Cryogenic electron tomographs of Coxsackievirus A9 treated with fatty-acid-free BSA
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ABSTRACT:
SUBMITTER: Sarah Jane Butcher
PROVIDER: EMPIAR-11371 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release.
Domanska Aušra A Plavec Zlatka Z Ruokolainen Visa V Löflund Benita B Marjomäki Varpu V Butcher Sarah J SJ
Journal of virology 20221130 24
Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditi ...[more]
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Project description:UnlabelledCoxsackievirus A9 (CVA9) is a member of the human enterovirus B species in the Enterovirus genus of the family Picornaviridae. According to earlier studies, CVA9 binds to αVβ3 and αVβ6 integrins on the cell surface and utilizes β2-microglobulin, dynamin, and Arf6 for internalization. However, the structures utilized by the virus for internalization and uncoating are less well understood. We show here, based on electron microscopy, that CVA9 is found in multivesicular structures 2 h postinfection (p.i.). A neutral red labeling assay revealed that uncoating occurs mainly around 2 h p.i., while double-stranded RNA is found in the cytoplasm after 3 h p.i. The biogenesis of multivesicular bodies (MVBs) is crucial for promoting infection, as judged by the strong inhibitory effect of the wild-type form of Hrs and dominant negative form of VPS4 in CVA9 infection. CVA9 infection is dependent on phospholipase C at the start of infection, whereas Rac1 is especially important between 1 and 3 h p.i., when the virus is in endosomes. Several lines of evidence implicate that low pH does not play a role in CVA9 infection. The infection is not affected by Bafilomycin A1. In addition, CVA9 is not targeted to acidic late endosomes or lysosomes, and the MVBs accumulating CVA9 have a neutral pH. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger neutral MVBs, which are important for virus infection.ImportanceWe demonstrate here that the enterovirus coxsackievirus A9 (CVA9) uses a nonclathrin and nonacidic pathway to infect cells. CVA9 does not accumulate in conventional late endosomes or lysosomes. We found that inhibitors of phospholipase C (PLC), Rac1, and the Na(+)/H(+) exchanger decreased CVA9 infection. The PLC inhibitor acts on early entry, the Rac1 inhibitor acts between 1 and 3 h, when the virus is in endosomes, and the Na(+)/H(+) exchange inhibitor acts during various steps during the virus life cycle. The infection depends on the formation of novel neutral multivesicular bodies (MVBs), which accumulate CVA9 during the first hours of entry. Thus, CVA9 is the second enterovirus demonstrated so far, after echovirus 1, that can trigger formation of neutral MVBs. The data show that these enteroviruses favor nonacidic conditions and complex MVBs to promote virus infection.
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Project description:Coxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin ?v family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for ?v?6, which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating.
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Project description:Coxsackievirus A9 (CVA9) is an enterically transmitted enterovirus and one of the most pathogenic type among human enteroviruses. CVA9 isolates use a distinctive RGD (Arg-Gly-Asp) motif within VP1 capsid protein that defines its ability to bind to integrin receptor(s) for cellular entry. To investigate CVA9 evolution and pathogenicity, genetic relationships and recombination events were analyzed between 54 novel clinical isolates of CVA9, as well as 21 previously published full length CVA9 sequences from GenBank. Samples were investigated by partial sequencing of the novel VP1 and 3Dpol genes, as well as including the corresponding areas from GenBank sequences. Phylogenetic analyses were combined with clinical data in a further attempt to analyze whether sequence evolution reflects CVA9 pathogenicity in the phylogenies. Furthermore, VP1 gene was also analyzed for receptor binding sites including the RGD motif and the putative heparan sulfate (HS) site. Analysis of the 559-nucleotide-long VP1 sequences identified six clades. Although most of the strains within each clade showed geographical clustering, the grouping pattern of the isolates in the analysis of the VP1 gene was strikingly different from grouping of 3Dpol, which suggests that recombination events may have occurred in the region encoding the nonstructural proteins. Inclusion of clinical data did not provide any evidence of symptom based phylogenetic clustering of CVA9 isolates. Amino acid sequence analysis of the VP1 polypeptide demonstrated that the RGD motif was fully conserved among the isolates while the putative HS binding site was only found in one isolate. These data suggest that integrin binding is essential for virus tropism, but do not explain the symptom repertoire.
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Project description:BACKGROUND: Coxsackievirus A9 (CA9) was one of the most prevalent serotype of enteroviral infections in Taiwan in 2011. After several patient series were reported in the 1960s and 1970s, few studies have focused on the clinical manifestations of CA9 infections. Our study explores and deepens the current understanding of CA9. METHODS: We analyzed the clinical presentations of 100 culture-proven CA9-infected patients in 2011 by reviewing their medical records and depicted the CA9 phylogenetic tree. RESULTS: Of the 100 patients with culture-proven CA9 infections, the mean (SD) age was 4.6 (3.4) years and the male to female ratio was 1.9. For clinical manifestations, 96 patients (96%) had fever and the mean (SD) duration of fever was 5.9 (3.4) days. Sixty one patients (61%) developed a skin rash, and the predominant pattern was a generalized non-itchy maculopapular rash without vesicular changes. While most patients showed injected throat, oral ulcers were found in only 19 cases (19%), among whom, 6 were diagnosed as herpangina. Complicated cases included: aseptic meningitis (n=8), bronchopneumonia (n=6), acute cerebellitis (n=1), and polio-like syndrome (n=1). Phylogenetic analysis for current CA9 strains is closest to the CA9 isolate 27-YN-2008 from the border area of mainland China and Myanmar. CONCLUSIONS: The most common feature of CA9 during the 2011 epidemic in Taiwan is generalized febrile exanthema rather than herpangina or hand, foot, and mouth disease. Given that prolonged fever and some complications are possible, caution should be advised in assessing patients as well as in predicting the clinical course.
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Project description:Background and objectivesHuman enteroviruses (EV) are the most common causes of neonatal sepsis-like disease. The frequencies of EV including coxsackievirus A, coxsackievirus B and Echovirus serotypes have been studied in young infants (younger than three months) with sepsis. So far, the role of enteroviruses among neonates with sepsis was not determined in Ahvaz, Iran. Therefore, this study was aimed to evaluate the frequency of EV among hospitalized young infants with clinical signs and symptoms of sepsis in Ahvaz.Materials and methodsBlood specimens from 128 neonates (younger than 90 days), including 56 (43.75%) girls and 72 (56.25%) boys, were collected from hospitalized neonates with clinical signs and symptoms of sepsis-like symptoms. All blood samples were negative for bacterial culture. RNA was extracted from all sera and tested for detection of 5'UTR (Untranslated Region) of the EV by RT-PCR. To determine specific strains of EV, positive 5'UTR samples were further tested for detection of the VP1 region of EV by RT-PCR.ResultsOverall, 50/128 (39.06%) specimens, including 24 (48%) girls and 26 (52%) boys, were positive for EV. 21/50 (42%) specimens were positive for the VP1 region. Randomly, 8 positive VP1 were selected and sequenced. Analysis of sequencing data showed 7/21 (33.33%) samples were positive for Echovirus 30 and 1/21 (4.76%) samples were positive for CVA9.ConclusionThe results of this survey indicate high prevalence of 39.06% of EV among young neonates with sepsis. A high prevalence of 33.3% Echoviruses 30 and a low rate of 4.76% coxsackievirus A9 infection has been observed in neonatal patients with viral sepsis. This outbreak is probably one of the first Enterovirus outbreaks to be reported in Ahvaz, Iran. The results of this survey will help to minimize unneeded use of antimicrobial drugs and reduce unnecessary hospitalization.
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