scholarly journals Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

scholarly journals Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

1998 ◽  
Vol 72 (5) ◽  
pp. 3684-3690 ◽  
Author(s):  
Huei-Chi Chou ◽  
Tsai-Yuan Hsieh ◽  
Gwo-Tarng Sheu ◽  
Michael M. C. Lai
Keyword(s):  
Nuclear Import ◽  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Binding Motif ◽  
Hepatitis Delta ◽  
Binding Motifs ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.

scholarly journals Hepatitis Delta Antigen Requires a Minimum Length of the Hepatitis Delta Virus Unbranched Rod RNA Structure for Binding

2009 ◽  
Vol 83 (9) ◽  
pp. 4548-4556 ◽  
Author(s):  
Dawn A. Defenbaugh ◽  
Matthew Johnson ◽  
Renxiang Chen ◽  
Ying Yi Zheng ◽  
John L. Casey
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Specific Binding ◽  
Micrococcal Nuclease ◽  
Minimum Length ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complexes ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

ABSTRACT 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 ∼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.

scholarly journals Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

2013 ◽  
Vol 87 (15) ◽  
pp. 8665-8674 ◽  
Author(s):  
L. H. Daigh ◽  
B. L. Griffin ◽  
A. Soroush ◽  
M. R. Mamedov ◽  
J. L. Casey
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Binding Activity ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Virus Rna ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

scholarly journals Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

1998 ◽  
Vol 18 (4) ◽  
pp. 1919-1926 ◽  
Author(s):  
Andrew G. Polson ◽  
Herbert L. Ley ◽  
Brenda L. Bass ◽  
John L. Casey
Keyword(s):  
Rna Editing ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Reading Frame ◽  
Adenosine Deaminases ◽  
Delta Antigen ◽  
Virus Rna ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

ABSTRACT RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

scholarly journals Characterization of RNA-binding domains of hepatitis delta antigen

1993 ◽  
Vol 74 (11) ◽  
pp. 2473-2478 ◽  
Author(s):  
F. Poisson ◽  
P. Roingeard ◽  
A. Baillou ◽  
F. Dubois ◽  
F. Bonelli ◽  
...  
Keyword(s):  
Rna Binding ◽  
Hepatitis Delta ◽  
Binding Domains ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Rna Binding Domains

scholarly journals Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity.

1988 ◽  
Vol 62 (7) ◽  
pp. 2403-2410 ◽  
Author(s):  
M F Chang ◽  
S C Baker ◽  
L H Soe ◽  
T Kamahora ◽  
J G Keck ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Activity ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Human Hepatitis ◽  
Hepatitis Delta Antigen ◽  
Nuclear Phosphoprotein
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