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.
| Identifer | oai:union.ndltd.org:ADTP/217787 |
| Date | January 2007 |
| Creators | Panjaworayan, Nattanan, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Nattanan Panjaworayan |
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