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 The Origin, Dynamic Morphology, and PI4P-Independent Formation of Encephalomyocarditis Virus Replication Organelles

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00420-18 ◽  
Author(s):  
C. E. Melia ◽  
H. M. van der Schaar ◽  
A. W. M. de Jong ◽  
H. R. Lyoo ◽  
E. J. Snijder ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Electron Tomography ◽  
Membrane Vesicles ◽  
Encephalomyocarditis Virus ◽  
Escape Mutant ◽  
Membrane Structures ◽  
Double Membrane ◽  
Single Membrane ◽  
Phosphatidylinositol 4

ABSTRACTPicornaviruses induce dramatic rearrangements of endomembranes in the cells that they infect to produce dedicated platforms for viral replication. These structures, termed replication organelles (ROs), have been well characterized for theEnterovirusgenus of thePicornaviridae. However, it is unknown whether the diverse RO morphologies associated with enterovirus infection are conserved among other picornaviruses. Here, we use serial electron tomography at different stages of infection to assess the three-dimensional architecture of ROs induced by encephalomyocarditis virus (EMCV), a member of theCardiovirusgenus of the family of picornaviruses that is distantly related. Ultrastructural analyses revealed connections between early single-membrane EMCV ROs and the endoplasmic reticulum (ER), establishing the ER as a likely donor organelle for their formation. These early single-membrane ROs appear to transform into double-membrane vesicles (DMVs) as infection progresses. Both single- and double-membrane structures were found to support viral RNA synthesis, and progeny viruses accumulated in close proximity, suggesting a spatial association between RNA synthesis and virus assembly. Further, we explored the role of phosphatidylinositol 4-phosphate (PI4P), a critical host factor for both enterovirus and cardiovirus replication that has been recently found to expedite enterovirus RO formation rather than being strictly required. By exploiting an EMCV escape mutant, we found that low-PI4P conditions could also be overcome for the formation of cardiovirus ROs. Collectively, our data show that despite differences in the membrane source, there are striking similarities in the biogenesis, morphology, and transformation of cardiovirus and enterovirus ROs, which may well extend to other picornaviruses.IMPORTANCELike all positive-sense RNA viruses, picornaviruses induce the rearrangement of host cell membranes to form unique structures, or replication organelles (ROs), that support viral RNA synthesis. Here, we investigate the architecture and biogenesis of cardiovirus ROs and compare them with those induced by enteroviruses, members of the well-characterized picornavirus genusEnterovirus. The origins and dynamic morphologies of cardiovirus ROs are revealed using electron tomography, which points to the endoplasmic reticulum as the donor organelle usurped to produce single-membrane tubules and vesicles that transform into double-membrane vesicles. We show that PI4P, a critical lipid for cardiovirus and enterovirus replication, is not strictly required for the formation of cardiovirus ROs, as functional ROs with typical morphologies are formed under phosphatidylinositol 4-kinase type III alpha (PI4KA) inhibition in cells infected with an escape mutant. Our data show that the transformation from single-membrane structures to double-membrane vesicles is a conserved feature of cardiovirus and enterovirus infections that likely extends to other picornavirus genera.

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 The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1030 ◽  
Author(s):  
Nicole Doyle ◽  
Philippa C. Hawes ◽  
Jennifer Simpson ◽  
Lorin H. Adams ◽  
Helena J. Maier
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Severe Diarrhea ◽  
The Usa ◽  
History Of ◽  
Host Cell Interactions ◽  
Positive Strand Rna

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus–host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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 Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Helena J. Maier ◽  
Philippa C. Hawes ◽  
Eleanor M. Cottam ◽  
Judith Mantell ◽  
Paul Verkade ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Infectious Bronchitis ◽  
Positive Sense ◽  
Infected Cells

ABSTRACTReplication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells ofex vivotracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCEAll positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

scholarly journals The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

2019 ◽  
Author(s):  
Nicole Doyle ◽  
Philippa C. Hawes ◽  
Jennifer Simpson ◽  
Lorin H. Adams ◽  
Helena J. Maier
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Synthesis ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Severe Diarrhea ◽  
The Usa ◽  
History Of ◽  
Host Cell Interactions ◽  
Positive Strand Rna

AbstractPorcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea and the virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to begin to address questions regarding virus-host cell interactions for this genera of coronavirus. Here we present a detailed study of PDCoV induced replication organelles. All positive strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta- and Gammacoronavirus genera have been characterized. However the structures induced by deltacoronaviruses, in particular the presence of convoluted membranes or double membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis and progeny particle release determined. Subsequently, virus induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double membrane vesicles. Significantly however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum and small associated tethered vesicles, double membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

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

2020 ◽  
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 ◽  
Critical Role ◽  
Membrane Vesicles ◽  
Genome Replication ◽  
Double Membrane ◽  
The Core ◽  
Novel Drug

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored micro-environment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and mRNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. Here, using cellular electron cryo-microscopy, we unveiled 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 critical role in coronavirus replication and thus constitutes a novel drug target

scholarly journals ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex

1998 ◽  
Vol 72 (8) ◽  
pp. 6689-6698 ◽  
Author(s):  
Yvonne van der Meer ◽  
Hans van Tol ◽  
Jacomine Krijnse Locker ◽  
Eric J. Snijder
Keyword(s):  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Membrane Association ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Replication Complex ◽  
Viral Proteases ◽  
Double Label ◽  
Membrane Bound ◽  
Triton X

ABSTRACT Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

scholarly journals Flavivirus RNA-Dependent RNA Polymerase Interacts with Genome UTRs and Viral Proteins to Facilitate Flavivirus RNA Replication

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 929 ◽  
Author(s):  
YanPing Duan ◽  
Miao Zeng ◽  
Bowen Jiang ◽  
Wei Zhang ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Current Knowledge ◽  
Antiviral Drug ◽  
Viral Proteins ◽  
Genome Replication ◽  
Human Pathogens ◽  
Replication Complex ◽  
Rdrp Domain ◽  
Methyltransferase Domain ◽  
Significant Public Health

Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses. Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.

scholarly journals Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.

1985 ◽  
Vol 101 (6) ◽  
pp. 2374-2382 ◽  
Author(s):  
M Bernstein ◽  
W Hoffmann ◽  
G Ammerer ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Fusion ◽  
Protein Assembly ◽  
Open Reading Frame ◽  
Cell Fractionation ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Cytosol Fraction ◽  
Reading Frame

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.

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