Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural view of the 2A protease from human rhinovirus C15.

ABSTRACT: The majority of outbreaks of the common cold are caused by rhinoviruses. The 2A protease (2Apro) of human rhinoviruses (HRVs) is known to play important roles in the propagation of the virus and the modulation of host signal pathways to facilitate viral replication. The 2Apro from human rhinovirus C15 (HRV-C15) has been expressed in Escherichia coli and purified by affinity chromatography, ion-exchange chromatography and gel-filtration chromatography. The crystals diffracted to 2.6?Å resolution. The structure was solved by molecular replacement using the structure of 2Apro from coxsackievirus A16 (CVA16) as the search model. The structure contains a conserved His-Asp-Cys catalytic triad and a Zn2+-binding site. Comparison with other 2Apro structures from enteroviruses reveals that the substrate-binding cleft of 2Apro from HRV-C15 exhibits a more open conformation, which presumably favours substrate binding.

SUBMITTER: Ling H

PROVIDER: S-EPMC5894110 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Structural view of the 2A protease from human rhinovirus C15.

Ling Hui H Yang Pan P Hou Hai H Sun Yao Y

Acta crystallographica. Section F, Structural biology communications 20180328 Pt 4

The majority of outbreaks of the common cold are caused by rhinoviruses. The 2A protease (2A<sup>pro</sup>) of human rhinoviruses (HRVs) is known to play important roles in the propagation of the virus and the modulation of host signal pathways to facilitate viral replication. The 2A<sup>pro</sup> from human rhinovirus C15 (HRV-C15) has been expressed in Escherichia coli and purified by affinity chromatography, ion-exchange chromatography and gel-filtration chromatography. The crystals diffracte ...[more]

PMID: 29633974

Similar Datasets

Solution structure of the 2A protease from a common cold agent, human rhinovirus C2, strain W12.

Project description:Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

| S-EPMC4061012 | biostudies-literature

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Project description:Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

| S-EPMC3255852 | biostudies-literature

Rhinovirus C15 Induces Airway Hyperresponsiveness via Calcium Mobilization in Airway Smooth Muscle.

Project description:Rhinovirus (RV) exposure evokes exacerbations of asthma that markedly impact morbidity and mortality worldwide. The mechanisms by which RV induces airway hyperresponsiveness (AHR) or by which specific RV serotypes differentially evoke AHR remain unknown. We posit that RV infection evokes AHR and inflammatory mediator release, which correlate with degrees of RV infection. Furthermore, we posit that rhinovirus C-induced AHR requires paracrine or autocrine mediator release from epithelium that modulates agonist-induced calcium mobilization in human airway smooth muscle. In these studies, we used an ex vivo model to measure bronchoconstriction and mediator release from infected airways in human precision cut lung slices to understand how RV exposure alters airway constriction. We found that rhinovirus C15 (RV-C15) infection augmented carbachol-induced airway narrowing and significantly increased release of IP-10 (IFN-γ-induced protein 10) and MIP-1β (macrophage inflammatory protein-1β) but not IL-6. RV-C15 infection of human airway epithelial cells augmented agonist-induced intracellular calcium flux and phosphorylation of myosin light chain in co-cultured human airway smooth muscle to carbachol, but not after histamine stimulation. Our data suggest that RV-C15-induced structural cell inflammatory responses are associated with viral load but that inflammatory responses and alterations in agonist-mediated constriction of human small airways are uncoupled from viral load of the tissue.

| S-EPMC7055698 | biostudies-literature

Human rhinovirus 2A proteinase mutant and its second-site revertants.

Project description:The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130-->Tyr, Phe-130-->Tyr/His-135-->Arg, Phe-130-->Tyr/His-137-->Arg, His-135-->Arg or His-137-->Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-I-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130-->Tyr 2A proteinase were reduced by about 17 degrees C compared with the wild-type enzyme. The presence of the additional mutations His-135-->Arg or His-137-->Arg in the Phe-130-->Tyr mutant increased temperature stability by 3 degrees C and 6 degrees C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.

| S-EPMC1217610 | biostudies-other

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view.

Project description:Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL-receptor family including the very low density lipoprotein (VLDL)-receptor (VLDL-R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL-R to 15 A resolution by cryo-electron microscopy. The receptor fragments, which include the first three ligand-binding repeats of the VLDL-R (V1-3), bind to the small star-shaped dome on the icosahedral 5-fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM-1 is located at the base of a depression around each 5-fold axis. Homology models of the three domains of V1-3 were used to explore the virus-receptor interaction. The footprint of VLDL-R on the viral surface covers the BC- and HI-loops on VP1.

| S-EPMC305862 | biostudies-literature

Human Rhinovirus 3C protease cleaves RIPK1, concurrent with caspase 8 activation.

Project description:Human Rhinovirus (HRV) is a pathogen of significant medical importance, being a major cause of upper respiratory tract infections (common colds) as well as causing the majority of virus-induced asthma exacerbations. We investigated whether HRV could modulate apoptosis, an innate antiviral response. Apoptotic signals are generated either extrinsically or intrinsically and are propagated via caspase cascades that lead to cell death, reducing viral replication, which relies on cellular machinery. Using HRV16 infected cells, in combination with chemical inducers and inhibitors of extrinsic apoptosis we show that HRV16 3C protease cleaves a key intermediate in extrinsic apoptosis. Receptor-interacting protein kinase-1 (RIPK1), an extrinsic apoptosis adaptor protein, was cleaved by caspase 8, as expected, during chemical induction of apoptosis. RIPK1 was cleaved in HRV infection albeit at a different site. Caspase 8 activation, which is associated with extrinsic apoptosis, was concurrent with HRV 3C protease mediated cleavage of RIPK1, and potentially increased the accessibility of the HRV 3C cleavage site within RIPK1 in-vitro. The caspase 8 mediated RIPK1 cleavage product has a pro-apoptotic function, and further cleavage of this pro-apoptotic cleavage product by HRV 3C may provide a mechanism by which HRV limits apoptosis.

| S-EPMC5785518 | biostudies-literature

Design of a Human Rhinovirus-14 3C Protease-Inducible Caspase-3.

Project description:The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.

| S-EPMC6571611 | biostudies-literature

Genome-scale CRISPR-Cas9 knockout screens for enterovirus D68 and rhinovirus C15

Project description:H1-HeLa cells were stably transduced with lentiCas9-Blast (Addgene, Plasmid #52962) and subsequently selected using blasticidin to generate constitutively expressing Cas9 H1-HeLa cells. A single Cas9-expressing H1-HeLa clone was then transduced with lentivirus without a selection marker to stably express CDHR3 C529Y (H1-HeLa+CDHR3). A single CDHR3-expressing H1-HeLa clone was then chosen based on RT-qPCR of CHDR3 expression and RV-C15 RNA levels for mutagenesis. 300 million of the H1-HeLa cells constitutively expressing CDHR3 and Cas9 were transduced with the lentiGuide-Puro from the GeCKO v2 library at a MOI of 0.3. Cells were selected using puromycin and heterogeneous H1-HeLa knockout cell populations were subsequently pooled together. The CRISPR genetic screens were started 10 days post transduction. >1000-fold coverage of mutagenized cells (libraries A and B) was infected with either RV-C15 (MOI=1 PFU/cell) or EV-D68 Missouri (MOI=1 PFU/cell). RV-C15 infection was repeated for an additional round at 6 days post-infection. As soon as appearance of visibly viable colonies was observed, populations of virus-resistant cells were pooled and harvested. Uninfected starting populations of mutagenized cells were used as the unselected reference. Total genomic DNA from both virus-resistant and uninfected cells was respectively extracted using QIAamp DNA Mini Kit (Qiagen). The inserted guide RNA sequences were retrieved from the genomic DNA by PCR amplification. The PCR products were then purified and subjected to NextSeq platform (Illumina) next-generation sequencing.

2019-07-10 | E-MTAB-8125 | biostudies-arrayexpress

Structure-activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors.

Project description:Human rhinovirus 3C protease (HRV 3C(pro)) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C(pro), a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C(pro), to determine the structure-activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC(50) value of 80nM. In addition, the binding mode of a novel analog, 19, with the 4-hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1 pocket.

| S-EPMC7126291 | biostudies-literature

Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells.

Project description:Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (?10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T125K) and replication (3A E41K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2-8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay.

| S-EPMC5110265 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data