Project Details
Abstract
Hepatitis delta virus (HDV) is a negative-stranded RNA virus using host RNA polymerase to
perform RNA-directed RNA replication. Therefore, HDV replicates in the nucleus. Hepatitis
delta antigen (HDAg) is the only viral-encoded protein with known biological functions.
HDAg mRNA is transcribed from HDV genomic RNA and the enzyme used is host RNA
polymerase II (pol). How the redirection from DNA to RNA templates occurs is an
interesting question remains to be investigated. The hypothesis regarding this redirection of
the template for pol II is that cellular proteins could be recruited to a promoter element in
HDV genomic RNA to form a complex, which in turn recognized by pol II to initiate
RNA-dependent RNA transcription. No clear evidence has emerged regarding what might be
a promoter element for HDV transcription. There is evidence that 5’-end of HDV mRNA is
both capped and located at a site very close to one end of the genomic rod-like RNA.
Although HDV cis element is an important issue for not only HDV transcription but also the
RNA synthesis in general in mammalian cells, the experimental system remains to be
established. HDV has been shown to be able to undergo RNA recombination. I have recently
constructed a genotype I and IIb chimeric HDV cDNA with junctions identified from
recombinants generated in the co-transfected cells. After transfection of the plasmid
containing this cDNA dimmer into cells, expression of HDAg was detected even this clone
is replication-incompetent. This finding prompted me to establish a plasmid containing a
0.6-mer HDV cDNA covering nt 889-1679/1/211 of genotype I sequence. The DNA
promoter drives the synthesis of HDV genomic RNA, which serves as template for
subsequent transcription of HDAg mRNA and the translation of HDAg. This experimental
system will provide a systematic way to characterize the promoter element for HDV RNA
transcription. Once the cis element has been identified, the trans factors binding to it will be
investigated.
Therefore, the Specific Aims of this grant proposal are:
1. Identification of the HDV RNA promoter for viral mRNA transcription
2. Investigation of the HDV RNA promoter-associated cellular proteins by biotinylated
RNA affinity chromatography and proteomic approaches
3. Functional analyses of RNA-proteins interactions in the regulation of HDAg mRNA
transcription by knockdown or over-expression experiments
Project IDs
Project ID:PC10001-1146
External Project ID:NSC99-2320-B182-004-MY3
External Project ID:NSC99-2320-B182-004-MY3
Status | Finished |
---|---|
Effective start/end date | 01/08/11 → 31/07/12 |
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