Identification of Cis and Trans Factors Required for the Mrna Transcription of Hepatitis Delta Antigen

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

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
StatusFinished
Effective start/end date01/08/1131/07/12

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