Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator.
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ABSTRACT:
SUBMITTER: Qing J
PROVIDER: S-EPMC4183573 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator.
Qing Jie J Wang Yaxin Y Sun Yuna Y Huang Jiaoyan J Yan Wenzhong W Wang Jinglan J Su Dan D Ni Cheng C Li Jian J Rao Zihe Z Liu Lei L Lou Zhiyong Z
PLoS pathogens 20141002 10
Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further rev ...[more]
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Project description:Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) in children, which can cause severe central nervous system disease and death. The capsids of EV71 consist of 60 copies of each of four viral structural proteins (VP1 to VP4), with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. VP1 plays a central role in particle assembly and cell entry. To gain insight into the role of positively charged residues in VP1 function in these processes, a charged-to-alanine scanning analysis was performed using an infectious cDNA clone of EV71. Twenty-seven mutants containing single charged-to-alanine changes were tested. Sixteen of them were not viable, seven mutants were replication defective, and the remaining four mutants were replication competent. By selecting revertants, second-site mutations which could at least partially restore viral infectivity were identified within VP1 for four defective mutations and two lethal mutations. The resulting residue pairs represent a network of intra- and intermolecular interactions of the VP1 protein which could serve as a potential novel drug target. Interestingly, mutation K215A in the VP1 GH loop led to a significant increase in thermal stability, demonstrating that conditional thermostable mutants can be generated by altering the charge characteristics of VP1. Moreover, all mutants were sensitive to the EV71 entry inhibitor suramin, which binds to the virus particle via the negatively charged naphthalenetrisulfonic acid group, suggesting that single charged-to-alanine mutation is not sufficient for suramin resistance. Taken together, these data highlight the importance of positively charged residues in VP1 for production of infectious particles.Infection with EV71 is more often associated with neurological complications in children and is responsible for the majority of fatalities. No licensed vaccines or antiviral therapies are currently available for the prevention or treatment of EV71 infection. Understanding the determinants of virion assembly and entry will facilitate vaccine development and drug discovery. Here, we identified 23 out of 27 positively charged residues in VP1 which impaired or blocked the production of infectious particles. The defect could be rescued by second-site mutations within the VP1 protein. Our findings highlight the importance of positively charged residues in VP1 during infectious particle production and reveal a potential strategy for blocking EV71 infections by inhibiting intra- or intermolecular interactions of the VP1 protein.
| S-EPMC4702679 | biostudies-literature
Project description:A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.
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Project description:The pathological manifestations of fatal cases of human hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) are characterized by inflammatory damage to the central nervous system (CNS). Here, the dynamic distribution of EV71 in the CNS and the subsequent pathological characteristics within different regions of neonatal rhesus macaque brain tissue were studied using a chimeric EV71 expressing green fluorescence protein. The results were compared with brain tissue obtained from the autopsies of deceased EV71-infected HFMD patients. These observations suggested that the virus was prevalent in areas around the blood vessels and nerve nuclei in the brain stem and showed a preference for astrocytes in the CNS. Interestingly, infected astrocytes within the in vivo and in vitro human and macaque systems exhibited increased expression of excitatory neurotransmitters and cytokines that also stimulated the neuronal secretion of the excitatory neurotransmitters noradrenalin and adrenalin, and this process most likely plays a role in the pathophysiological events that occur during EV71 infection.
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Project description:Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR) in host cells. This study found that enterovirus A71 (EVA71) utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), a key endoplasmic reticulum protein (ER) involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs) on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice). These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity.
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Project description:Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.
| S-EPMC3341398 | biostudies-literature
Project description:Human enterovirus 71 is a picornavirus causing hand, foot, and mouth disease that may progress to fatal encephalitis in infants and small children. As of now, no cure is available for enterovirus 71 infections. Small molecule inhibitors binding into a hydrophobic pocket within capsid viral protein 1 were previously shown to effectively limit infectivity of many picornaviruses. Here we report a 3.2-Å-resolution X-ray structure of the enterovirus 71 virion complexed with the capsid-binding inhibitor WIN 51711. The inhibitor replaced the natural pocket factor within the viral protein 1 pocket without inducing any detectable rearrangements in the structure of the capsid. Furthermore, we show that the compound stabilizes enterovirus 71 virions and limits its infectivity, probably through restricting dynamics of the capsid necessary for genome release. Thus, our results provide a structural basis for development of antienterovirus 71 capsid-binding drugs.
| S-EPMC3619292 | biostudies-literature
Project description:AimTo characterise the viral kinetics of enterovirus 71 (EV71).MethodsIn this study, human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI). After infection, the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0, 6, 12, 24 h post infection). Cell growth and viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point. EV71 replication kinetics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR). Also, the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion. The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting.ResultsEV71 infection induced a significant CPE as early as 6 h post infection (p.i.) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1). In EV71 infected cells, the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection. EV71 virions were uncoated immediately after entry. The intracellular viral RNA began to increase at as early as 3 h p.i. and the exponential increase was found between 3 h to 6 h p.i. in both infected groups. For viral structure protein synthesis, results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h p.i. in the cells infected at either MOI 1 or MOI 10; and reached the peak at 9 h p.i. in the cells infected with EV71 at both MOI 1 and MOI 10. Simultaneously, the viral package and secretion were also actively processed as the virus underwent rapid replication. The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups. It was observed that at 3 h p.i, the intracellular virions obviously decreased, thereafter, the intracellular virions began to increase and enter into the exponential phase until 12 h p.i. The total amounts of intracellular virons were decreased from 12 to 24 h p.i. Consistent with this result, the increase of virus secretion occurred during 6 to 12 h p.i.ConclusionThe viral kinetics of EV71 were established by analyzing viral replication, package and secretion in RD cells.
| S-EPMC3203367 | biostudies-literature