scholarly journals Phosphorylation of Serine 177 of the Small Hepatitis Delta Antigen Regulates Viral Antigenomic RNA Replication by Interacting with the Processive RNA Polymerase II

2009 ◽  
Vol 84 (3) ◽  
pp. 1430-1438 ◽  
Author(s):  
Shiao-Ya Hong ◽  
Pei-Jer Chen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Posttranslational Modifications ◽  
Transcription Initiation ◽  
Kinase Inhibitor ◽  
Rna Replication ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Rnap Ii

ABSTRACT Recent studies revealed that posttranslational modifications (e.g., phosphorylation and methylation) of the small hepatitis delta antigen (SHDAg) are required for hepatitis delta virus (HDV) replication from antigenomic to genomic RNA. The phosphorylation of SHDAg at serine 177 (Ser177) is involved in this step, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser177 interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser177-phosphorylated counterpart (pSer177-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer177-SHDAg was hyperphosphorylated at both the Ser2 and Ser5 residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) not only blocked the interaction between pSer177-SHDAg and RNAP IIO but also inhibited HDV antigenomic replication. Our results suggest that the phosphorylation of SHDAg at Ser177 shifted its affinity toward the RNA RNAP IIO isoform and thus is a switch for HDV antigenomic RNA replication from the initiation to the elongation stage.

scholarly journals Nucleolar Targeting of Hepatitis Delta Antigen Abolishes Its Ability To Initiate Viral Antigenomic RNA Replication

2007 ◽  
Vol 82 (2) ◽  
pp. 692-699 ◽  
Author(s):  
Wen-Hung Huang ◽  
Yen-Shun Chen ◽  
Pei-Jer Chen
Keyword(s):  
Subcellular Localization ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Rna Replication ◽  
Genomic Rna ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.

Hepatitis delta antigen binds to the clamp of RNA polymerase II and affects transcriptional fidelity

2007 ◽  
Vol 12 (7) ◽  
pp. 863-875 ◽  
Author(s):  
Yuki Yamaguchi ◽  
Takashi Mura ◽  
Sittinan Chanarat ◽  
Sachiko Okamoto ◽  
Hiroshi Handa
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

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 The RNA Polymerase II Kinase Ctk1 Regulates Positioning of a 5′ Histone Methylation Boundary along Genes

2006 ◽  
Vol 27 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Tiaojiang Xiao ◽  
Yoichiro Shibata ◽  
Bhargavi Rao ◽  
R. Nicholas Laribee ◽  
Rose O'Rourke ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Histone Deacetylation ◽  
H3k4 Methylation ◽  
H3k36 Methylation ◽  
Transcriptional Initiation ◽  
Spurious Transcription ◽  
Transcriptional Stress ◽  
Rnap Ii

ABSTRACT In yeast and other eukaryotes, the histone methyltransferase Set1 mediates methylation of lysine 4 on histone H3 (H3K4me). This modification marks the 5′ end of transcribed genes in a 5′-to-3′ tri- to di- to monomethyl gradient and promotes association of chromatin-remodeling and histone-modifying enzymes. Here we show that Ctk1, the serine 2 C-terminal domain (CTD) kinase for RNA polymerase II (RNAP II), regulates H3K4 methylation. We found that CTK1 deletion nearly abolished H3K4 monomethylation yet caused a significant increase in H3K4 di- and trimethylation. Both in individual genes and genome-wide, loss of CTK1 disrupted the H3K4 methylation patterns normally observed. H3K4me2 and H3K4me3 spread 3′ into the bodies of genes, while H3K4 monomethylation was diminished. These effects were dependent on the catalytic activity of Ctk1 but are independent of Set2-mediated H3K36 methylation. Furthermore, these effects are not due to spurious transcription initiation in the bodies of genes, to changes in RNAP II occupancy, to changes in serine 5 CTD phosphorylation patterns, or to “transcriptional stress.” These data show that Ctk1 acts to restrict the spread of H3K4 methylation through a mechanism that is independent of a general transcription defect. The evidence presented suggests that Ctk1 controls the maintenance of suppressive chromatin in the coding regions of genes by both promoting H3K36 methylation, which leads to histone deacetylation, and preventing the 3′ spread of H3K4 trimethylation, a mark associated with transcriptional initiation.

scholarly journals Large Hepatitis Delta Antigen Is Not a Suppressor of Hepatitis Delta Virus RNA Synthesis once RNA Replication Is Established

2002 ◽  
Vol 76 (19) ◽  
pp. 9910-9919 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
State Level ◽  
Rna Replication ◽  
Replication Cycle ◽  
Steady State Level ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen ◽  
Delta Virus

ABSTRACT Moderation of hepatitis delta virus (HDV) replication is a likely prerequisite in the establishment of chronic infections and is thought to be mediated by the intracellular accumulation of large hepatitis delta antigen (L-HDAg). The regulatory role of this protein was suggested from several studies showing that cotransfection of plasmid cDNAs expressing both L-HDAg and HDV RNA results in a potent inhibition of HDV RNA replication. However, since this approach differs significantly from natural HDV infections, where HDV RNA replication is initiated from an RNA template, and L-HDAg appears only late in the replication cycle, it remains unclear whether L-HDAg can modulate HDV RNA replication in the natural HDV replication cycle. In this study, we investigated the effect of L-HDAg, produced as a result of the natural HDV RNA editing event, on HDV RNA replication. The results showed that following cDNA-free HDV RNA transfection, a steady-state level of RNA was established at 3 to 4 days posttransfection. The same level of HDV RNA was reached when a mutant HDV genome unable to make L-HDAg was used, suggesting that L-HDAg did not play a role. The rates of HDV RNA synthesis, as measured by metabolic labeling experiments, were identical at 4 and 8 days posttransfection and in the wild type and the L-HDAg-deficient mutant. We further examined the effect of overexpression of L-HDAg at various stages of the HDV replication cycle, showing that HDV RNA synthesis was resistant to L-HDAg when it was overexpressed 3 days after HDV RNA replication had initiated. Finally, we showed that, contrary to conventional thinking, L-HDAg alone, at a certain molar ratio with HDV RNA, can initiate HDV RNA replication. Thus, L-HDAg does not inherently inhibit HDV RNA synthesis. Taken together, these results indicated that L-HDAg affects neither the rate of HDV RNA synthesis nor the final steady-state level of HDV RNA and that L-HDAg is unlikely to act as an inhibitor of HDV RNA replication in the natural HDV replication cycle.

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