scholarly journals Transcription polymerase–catalyzed emergence of novel RNA replicons

Science ◽  
2020 ◽  
Vol 368 (6487) ◽  
pp. eaay0688 ◽  
Author(s):  
Nimit Jain ◽  
Lucas R. Blauch ◽  
Michal R. Szymanski ◽  
Rhiju Das ◽  
Sindy K. Y. Tang ◽  
...  
Keyword(s):  
Experimental Model ◽  
Hepatitis Delta Virus ◽  
De Novo ◽  
Rna Replication ◽  
Hereditary Material ◽  
Rolling Circle ◽  
Naturally Occurring ◽  
T7 Bacteriophage ◽  
Delta Virus

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

scholarly journals Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication

2004 ◽  
Vol 78 (23) ◽  
pp. 13325-13334 ◽  
Author(s):  
Yi-Jia Li ◽  
Michael R. Stallcup ◽  
Michael M. C. Lai
Keyword(s):  
Subcellular Localization ◽  
Posttranslational Modification ◽  
Hepatitis Delta Virus ◽  
Rna Binding ◽  
Rna Replication ◽  
Genomic Rna ◽  
Hepatitis Delta ◽  
Arginine Residues ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.

scholarly journals Genomic but Not Antigenomic Hepatitis Delta Virus RNA Is Preferentially Exported from the Nucleus Immediately after Synthesis and Processing

2002 ◽  
Vol 76 (8) ◽  
pp. 3928-3935 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis Delta Virus ◽  
Large Fraction ◽  
Rna Replication ◽  
Metabolic Labeling ◽  
Rolling Circle Replication ◽  
Cell Region ◽  
Hepatitis Delta ◽  
Rolling Circle ◽  
Rna Export ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [32P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominately in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.

scholarly journals Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases

2002 ◽  
Vol 76 (8) ◽  
pp. 3920-3927 ◽  
Author(s):  
Thomas B. Macnaughton ◽  
Stephanie T. Shi ◽  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Rna Polymerases ◽  
Rolling Circle Replication ◽  
Hepatitis Delta ◽  
Rolling Circle ◽  
System L ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains a viroid-like circular RNA that is presumed to replicate via a rolling circle replication mechanism mediated by cellular RNA polymerases. However, the exact mechanism of rolling circle replication for HDV RNA and viroids is not clear. Using our recently described cDNA-free transfection system (L. E. Modahl and M. M. Lai, J. Virol. 72:5449-5456, 1998), we have succeeded in detecting HDV RNA replication by metabolic labeling with [32P]orthophosphate in vivo and obtained direct evidence that HDV RNA replication generates high-molecular-weight multimeric species of HDV RNA, which are processed into monomeric and dimeric forms. Thus, these multimeric RNAs are the true intermediates of HDV RNA replication. We also found that HDV RNA synthesis is highly temperature sensitive, occurring most efficiently at 37 to 40°C and becoming virtually undetectable at temperatures below 30°C. Moreover, genomic HDV RNA synthesis was found to occur at a rate roughly 30-fold higher than that of antigenomic RNA synthesis. Finally, in lysolecithin-permeabilized cells, the synthesis of full-length antigenomic HDV RNA was completely resistant to high concentrations (100 μg/ml) of α-amanitin. In contrast, synthesis of genomic HDV RNA was totally inhibited by α-amanitin at concentrations as low as 2.5 μg/ml. Thus, these results suggest that genomic and antigenomic HDV RNA syntheses are performed by two different host cell enzymes. This observation, combined with our previous finding that hepatitis delta antigen mRNA synthesis is likely performed by RNA polymerase II, suggests that the different HDV RNA species are synthesized by different cellular transcriptional machineries.

scholarly journals Initiation of Hepatitis Delta Virus Genome Replication

1998 ◽  
Vol 72 (6) ◽  
pp. 4783-4788 ◽  
Author(s):  
Kate Dingle ◽  
Vadim Bichko ◽  
Harmon Zuccola ◽  
James Hogle ◽  
John Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Virus Genome ◽  
Northern Analysis ◽  
Genome Replication ◽  
Hepatitis Delta ◽  
Delta Virus

ABSTRACT The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (δAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant δAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input δAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of δAg-S. It is this second source of δAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for δAg-S with truncated or modified forms of the δAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of δAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

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.

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 Selection in vitro of Trans -Acting Genomic Human Hepatitis Delta Virus (HDV) Ribozymes

1996 ◽  
Vol 237 (3) ◽  
pp. 712-718 ◽  
Author(s):  
Fumiko Nishikawa ◽  
Junji Kawakami ◽  
Atsushi Chiba ◽  
Makoto Shirai ◽  
Penmetcha K. R. Kumar ◽  
...  
Keyword(s):  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Human Hepatitis ◽  
Delta Virus

scholarly journals The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication

2000 ◽  
Vol 74 (16) ◽  
pp. 7375-7380 ◽  
Author(s):  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Life Cycle ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Dominant Negative ◽  
Genomic Rna ◽  
Inhibitory Effects ◽  
Hepatitis Delta ◽  
Artificial Dna ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.

Farnesoid X receptor alpha ligands inhibit hepatitis delta virus replication in vitro independently of their effect on hepatitis B virus

2020 ◽  
Vol 73 ◽  
pp. S834-S835
Author(s):  
Benoît Lacombe ◽  
Julie Lucifora ◽  
Camille Ménard ◽  
Michelet Maud ◽  
Adrien Foca ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Hepatitis Delta Virus ◽  
Farnesoid X Receptor ◽  
Hepatitis Delta ◽  
Receptor Alpha ◽  
B Virus ◽  
Delta Virus

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.

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