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 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 BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

2016 ◽  
Vol 90 (15) ◽  
pp. 6864-6883 ◽  
Author(s):  
Morgan R. Herod ◽  
Cristina Ferrer-Orta ◽  
Eleni-Anna Loundras ◽  
Joseph C. Ward ◽  
Nuria Verdaguer ◽  
...  
Keyword(s):  
Disease Virus ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Rna Replication ◽  
Mouth Disease ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Complex ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

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 An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis

2007 ◽  
Vol 81 (17) ◽  
pp. 9426-9436 ◽  
Author(s):  
Nancy Beerens ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Critical Role ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Replicase Gene ◽  
Loop Structure ◽  
Minus Strand ◽  
Stem Loop ◽  
Loop Region ◽  
Stem Loop Structure

ABSTRACT In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

scholarly journals Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis

2016 ◽  
Vol 90 (11) ◽  
pp. 5384-5398 ◽  
Author(s):  
Long Liu ◽  
Jiao Tian ◽  
Hao Nan ◽  
Mengmeng Tian ◽  
Yuan Li ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Continuous Extension ◽  
Rna Helicase ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Nascent Transcript ◽  
Multifunctional Protein ◽  
N Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.IMPORTANCEIt is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

scholarly journals Complementary Mutations in the N and L Proteins for Restoration of Viral RNA Synthesis

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Weike Li ◽  
Ryan H. Gumpper ◽  
Yusuf Uddin ◽  
Ingeborg Schmidt-Krey ◽  
Ming Luo
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Rna Synthesis ◽  
Large Subunit ◽  
Viral Rna ◽  
Structural Motif ◽  
General Mechanism ◽  
Rna Dependent Rna Polymerase ◽  
N Protein ◽  
Rna Genome

ABSTRACTDuring viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif—Glu169, Phe171, and Leu174—to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCEDuring viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

scholarly journals The Matrix Protein of Measles Virus Regulates Viral RNA Synthesis and Assembly by Interacting with the Nucleocapsid Protein

2009 ◽  
Vol 83 (20) ◽  
pp. 10374-10383 ◽  
Author(s):  
Masaharu Iwasaki ◽  
Makoto Takeda ◽  
Yuta Shirogane ◽  
Yuichiro Nakatsu ◽  
Takanori Nakamura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Rna Synthesis ◽  
Matrix Protein ◽  
Viral Rna ◽  
M Protein ◽  
Ribonucleoprotein Complex ◽  
N Protein ◽  
The Matrix

ABSTRACT The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, the M protein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.

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