Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease

ABSTRACT Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

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The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor

Journal of Virology ◽

10.1128/jvi.78.22.12218-12224.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12218-12224 ◽

Cited By ~ 119

Author(s):

Kanchan Bhardwaj ◽

Linda Guarino ◽

C. Cheng Kao

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Biochemical Characterization ◽

Nonstructural Protein ◽

Tryptophan Fluorescence ◽

Conformation Change ◽

Activity Concentrations ◽

Level Of Activity ◽

Novel Coronavirus ◽

Endoribonuclease Activity

ABSTRACT Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

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Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors

Journal of Virology ◽

10.1128/jvi.00490-19 ◽

2019 ◽

Vol 93 (18) ◽

Author(s):

Adriaan H. de Wilde ◽

A. Linda Boomaars-van der Zanden ◽

Anja W. M. de Jong ◽

Montserrat Bárcena ◽

Eric J. Snijder ◽

...

Keyword(s):

Rna Synthesis ◽

Rna Viruses ◽

Nonstructural Protein ◽

Equine Arteritis Virus ◽

Adaptive Mutation ◽

Resistance Mutations ◽

Adaptive Mutations ◽

Virus Rna ◽

Future Drug

ABSTRACTPreviously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the orderNidovirales. In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV’s CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in anin vitroassay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCECurrently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.

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Inhibition of arterivirus RNA synthesis by cyclophilin inhibitors is counteracted by mutations in replicase transmembrane subunits

10.1101/587261 ◽

2019 ◽

Cited By ~ 1

Author(s):

Adriaan H. de Wilde ◽

A. Linda Boomaars-van der Zanden ◽

Anja W. M. de Jong ◽

Montserrat Barcéna ◽

Eric J. Snijder ◽

...

Keyword(s):

Cyclosporin A ◽

Rna Synthesis ◽

Rna Viruses ◽

Nonstructural Protein ◽

Equine Arteritis Virus ◽

Adaptive Mutation ◽

Resistance Mutations ◽

Adaptive Mutations ◽

Future Drug

AbstractPreviously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the orderNidovirales. Here we aimed to identify arterivirus proteins involved in the mode-of-action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to a ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV’s CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA-resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in anin vitroassay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely-related drug ALV CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.ImportanceCurrently, no approved treatments are available to combat infections with nidoviruses, a group of plus-stranded RNA viruses including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and may thus represent a class of pan-nidovirus inhibitors. Here, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two trans-membrane subunits of the viral replication complex, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of nidoviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and other RNA viruses inhibited by these compounds.

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Biochemical Characterization of the Equine Arteritis Virus Helicase Suggests a Close Functional Relationship between Arterivirus and Coronavirus Helicases

Journal of Virology ◽

10.1128/jvi.74.20.9586-9593.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9586-9593 ◽

Cited By ~ 59

Author(s):

Anja Seybert ◽

Leonie C. van Dinten ◽

Eric J. Snijder ◽

John Ziebuhr

Keyword(s):

Functional Relationship ◽

Biochemical Characterization ◽

Rna Virus ◽

Nonstructural Protein ◽

Biochemical Properties ◽

Equine Arteritis Virus ◽

Helicase Domain ◽

Rna And Dna ◽

Combined Data

ABSTRACT The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5′ single-stranded regions on the partial-duplex substrates, indicating a 5′-to-3′ polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.

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Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

Veterinary Pathology ◽

10.1177/030098588502200413 ◽

1985 ◽

Vol 22 (4) ◽

pp. 375-386 ◽

Cited By ~ 6

Author(s):

H. C. Wimberly ◽

D. O. Slauson ◽

N. R. Neilsen

Keyword(s):

Functional Activity ◽

Biochemical Characterization ◽

Platelet Activating Factor ◽

Biochemical Properties ◽

Naturally Occurring ◽

Rabbit Platelets ◽

Rapid Reaction ◽

Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01967-16 ◽

2016 ◽

Vol 61 (3) ◽

Cited By ~ 24

Author(s):

Gaofei Lu ◽

Gregory R. Bluemling ◽

Paul Collop ◽

Michael Hager ◽

Damien Kuiper ◽

...

Keyword(s):

Rna Polymerase ◽

Zika Virus ◽

Nonstructural Protein ◽

Rna Dependent Rna Polymerase ◽

Antiviral Compounds ◽

Double Stranded Dna ◽

Virus Rna ◽

Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.427 ◽

1985 ◽

Vol 101 (2) ◽

pp. 427-440 ◽

Cited By ~ 40

Author(s):

E Bartnik ◽

M Osborn ◽

K Weber

Keyword(s):

Positive Reaction ◽

Intermediate Filaments ◽

Biochemical Characterization ◽

Helix Pomatia ◽

Molar Ratio ◽

Negative Stain ◽

Epithelial Morphology ◽

Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

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