Competition between the Sendai Virus N mRNA Start Site and the Genome 3′-End Promoter for Viral RNA Polymerase

ABSTRACT The genomic and antigenomic 3′-end replication promoters of Sendai virus are bipartite in nature and symmetrical, composed of le or tr sequences; a gene start or gene end site, respectively; and a simple hexameric repeat. The relative strengths of these 3′-end promoters determines the ratios of genomes and antigenomes formed during infection and whether model mini-genomes can be rescued from DNA by nondefective helper viruses. Using these tests of promoter strength, we have confirmed that tr is stronger than le in this respect. We have also found that the presence of a gene start site within either 3′-end promoter strongly reduces 3′-end promoter strength. The negative effects of the gene start site on the 3′-end promoter suggest that these closely spaced RNA start sites compete with each other for a common pool of viral RNA polymerase. The manner in which this competition could occur for polymerase off the template (in trans) and polymerase on the template (in cis) adds insight into how the viral RNA polymerase switches between its dual functions as transcriptase and replicase.

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Nature of a paramyxovirus replication promoter influences a nearby transcription signal

Journal of General Virology ◽

10.1099/vir.0.80435-0 ◽

2005 ◽

Vol 86 (1) ◽

pp. 171-180 ◽

Cited By ~ 12

Author(s):

Diane Vulliémoz ◽

Samuel Cordey ◽

Geneviève Mottet-Osman ◽

Laurent Roux

Keyword(s):

Transcription Initiation ◽

Sendai Virus ◽

Initiation Site ◽

Essential Elements ◽

Replication Initiation ◽

Start Site ◽

Mrna Synthesis ◽

Gene Start ◽

Or Gene ◽

Transcription And Replication

The genomic and antigenomic 3′ ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3′ end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.

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Reovirus Low-Density Particles Package Cellular RNA

Viruses ◽

10.3390/v13061096 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1096

Author(s):

Timothy W. Thoner ◽

Xiang Ye ◽

John Karijolich ◽

Kristen M. Ogden

Keyword(s):

Rna Polymerase ◽

Viral Proteins ◽

Viral Rna ◽

Low Density ◽

Rna Packaging ◽

Genome Packaging ◽

Double Stranded Rna ◽

Viral Genomes ◽

Mammalian Orthoreovirus ◽

Insight Into

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

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Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World

Journal of Virology ◽

10.1128/jvi.73.8.6424-6429.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6424-6429 ◽

Cited By ~ 20

Author(s):

Robert W. Siegel ◽

Laurent Bellon ◽

Leonid Beigelman ◽

C. Cheng Kao

Keyword(s):

Rna Polymerase ◽

Mosaic Virus ◽

Rna Synthesis ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Competition Assay ◽

Rna Dependent Rna Polymerase ◽

Dna Molecule ◽

Mechanism Of Catalysis ◽

Insight Into

ABSTRACT All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.

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

Journal of Virology ◽

10.1128/jvi.02017-21 ◽

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.

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A mechanism for prime-realignment during influenza A virus replication

10.1101/138487 ◽

2017 ◽

Cited By ~ 2

Author(s):

Judith Oymans ◽

Aartjan J.W. te Velthuis

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Severe Disease ◽

Viral Rna ◽

Insight Into

AbstractThe influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2 and PA. To copy the viral RNA (vRNA) genome segments and the complementary RNA (cRNA) segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two differentde novoinitiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3’ terminus, while on the cRNA promoter the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into prime-realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.ImportanceInfluenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

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A Mechanism for Priming and Realignment during Influenza A Virus Replication

Journal of Virology ◽

10.1128/jvi.01773-17 ◽

2017 ◽

Vol 92 (3) ◽

Cited By ~ 8

Author(s):

Judith Oymans ◽

Aartjan J. W. te Velthuis

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Viral Replication ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Severe Disease ◽

Viral Rna ◽

Insight Into

ABSTRACTThe influenza A virus genome consists of eight segments of single-stranded RNA. These segments are replicated and transcribed by a viral RNA-dependent RNA polymerase (RdRp) that is made up of the influenza virus proteins PB1, PB2, and PA. To copy the viral RNA (vRNA) genome segments and the cRNA segments, the replicative intermediate of viral replication, the RdRp must use two promoters and two differentde novoinitiation mechanisms. On the vRNA promoter, the RdRp initiates on the 3′ terminus, while on the cRNA promoter, the RdRp initiates internally and subsequently realigns the nascent vRNA product to ensure that the template is copied in full. In particular, the latter process, which is also used by other RNA viruses, is not understood. Here we provide mechanistic insight into priming and realignment during influenza virus replication and show that it is controlled by the priming loop and a helix-loop-helix motif of the PB1 subunit of the RdRp. Overall, these observations advance our understanding of how the influenza A virus initiates viral replication and amplifies the genome correctly.IMPORTANCEInfluenza A viruses cause severe disease in humans and are considered a major threat to our economy and health. The viruses replicate and transcribe their genome by using an enzyme called the RNA polymerases. To ensure that the genome is amplified faithfully and that abundant viral mRNAs are made for viral protein synthesis, the RNA polymerase must work correctly. In this report, we provide insight into the mechanism that the RNA polymerase employs to ensure that the viral genome is copied correctly.

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Ambisense Sendai Viruses Are Inherently Unstable but Are Useful To Study Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.76.11.5492-5502.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5492-5502 ◽

Cited By ~ 24

Author(s):

Philippe Le Mercier ◽

Dominique Garcin ◽

Stéphane Hausmann ◽

Daniel Kolakofsky

Keyword(s):

Rna Synthesis ◽

Sendai Virus ◽

Viral Rna ◽

Start Site ◽

Mrna Editing ◽

Nascent Chain ◽

Vesicular Stomatitis ◽

Chicken Eggs ◽

Modest Level ◽

Selection Of

ABSTRACT Ambisense Sendai virus (SeV) was prepared in order to study the control of viral RNA synthesis. In these studies, we found that the relative ratios of genomes/antigenomes formed during infection are largely determined by the relative strengths of the replication promoters, independent of the presence of a functional mRNA start site. We also found that the ability of the viral polymerase (vRdRP) to respond to an mRNA editing site requires prior (re)initiation at an mRNA start site, similar to the acquisition of vRdRP processivity in the absence of nascent chain coassembly. During these studies, the inherent instability of ambisense SeV upon passage in embryonated chicken eggs was noted and was found to be associated with a point mutation in the ambisense mRNA (ambi-mRNA) start site that severely limited its expression. Since the interferon (IFN)-induced antiviral state is mediated in part via double-stranded RNA (dsRNA), the efficiency of the ambi-mRNA poly(A)/stop site was examined. This site was found to operate in a manner similar to that of other SeV mRNA poly(A)/stop sites, i.e., at ∼95% efficiency. This modest level of vRdRP read-through is apparently tolerable for natural SeV because the potential to form dsRNA during infection remains limited. However, when mRNAs are expressed from ambisense SeV antigenomes, vRdRP read-through of the ambi-mRNA poly(A)/stop site creates a capped transcript that can potentially extend the entire length of the antigenome, since there are no further poly(A)/stop sites here. In support of this hypothesis, loss of ambi-mRNA expression during passage of ambisense SeV stocks in eggs is also characterized by conversion of virus that grows poorly in IFN-sensitive cultures and is relatively IFN sensitive to virus that grows well even in IFN-pretreated cells that restrict vesicular stomatitis virus replication, i.e., the wild-type SeV phenotype. The selection of mutants unable to express ambi-mRNA on passage in chicken eggs is presumably due to increased levels of dsRNA during infection. How natural ambisense viruses may deal with this dilemma is discussed.

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