Titration of Human Coronaviruses, HCoV-229E and HCoV-OC43, by an Indirect Immunoperoxidase Assay

Jacomy, Hélène; Marceau, Gabriel; Talbot, Pierre J.

doi:10.1007/978-1-59745-181-9_8

Cited by 36 publications

(46 citation statements)

References 15 publications

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“…The titers of infectious HCoV-OC43 virions were determined by IPA as previously reported (30,31). Briefly, RD cells were seeded in 96-well plates and infected with each virus in a serial dilution of 10 Ϫ1 to 10 Ϫ7 .…”

Section: Cells and Virusesmentioning

confidence: 99%

“…RD cells were infected with recombinant WT or ⌬ns12.9 viruses at an MOI of 1 for 24 h. The cells were washed with PBS and fixed with 2% glutaraldehyde overnight at 4°C. The samples then were treated with 2% osmium tetroxide for 1 h, dehydrated gradually through an ethanol series (30,50,70,90, and 100%), and embedded in Epon 812 resin. The infiltrated samples were polymerized at 60°C for 48 h. Ultrathin sections (70 nm) of the cells were produced using an EM UC6 ultramicrotome (Leica) and stained with 2% uranyl acetate and 1% lead citrate.…”

Section: Cells and Virusesmentioning

confidence: 99%

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The ns12.9 Accessory Protein of Human Coronavirus OC43 Is a Viroporin Involved in Virion Morphogenesis and Pathogenesis

Wang

Ping

et al. 2015

J Virol

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An accessory gene between the S and E gene loci is contained in all coronaviruses (CoVs), and its function has been studied in some coronaviruses. This gene locus in human coronavirus OC43 (HCoV-OC43) encodes the ns12.9 accessory protein; however, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein (HCoV-OC43-⌬ns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-⌬ns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo. Intriguingly, exogenous ns12.9 and heterologous viroporins with ion channel activity could compensate for the production of HCoV-OC43-⌬ns12.9, indicating that the ion channel activity of ns12.9 plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication revealed that ns12.9 protein had no measurable effect on virus entry, subgenomic mRNA (sgmRNA) synthesis, and protein expression. Further characterization revealed that HCoV-OC43-⌬ns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus (HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response, and virulence in HCoV-OC43-⌬ns12.9-infected mice were observed compared to the levels for HCoV-OC43-WT-infected mice. Taken together, our results demonstrated that the ns12.9 accessory protein functions as a viroporin and is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection. IMPORTANCEHCoV-OC43 was isolated in the 1960s and is a major agent of the common cold. The functions of HCoV-OC43 structural proteins have been well studied, but few studies have focused on its accessory proteins. In the present study, we demonstrated that the ns12.9 protein is a newly recognized viroporin, and the ns12.9 gene knockout virus (HCoV-OC43-⌬ns12.9) presents a growth defect in vitro and in vivo. We identified the important functions of the ns12.9 viroporin in virion morphogenesis during HCoV-OC43 infection. Furthermore, mice infected with HCoV-OC43-⌬ns12.9 exhibited reduced inflammation and virulence accompanied by a lower titer in the brain than that of wild-type-infected mice, suggesting the ns12.9 viroporin influences virus pathogenesis. Therefore, our findings revealed that the ns12.9 viroporin facilitates virion morphogenesis to enhance viral production, and these results provided a deeper understanding of HCoV-OC43 pathogenesis.T he coronaviruses (CoVs) belong to the Coronaviridae family of the order Nidovirales and are distributed widely among animals, birds, and humans (1). Members of the CoVs are further classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (2). Human coronavirus OC43 (HCoV-OC43) was isolated from a patient with upper respiratory tract disease ...

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Section: Cells and Virusesmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

The ns12.9 Accessory Protein of Human Coronavirus OC43 Is a Viroporin Involved in Virion Morphogenesis and Pathogenesis

Wang

Ping

et al. 2015

J Virol

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“…Quantitation of infectious virus titers by IPA. The immunoperoxidase assay (IPA) was performed on L132 cells as previously described (13). Briefly, the primary antibody used was monoclonal antibody (MAb) 5-11H.6 directed against the S protein of HCoV-229E.…”

Section: In Vitro Translationsmentioning

confidence: 99%

“…The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (KPL). Immune complexes were detected by incubation with 0.025% (wt/vol) 3,3Ј-diaminobenzidine tetrahydrochloride (Bio-Rad) and 0.01% hydrogen peroxide in PBS, and infectious virus titers were calculated by the Karber method as previously described (13).…”

Section: In Vitro Translationsmentioning

confidence: 99%

Blocking eIF4E-eIF4G Interaction as a Strategy To Impair Coronavirus Replication

Cencic

Desforges

Hall

et al. 2011

J Virol

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Coronaviruses are a family of enveloped single-stranded positive-sense RNA viruses causing respiratory, enteric, and neurologic diseases in mammals and fowl. Human coronaviruses are recognized to cause up to a third of common colds and are suspected to be involved in enteric and neurologic diseases. Coronavirus replication involves the generation of nested subgenomic mRNAs (sgmRNAs) with a common capped 5 leader sequence. The translation of most of the sgmRNAs is thought to be cap dependent and displays a requirement for eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex needed for the recruitment of 40S ribosomes. We recently reported on an ultrahigh-throughput screen to discover compounds that inhibit eIF4F activity by blocking the interaction of two of its subunits (R. Cencic et al., Proc. Natl. Acad. Sci. U. S. A. 108:1046-1051, 2011). Herein we describe a molecule from this screen that prevents the interaction between eIF4E (the cap-binding protein) and eIF4G (a large scaffolding protein), inhibiting cap-dependent translation. This inhibitor significantly decreased human coronavirus 229E (HCoV-229E) replication, reducing the percentage of infected cells and intra-and extracellular infectious virus titers. Our results support the strategy of targeting the eIF4F complex to block coronavirus infection.

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“…Although all known HCoVs have viral tropism targeted at the human respiratory tract, lung cell lines infected by a broad range of HCoVs have not been defined. A key feature of SARS-CoV and MERS-CoV is that highly pathogenic coronaviruses grow to higher viral titer on a wider range of cell lines than the other mildly pathogenic coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 [40][41][42][43][44]. High throughput approaches to screen compound libraries for targeted activity against coronaviruses have been underdeveloped and limited in the number of viral strains used [45][46][47][48][49][50][51][52].…”

Section: Cell-based Systemsmentioning

confidence: 99%

Broad-spectrum coronavirus antiviral drug discovery

Totura

Bavari

2019

Expert Opinion on Drug Discovery

183

170

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Introduction: The highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are lethal zoonotic viruses that have emerged into human populations these past 15 years. These coronaviruses are associated with novel respiratory syndromes that spread from person-to-person via close contact, resulting in high morbidity and mortality caused by the progression to Acute Respiratory Distress Syndrome (ARDS). Areas covered: The risks of re-emergence of SARS-CoV from bat reservoir hosts, the persistence of MERS-CoV circulation, and the potential for future emergence of novel coronaviruses indicate antiviral drug discovery will require activity against multiple coronaviruses. In this review, approaches that antagonize viral nonstructural proteins, neutralize structural proteins, or modulate essential host elements of viral infection with varying levels of efficacy in models of highly pathogenic coronavirus disease are discussed. Expert opinion: Treatment of SARS and MERS in outbreak settings has focused on therapeutics with general antiviral activity and good safety profiles rather than efficacy data provided by cellular, rodent, or nonhuman primate models of highly pathogenic coronavirus infection. Based on lessons learned from SARS and MERS outbreaks, lack of drugs capable of pan-coronavirus antiviral activity increases the vulnerability of public health systems to a highly pathogenic coronavirus pandemic. ARTICLE HISTORY

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Titration of Human Coronaviruses, HCoV-229E and HCoV-OC43, by an Indirect Immunoperoxidase Assay

Cited by 36 publications

References 15 publications

The ns12.9 Accessory Protein of Human Coronavirus OC43 Is a Viroporin Involved in Virion Morphogenesis and Pathogenesis

The ns12.9 Accessory Protein of Human Coronavirus OC43 Is a Viroporin Involved in Virion Morphogenesis and Pathogenesis

Blocking eIF4E-eIF4G Interaction as a Strategy To Impair Coronavirus Replication

Broad-spectrum coronavirus antiviral drug discovery

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