Hepatitis delta antigen requires a minimum length of the hepatitis delta virus unbranched rod RNA structure for binding.
Hepatitis delta virus (HDV) is a subviral pathogen that increases the severity of liver disease caused by hepatitis B virus. Both the small circular RNA genome and its complement, the antigenome, form a characteristic unbranched rod structure in which approximately 70% of the nucleotides are base paired. These RNAs are associated with the sole virally encoded protein, hepatitis delta antigen (HDAg), in infected cells; however, the nature of the ribonucleoprotein complexes (RNPs) is not well understood. Previous analyses of binding in vitro using native, bacterially expressed HDAg have been hampered by a lack of specificity for HDV RNA. Here, we show that removal of the C-terminal 35 amino acids of HDAg yields a native, bacterially expressed protein, HDAg-160, that specifically binds HDV unbranched rod RNA with high affinity. In an electrophoretic mobility shift assay, this protein produced a discrete, micrococcal nuclease-resistant complex with an approximately 400-nucleotide (nt) segment of HDV unbranched rod RNA. Binding occurred with several segments of HDV RNA, although with various affinities and efficiencies. Analysis of the effects of deleting segments of the unbranched rod indicated that binding did not require one or two specific binding sites within these RNA segments. Rather, a minimum-length HDV RNA unbranched rod approximately 311 nt was essential for RNP formation. The results are consistent with a model in which HDAg binds HDV unbranched rod RNA as multimers of fixed size rather than as individual subunits.
Defenbaugh
Dawn A
DA
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA.
Johnson
Matthew
M
Chen
Renxiang
R
Zheng
Ying Yi
YY
Casey
John L
JL
D002460:Cell Line; D003698:Hepatitis Delta Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D035923:Hepatitis delta Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D035923:Hepatitis delta Antigens; D012367:RNA, Viral; D008836:Micrococcal Nuclease