Identification of a binding site for ASF/SF2 on an RNA fragment derived from the hepatitis delta virus genome.
The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada.
Dorota
D
Sikora
Dajiang
D
Zhang
Teodora
T
Bojic
Yasnee
Y
Beeharry
Ali
A
Tanara
Martin
M
Pelchat
D001665:Binding Sites; D016679:Genome, Viral; D057809:HEK293 Cells; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014779:Virus Replication / Q000235:genetics
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; D012319:RNA Polymerase II