A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage

Emmott, Edward; Sweeney, Trevor R.; Goodfellow, Ian

doi:10.1074/jbc.m115.688234

Cited by 29 publications

(36 citation statements)

References 48 publications

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“…This continuous assay allows gathering of valuable information concerning the initial period of the reaction and can be easily used in a high-throughput format. The FRET-based assay can also use recombinant protein based substrates, such as a pair of fluorescent protein joined by a cleavable linker (40). However, as CHIKV nsP2 cleaves recombinant protein and properly designed peptide substrates with similar efficiencies (K. Rausalu, A. Utt, T. Quirin, F. S. Varghese, E. Žusinaite, P. K. Das, T. Ahola, and A.…”

Section: Molecular Designmentioning

confidence: 99%

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Das

Puusepp

Varghese

et al. 2016

Antimicrob Agents Chemother

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dChikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC 50 ) of the most potent inhibitor being ϳ1.5 M. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism. C hikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) is the causative agent of chikungunya fever, a disease that has affected millions in the last decade. It is characterized by high fever, arthralgia, myalgia, headache, and rash. The disease is usually self-limiting; however long-lasting chronic symptoms are observed in nearly 50% of CHIKV-infected patients (1). As there is no approved vaccine or specific licensed antiviral compounds (2), the treatment of CHIKV infection is largely based on relief of symptoms.CHIKV infection can be inhibited by targeting host factors essential for virus infection. Targeting of several metabolic pathways has revealed anti-CHIKV effects of several licensed drugs (3). Compounds most likely targeting virus entry or host cell-specific components required for virus infection have been described (4-8). Several nucleosides or nucleotides, acting as pseudosubstrates for CHIKV RNA-dependent RNA polymerase and/or using another, more general mechanism of action, have shown anti-CHIKV activity (9). In addition, anti-CHIKV effects have been described for several groups of novel synthetic compounds (10-12).Computer-aided design based on molecular docking and molecular dynamics simulations and pharmacophore approach allows identifying potential in silico hits as active inhibitors for different CHIKV replicase proteins. This approach, however, requires the three-dimensional (3D) structure...

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Section: Molecular Designmentioning

confidence: 99%

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Das

Puusepp

Varghese

et al. 2016

Antimicrob Agents Chemother

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“…MNV possesses a genome of ~7.5kb in length, capped by a viral genome-linked protein (VPg) covalently attached at the 5' end and polyadenylated at the 3' end. The VPg protein directs the translation of the MNV ORF1-4 by interacting directly with eIFs (41,42) generating polyproteins subsequently proteolytically processed into three structural and seven non-structural proteins (43).…”

Section: Introductionmentioning

confidence: 99%

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

Brocard

Iadevaia

Klein

et al. 2018

Preprint

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During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation.We show that while MNV infection represses host cell translation, it results in the assembly of virusspecific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

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“…Cell lysates were resolved by SDS-PAGE and then probed using anti-GAPDH (Ambion, AM4300), anti-NS7, anti-ATP5a (Abcam, ab14748), or a mouse monoclonal anti-VF1 (Abmart, 4K5) antibodies. The anti-NS7 antibody was described previously [24]. Goat anti-rabbit (Santa Cruz, sc-2030) and goat anti-mouse (Santa Cruz, sc-2031) secondary antibodies conjugated with horseradish peroxidase were used for detection by chemiluminescence using the Amersham ECL Prime Kit (GE Healthcare).…”

Section: Immunoblottingmentioning

confidence: 99%

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

Borg

Jahun

Thorne

et al. 2019

Preprint

Self Cite

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Background: Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and established that it acts as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Methods:Using reverse genetics, we generated VF1-knockout MNV-3 that contained a single or a triple termination codon inserted in the VF1 open reading frame.Results: VF1-knockout MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. Mice infected with VF1-knockout MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a three-month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Conclusion:Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonise the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilise multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

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A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage

Cited by 29 publications

References 48 publications

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

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