scholarly journals Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

2005 ◽  
Vol 79 (10) ◽  
pp. 6312-6324 ◽  
Author(s):  
Erwin van den Born ◽  
Clara C. Posthuma ◽  
Alexander P. Gultyaev ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Proximal Part ◽  
Antisense Transcription ◽  
Proximal Region ◽  
Leader Sequence ◽  
Equine Arteritis Virus ◽  
Hairpin Structure ◽  
Original Position ◽  
Genome Replication ◽  
Subgenomic Rna

ABSTRACT Nidoviruses produce an extensive 3′-coterminal nested set of subgenomic (sg) mRNAs, which are used to express structural proteins and sometimes accessory proteins. In arteriviruses and coronaviruses, these mRNAs contain a common 5′ leader sequence, derived from the genomic 5′ end. The joining of the leader sequence to different segments derived from the 3′-proximal part of the genome (mRNA bodies) presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis in which base pairing between sense and antisense transcription-regulating sequences (TRSs) plays an essential role. The leader TRS is present in the loop of a hairpin structure that functions in sg mRNA synthesis. In this study, the minimal sequences in the 5′-proximal region of the Equine arteritis virus genome that are required for sg RNA synthesis were delimited through mutagenesis. A full-length cDNA clone was engineered in which this domain was duplicated, allowing us to make mutations and monitor their effects on sg RNA synthesis without seriously affecting genome replication and translation. The leader TRS present in the duplicated sequence was used and yielded novel sg mRNAs with significantly extended leaders. Our combined findings suggest that the leader TRS hairpin (LTH) and its immediate flanking sequences are essential for efficient sg RNA synthesis and form an independent functional entity that could be moved 300 nucleotides downstream of its original position in the genome. We hypothesize that a conformational switch in the LTH region regulates the role of the 5′-proximal region of the arterivirus genome in subgenomic RNA synthesis.

scholarly journals The Stability of the Duplex between Sense and Antisense Transcription-Regulating Sequences Is a Crucial Factor in Arterivirus Subgenomic mRNA Synthesis

2003 ◽  
Vol 77 (2) ◽  
pp. 1175-1183 ◽  
Author(s):  
Alexander O. Pasternak ◽  
Erwin van den Born ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Proximal Part ◽  
The Body ◽  
Equine Arteritis Virus ◽  
Strand Transfer ◽  
Minus Strand ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Subgenomic Rna ◽  
The Stability

ABSTRACT Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a “body” part of variable size, which are derived from the 5′- and 3′-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the subgenomic mRNA synthesis of the arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus subgenomic mRNA synthesis.

scholarly journals Regulation of Relative Abundance of Arterivirus Subgenomic mRNAs

2004 ◽  
Vol 78 (15) ◽  
pp. 8102-8113 ◽  
Author(s):  
Alexander O. Pasternak ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Mutual Influence ◽  
Proximal Region ◽  
Equine Arteritis Virus ◽  
Strand Transfer ◽  
Minus Strand ◽  
Subgenomic Rna ◽  
Molar Ratios ◽  
Flanking Sequences ◽  
Discontinuous Transcription

ABSTRACT The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5′- and 3′-coterminal nested set with the viral genome. Their 5′ common leader sequence is derived from the genomic 5′-proximal region. Fusion of sg RNA leader and “body” segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.

scholarly journals The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription

2000 ◽  
Vol 81 (10) ◽  
pp. 2491-2496 ◽  
Author(s):  
Richard Molenkamp ◽  
Hans van Tol ◽  
Babette C. D. Rozier ◽  
Yvonne van der Meer ◽  
Willy J. M. Spaan ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Point Mutations ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Structural Proteins ◽  
Genome Replication ◽  
Mrna Transcription ◽  
Replication Complex ◽  
N Protein ◽  
Wild Type Phenotype

Equine arteritis virus (EAV) (Arteriviridae) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

scholarly journals The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis

2000 ◽  
Vol 74 (11) ◽  
pp. 5213-5223 ◽  
Author(s):  
Leonie C. van Dinten ◽  
Hans van Tol ◽  
Alexander E. Gorbalenya ◽  
Eric J. Snijder
Keyword(s):  
Metal Binding ◽  
Rna Synthesis ◽  
Proteolytic Processing ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Zinc Binding ◽  
Progeny Virus ◽  
Genome Replication ◽  
Mrna Synthesis ◽  
Mrna Transcription

ABSTRACT Equine arteritis virus (EAV), the prototypeArterivirus, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429→Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429→Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of “hinge spacer,” which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.

scholarly journals Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle

2006 ◽  
Vol 80 (4) ◽  
pp. 1653-1661 ◽  
Author(s):  
Clara C. Posthuma ◽  
Danny D. Nedialkova ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Jeroen H. Blokhuis ◽  
Alexander E. Gorbalenya ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Pleiotropic Effects ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Genome Replication ◽  
Conserved Residues

ABSTRACT The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.

scholarly journals Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

2015 ◽  
Vol 89 (11) ◽  
pp. 6033-6047 ◽  
Author(s):  
Kelley R. Hurst-Hess ◽  
Lili Kuo ◽  
Paul S. Masters
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Proteolytic Processing ◽  
Alternative Mechanism ◽  
Genome Replication ◽  
Structural Domains ◽  
Subgenomic Rna ◽  
Amino Terminal ◽  
Viral Nucleocapsid

ABSTRACTCoronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis.IMPORTANCEThe largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as to combat host innate immunity. We initiated a dissection of MHV nsp3 aimed at identifying those activities or structures in this huge molecule that are essential to replicase activity. We found that both PLP1 and ADRP could be entirely deleted, provided that the requirement for proteolytic processing by PLP1 was offset by an alternative mechanism. This demonstrated that neither PLP1 nor ADRP plays an essential role in coronavirus RNA synthesis.

scholarly journals Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles Which Carry the Viral Replication Complex

1999 ◽  
Vol 73 (3) ◽  
pp. 2016-2026 ◽  
Author(s):  
Ketil W. Pedersen ◽  
Yvonne van der Meer ◽  
Norbert Roos ◽  
Eric J. Snijder
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Genome Replication ◽  
Electron Microscopic ◽  
Double Membrane ◽  
Reading Frame ◽  
Replication Complex

ABSTRACT The replicase of equine arteritis virus (EAV; familyArteriviridae, order Nidovirales) is expressed in the form of two polyproteins (the open reading frame 1a [ORF1a] and ORF1ab proteins). Three viral proteases cleave these precursors into 12 nonstructural proteins, which direct both genome replication and subgenomic mRNA transcription. Immunofluorescence assays showed that most EAV replicase subunits localize to membranes in the perinuclear region of the infected cell. Using replicase-specific antibodies and cryoimmunoelectron microscopy, unusual double-membrane vesicles (DMVs) were identified as the probable site of EAV RNA synthesis. These DMVs were previously observed in cells infected with different arteriviruses but were never implicated in viral RNA synthesis. Extensive electron microscopic analysis showed that they appear to be derived from paired endoplasmic reticulum membranes and that they are most likely formed by protrusion and detachment of vesicular structures with a double membrane. Interestingly, very similar membrane rearrangements were observed upon expression of ORF1a-encoded replicase subunits nsp2 to nsp7 from an alphavirus-based expression vector. Apparently, the formation of a membrane-bound scaffold for the replication complex is a distinct step in the arterivirus life cycle, which is directed by the ORF1a protein and does not depend on other viral proteins and/or EAV-specific RNA synthesis.

scholarly journals Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Qiang Ding ◽  
Ila Nimgaonkar ◽  
Nicholas F. Archer ◽  
Yaron Bram ◽  
Brigitte Heller ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Regulatory Element ◽  
Hepatitis E ◽  
Regulatory Elements ◽  
Genome Replication ◽  
Genomic Rna ◽  
Chronic Infections ◽  
Particle Assembly ◽  
Subgenomic Rna ◽  
General Importance

ABSTRACT Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis -acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen. IMPORTANCE HEV is an emerging pathogen causing severe liver disease. The genetic information of HEV is encoded in RNA. The genomic RNA is initially copied into a complementary, antigenomic RNA that is a template for synthesis of more genomic RNA and for so-called subgenomic RNA. In this study, we identified the precise region within the HEV genome at which the synthesis of the subgenomic RNA is initiated. The nucleotides within this region are conserved across genetically distinct variants of HEV, highlighting the general importance of this segment for the virus. To identify this regulatory element, we developed a new experimental system that is a powerful tool with broad utility to mechanistically dissect many other poorly understood functional elements of HEV.

scholarly journals A molecular pore spans the double membrane of the coronavirus replication organelle

Science ◽  
2020 ◽  
Vol 369 (6509) ◽  
pp. 1395-1398 ◽  
Author(s):  
Georg Wolff ◽  
Ronald W. A. L. Limpens ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Ulrike Laugks ◽  
Shawn Zheng ◽  
...  
Keyword(s):  
Drug Target ◽  
Rna Synthesis ◽  
Transmembrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Genome Replication ◽  
Messenger Rnas ◽  
Double Membrane ◽  
The Core ◽  
Cryo Electron Microscopy

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.

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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