title: Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication.
abstract: To understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582-1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223-1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599-1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670-1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662-1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351-1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351-1352) or 3 nt mutations (I5-m3, nt 1353-1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
authors.forename: Fu-Tien
authors.initials: FT
authors.lastname: Liao
authors.affiliation: Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC.
authors.forename: Li-Sung
authors.initials: LS
authors.lastname: Hsu
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
authors.forename: Jiunn-Liang
authors.initials: JL
authors.lastname: Ko
authors.affiliation: Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC.|Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
authors.forename: Chun-Che
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC.
authors.forename: Gwo-Tarng
authors.initials: GT
authors.lastname: Sheu
mesh_terms: D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism*; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral; D012319:RNA Polymerase II