Hepatitis delta virus genome RNA synthesis initiates at position 1646 with a non-templated guanosine

2021 ◽  
Author(s):  
Susannah Stephenson-Tsoris ◽  
John L. Casey
Keyword(s):  
Liver Disease ◽  
Rna Polymerase ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Start Site ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Infected Cells ◽  
Delta Virus

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense ssRNA virus that produces three RNAs in infected cells: genome, antigenome and mRNA; the latter encodes hepatitis delta antigen, the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5’ end of the genome, including the synthesis start site and its chemical composition, are not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling detection of the 5’ end of the genome RNA. The 5’ end of this RNA is capped, as expected for a Pol II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA Polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how synthesis of the viral RNA begins, nor even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed hairpin-like structure of the viral RNA. The 5’ end of the RNA is capped, as expected for Pol II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses Pol II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

scholarly journals RNA-Templated Replication of Hepatitis Delta Virus: Genomic and Antigenomic RNAs Associate with Different Nuclear Bodies

2006 ◽  
Vol 80 (13) ◽  
pp. 6478-6486 ◽  
Author(s):  
Yi-Jia Li ◽  
Thomas Macnaughton ◽  
Lu Gao ◽  
Michael M. C. Lai
Keyword(s):  
Rna Polymerase ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Nuclear Bodies ◽  
Genomic Rna ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Pol I ◽  
Rna Labeling ◽  
Delta Virus

ABSTRACT Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to α-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both α-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was α-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was α-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.

scholarly journals Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

2007 ◽  
Vol 82 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Jinhong Chang ◽  
Xingcao Nie ◽  
Ho Eun Chang ◽  
Ziying Han ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Cell System ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Delta Antigen ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

scholarly journals Origin of Hepatitis Delta Virus mRNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7204-7210 ◽  
Author(s):  
Severin Gudima ◽  
Shwu-Yuan Wu ◽  
Cheng-Ming Chiang ◽  
Gloria Moraleda ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Genome Replication ◽  
Rna Transcription ◽  
Hepatitis Delta ◽  
New Findings ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5′ end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5′-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5′ ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5′ end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3′-end addition to the template. These new findings support the interpretation that the 5′ end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

scholarly journals Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Kaneemozhe Harichandran ◽  
Yiran Shen ◽  
Susannah Stephenson Tsoris ◽  
See-Chi Lee ◽  
John L. Casey
Keyword(s):  
Liver Disease ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
B Virus ◽  
Hepatitis Delta Antigen ◽  
Infected Cells ◽  
Delta Virus

ABSTRACTHepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated usingin vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCEHepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

scholarly journals The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

Virology ◽  
2004 ◽  
Vol 319 (1) ◽  
pp. 60-70 ◽  
Author(s):  
Jung-Jung Mu ◽  
Yeou-Guang Tsay ◽  
Li-Jung Juan ◽  
Tsai-Feng Fu ◽  
Wen-Hung Huang ◽  
...  
Keyword(s):  
Hepatitis Delta Virus ◽  
Cellular Localization ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Delta Virus

scholarly journals Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication

2012 ◽  
Vol 93 (3) ◽  
pp. 577-587 ◽  
Author(s):  
Fu-Tien Liao ◽  
Li-Sung Hsu ◽  
Jiunn-Liang Ko ◽  
Chun-Che Lin ◽  
Gwo-Tarng Sheu
Keyword(s):  
Hepatitis Delta Virus ◽  
Promoter Activity ◽  
Rna Synthesis ◽  
Stop Codon ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Rolling Circle ◽  
Delta Virus

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.

scholarly journals Intracellular Localization of Hepatitis Delta Virus Proteins in the Presence and Absence of Viral RNA Accumulation

2009 ◽  
Vol 83 (13) ◽  
pp. 6457-6463 ◽  
Author(s):  
Ziying Han ◽  
Carolina Alves ◽  
Severin Gudima ◽  
John Taylor
Keyword(s):  
Rna Polymerase Ii ◽  
Protein Function ◽  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Intracellular Localization ◽  
Genome Replication ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Presence And Absence ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (δAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that δAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, δAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of δAg in the presence and absence of replicating and nonreplicating HDV RNAs. When δAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both δAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, δAg moved to the nucleoplasm, but nucleolin was unchanged. When δAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of δAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of δAg altered at specific locations considered to be essential for protein function. These studies provide evidence that δAg does not interact directly with either Pol II or nucleolin and that forms of δAg which support replication are also capable of prior nucleolar transit.

Hepatitis Delta Virus cDNA Monomer Can Be Used in Transfection Experiments to Initiate Viral RNA Replication

Virology ◽  
1993 ◽  
Vol 197 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Fei-Ping Tai ◽  
Pei-Jer Chen ◽  
Fu-Lin Chang ◽  
Ding-Shinn Chen
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Hepatitis Delta ◽  
Virus Cdna ◽  
Delta Virus

scholarly journals Delta hepatitis: molecular biology and clinical and epidemiological features.

1993 ◽  
Vol 6 (3) ◽  
pp. 211-229 ◽  
Author(s):  
L B Polish ◽  
M Gallagher ◽  
H A Fields ◽  
S C Hadler
Keyword(s):  
Liver Disease ◽  
High Risk ◽  
Hepatitis B ◽  
Hepatitis Delta Virus ◽  
Hepatitis B Infection ◽  
Hepatitis Delta ◽  
Hepatitis Delta Virus Infection ◽  
B Virus ◽  
The World ◽  
Delta Virus

Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in hepatocytes and is an important cause of acute, fulminant, and chronic liver disease in many regions of the world. Because of the helper function of hepatitis delta virus, infection with it occurs either as a coinfection with hepatitis B or as a superinfection of a carrier of hepatitis B surface antigen. Although the mechanisms of transmission are similar to those of hepatitis B virus, the patterns of transmission of delta virus vary widely around the world. In regions of the world in which hepatitis delta virus infection is not endemic, the disease is confined to groups at high risk of acquiring hepatitis B infection and high-risk hepatitis B carriers. Because of the propensity of this viral infection to cause fulminant as well as chronic liver disease, continued incursion of hepatitis delta virus into areas of the world where persistent hepatitis B infection is endemic will have serious implications. Prevention depends on the widespread use of hepatitis B vaccine. This review focuses on the molecular biology and the clinical and epidemiologic features of this important viral infection.

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