| Summary: | 博士 === 中山醫學大學 === 醫學研究所 === 100 === Objective: Hepatitis delta virus (HDV) is a major cause of severe liver disease. HDV is a negative single-stranded RNA with a circular conformation and has the ability to fold as a unique double-stranded, rod-like structure on itself. There are two different forms of hepatitis delta antigens (HDAgs) are produced during life cycle of HDV: small HDAg (S-HDAg) and large HDAg (L-HDAg). HDAgs are indispensible for HDV life cycle. Both HDAgs are transcriptional inducers and capable of regulating cellular gene expression. However, replication of HDV RNA is dependent of cellular RNA polymerases and does not involve any DNA intermediates. How cellular polymerases change the template from DNA to RNA and synthesize HDV RNA is a very important issue and requires further investigation. Therefore, the two objectives of this study are: (1) to analyse the cellular genes regulated by HDAgs and to characterize the gene function in HDV infection, (2) to investigate the cis-elements in HDV genome that are critical for RNA-dependent RNA replication.
Materials and Methods: The S- or L-HDAg-expressing Huh7 cells (Huh-S and Huh-L) were established to identify genes activated by S-HDAg or L-HDAg using Agilent Human Whole Genome Oligo 4×44 K Microarrays. Several up-regulated genes were further examined by real-time PCR and Western blots. The luciferase reporter gene assay and chromatin immunoprecipitation assay (ChIP assay) were used to examine how the genes regulated by HDAgs and by HDV. In addition, we screened various fragments of putative HDV promoter by luciferase reporter gene assay. We also examined the promoter activities regulated by HDAgs. The WT and mutated HDV RNAs were synthesized by in vitro transcription and transfected into Huh-S cells to characterize the HDV de novo RNA synthesis on Northern blots and Western blots.
Results: We found: (1) Nearly 20,000 genes are affected in Huh7 cells that stably expressing HDAgs. We selected 11 genes and further identified by real-time PCR and Western blots. (2) CLU expression is upregulated by HDAgs and positively associated with HDV replication. HDAgs enhance histone H3 acetylation of clusterin promoter to increase gene expression and decrease drug sensitivity to adriamycin treatment. We also found several evidences of the cis-elements in HDV genome associated with HDV RNA replication: (1) Two HDV cDNA fragments (TR-P1, nt 1582~1683 and TR-I5, nt 1223~1363) containing bidirectional promoter activity and enhanced by HDAgs to differing degrees. (2) The AUG codon at HDAg-ORF of HDV RNA (nt 1599~1601) that mutates to UAG (Amber) results in loss of dimeric but not monomeric HDV RNA synthesis. (3) Mutagenesis of TR-P1 and TR-I5 affect HDV RNA replication. (4) RNA editing event occurs in the monomeric HDV RNA and independent of double rolling-circle RNA synthesis.
Conclusion: We demonstrated that clusterin is an authentic HDV-regulated gene, which is specifically transcriptional induced by S-HDAg and L-HDAg via histone hyperacetylation in Huh7 cells. However, how many genes are regulated by HDAgs via epigenetic-mechanism remains unclear. Cytoprotective genes, like CLU, are induced in HDV infected cells may increase cell survival and further increase oncogenic potential in the liver of HDV carrier. We also provide evidences that TR-P1 region is most likely the authentic HDV RNA promoter for mRNA, genomic, and antigenomic RNA synthesis. Although TR-I5 is not an RNA promoter but it is associated with RNA double rolling-circle replication. The integrity of HDAg-ORF is also essential for HDV RNA double rolling-circle replication. These results provide evidences to unmask the mechanisms of transactivation and RNA-dependent RNA replication with hepatitis delta virus.
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