative capability, relative to its co-infecting strains, while both HBV/A/G strains had levels of secreted HBV DNA greater than HBV/A alone, suggesting a modulating effect due to recombination. Replication marker levels revealed possible reasons for a co-infection requirement during HBV/G infection such as HBeAg for chronicity. These observations demonstrate the potential interactions of HBV/G with its co-infecting HBV genotype and provide the first reported phenotypic analysis of a HBV recombinant.

In 2010, the Hepatitis B virus (HBV) infected 1.2 million people in the United States, many of whom were unaware of their infection (CDC, 2010). The available research on HBV infection is predominately among Asian American, Native Hawaiian, and other Pacific Islander. HBV infection and Human Immunodeficiency Virus (HIV) infection share similar modes of transmission. Very little HBV research has been dedicated to the African American females; who accounted for 29% of the new HIV cases among young adolescents in 2010 (CDC, 2010). Due to the common mode of transmission of HIV and Hepatitis B many persons at risk for HIV are also at risk for contracting Hepatitis B. One’s risk for acquisition of HBV can be mitigated or eliminated by vaccination or naturally acquired immunity. In the absence of both, an individual is susceptible to acquisition of HBV. The aims of this study are to define susceptibility of non-Hispanic, blacks to Hepatitis B infection compared to other races as well as defining possible risk factors that may increase or decrease their susceptibility.

Hepatitis B virus genotype G (HBV/G) is a unique genotype of HBV which contains a 36-nucleotide insertion in the Core gene as well as 2 mutations that lead to stop codons in the Pre-Core coding region. Chronic infection with HBV/G is not known to occur without a co-infecting HBV genotype, suggesting that it is defective on its own. This study aims to look at the replication capacity of HBV/G, HBV/A, and HBV/A/G recombinant strains circulating in Canada and to determine the relationship between co-infecting strains. Four full-length HBV genomes were isolated from 2 different patients and transiently transfected into the HepG2 human hepatoma cell line for phenotypic analysis of each strain. HBV/G, HBV/A and HBV/A/G recombinant strains were isolated from Patient 1, while a different HBV/A/G recombinant strain was isolated from Patient 2. HBV replication capacity was measured using a quantitative real time PCR assay. Markers of replication, such as secreted HBsAg and HBeAg, intracellular core particles and replicative DNA intermediates were measured by ELISA, Western blot and Southern blot, respectively. HBV/G demonstrated a higher replicative capability, relative to its co-infecting strains, while both HBV/A/G strains had levels of secreted HBV DNA greater than HBV/A alone, suggesting a modulating effect due to recombination. Replication marker levels revealed possible reasons for a co-infection requirement during HBV/G infection such as HBeAg for chronicity. These observations demonstrate the potential interactions of HBV/G with its co-infecting HBV genotype and provide the first reported phenotypic analysis of a HBV recombinant.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma and liver cirrhosis worldwide. HBV vaccination can prevent new infections, but effective antiviral drugs are not available for a large number of HBV infected patients. To develop novel antiviral drugs, a better understanding of the regulation of HBV gene expression is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNAs from the nucleus of a cell to the site of incorporation into new HBV particles. The HBV post-transcriptional regulatory element (HBV PRE) is a cis acting RNA element found in all HBV transcripts. It has been reported to play an important role in the nuclear export of HBV mRNAs. Moreover, it has the ability to enhance expression of intronless as well as unspliced transcripts. Despite concerted investigations, the functional core element of HBV PRE remains unknown and the exact mechanism of how HBV PRE mediates nuclear export is unclear. This project first produced a complete HBV genome with comprehensive annotation of both coding regions and regulatory signals, which was then used for comparative genomic analysis. The functional elements of the HBV PRE were first subjected to analysis in silico. The HBV PRE is highly conserved among HBVs. Based on this sequence conservation and prediction of conserved RNA secondary structure, potentially functional HBV PRE elements including the previously reported elements (HBV SLα and HBV SLβ) were identified. Experimental deletion analysis of the HBV PRE sequence showed that the effect of each of these elements on the intronless reporter gene�s expression was similar to that of the entire full length HBV PRE. Thus, the results suggested that overall HBV PRE function was not due to additive effects from the individual elements. Surprisingly, a specific sub-section of HBV PRE decreased the level of reporter gene expression. This sub-section has not been identified previously, thus it is a novel HBV PRE inhibitory element. Further analyses using specific reporter assays revealed that the HBV PRE enhanced expression of an unspliced reporter gene whereas the RNA nuclear export elements of retroviruses, CTE (in MPMV) and RRE (in HIV-1) were not able to. Therefore, these results indicate that HBV PRE is involved in inhibition of splicing and it utilizes a different mechanism from CTE and RRE. Interestingly, HBV PRE was observed to be unable to enhance the expression of an intronless luciferase gene. Therefore, HBV PRE is not able to enhance cytoplasmic expression of all intronless transcripts. This project also addressed the idea that the RNA-binding protein, polypyrimidine tract binding protein (PTB) is a positive trans-acting factor for HBV PRE function. Transient expression of exogenous PTB in cultured cells showed no specific effect on constructs containing HBV PRE. Moreover, reduction of endogenous PTB by RNAi did not affect HBV PRE function. Therefore, the results presented in this project do not support the hypothesis that PTB plays a role in HBV PRE function. Given that HBV PRE is highly conserved and present in all HBV transcripts, it makes a good target site for novel molecular therapeutic treatments such as siRNA. To identify potential siRNA target sites within HBV PRE, an RNAi study using a plasmid expressing shRNA against HBV PRE was done. The results from the RNAi study revealed that the expression of a reporter gene could be significantly reduced by siRNA targeted to the HBV PRE. Overall, this project produced a highly annotated HBV genome that can be used as the reference sequence for comparative genomic analysis. Moreover, this work identified novel regulatory elements within HBV PRE that are likely to play an important role in HBV gene expression. Furthermore, the study also identified an excellent siRNA target site within HBV PRE that may inhibit HBV gene expression.

Thesis (PhD)--University of South Australia, 2003.