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Scientific Reports Feb 2022Echovirus 9 (E9) belongs to the species Enterovirus B. So far, 12 whole genome sequences of E9 are available in GenBank. In this study, we determined the whole genomic...
Echovirus 9 (E9) belongs to the species Enterovirus B. So far, 12 whole genome sequences of E9 are available in GenBank. In this study, we determined the whole genomic sequences of five E9 strains isolated from the stools of patients with hand-foot-and-mouth disease in Kunming, Yunnan Province, China, in 2019. Their nucleotide and amino acid sequences shared 80.8-80.9% and 96.4-96.8% identity with the prototype Hill strain, respectively, and shared 99.3-99.9% and 99.1-99.8% mutual identity, respectively. Recombination analyses revealed that intertype recombination had occurred in the 2C and 3D regions of the five Yunnan E9 strains with coxsackieviruses B5 and B4, respectively. This study augmented the whole genome sequences of E9 in the GenBank database and extended the molecular characterization of this virus in China.
Topics: Child, Preschool; China; Databases, Nucleic Acid; Echovirus 9; Enterovirus B, Human; Genome, Viral; Hand, Foot and Mouth Disease; Humans; Infant; Phylogeny; Recombination, Genetic; Whole Genome Sequencing
PubMed: 35145190
DOI: 10.1038/s41598-022-06309-1 -
Molecular Biology and Evolution Jan 2021Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus...
Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.
Topics: Amino Acid Substitution; Biological Evolution; Capsid Proteins; Enterovirus B, Human; Genetic Fitness; Virus Shedding
PubMed: 32810259
DOI: 10.1093/molbev/msaa208 -
Viruses Mar 2022Rigvir is a cell-adapted, oncolytic virotherapy enterovirus, which derives from an echovirus 7 (E7) isolate. While it is claimed that Rigvir causes cytolytic infection...
Rigvir is a cell-adapted, oncolytic virotherapy enterovirus, which derives from an echovirus 7 (E7) isolate. While it is claimed that Rigvir causes cytolytic infection in several cancer cell lines, there is little molecular evidence for its oncolytic and oncotropic potential. Previously, we genome-sequenced Rigvir and five echovirus 7 isolates, and those sequences are further analyzed in this paper. A phylogenetic analysis of the full-length data suggested that Rigvir was most distant from the other E7 isolates used in this study, placing Rigvir in its own clade at the root of the phylogeny. Rigvir contained nine unique mutations in the viral capsid proteins VP1-VP4 across the whole data set, with a structural analysis showing six of the mutations concerning residues with surface exposure on the cytoplasmic side of the viral capsid. One of these mutations, E/Q/N162G, was located in the region that forms the contact interface between decay-accelerating factor (DAF) and E7. Rigvir and five other isolates were also subjected to cell infectivity assays performed on eight different cell lines. The used cell lines contained both cancer and non-cancer cell lines for observing Rigvir's claimed properties of being both oncolytic and oncotropic. Infectivity assays showed that Rigvir had no discernable difference in the viruses' oncolytic effect when compared to the Wallace prototype or the four other E7 isolates. Rigvir was also seen infecting non-cancer cell lines, bringing its claimed effect of being oncotropic into question. Thus, we conclude that Rigvir's claim of being an effective treatment against multiple different cancers is not warranted under the evidence presented here. Bioinformatic analyses do not reveal a clear mechanism that could elucidate Rigvir's function at a molecular level, and cell infectivity tests do not show a discernable difference in either the oncolytic or oncotropic effect between Rigvir and other clinical E7 isolates used in the study.
Topics: DNA Viruses; Enterovirus B, Human; Humans; Neoplasms; Oncolytic Virotherapy; Oncolytic Viruses; Phylogeny; Viruses, Unclassified
PubMed: 35336934
DOI: 10.3390/v14030525 -
Communications Biology Feb 2021Enteroviruses pose a persistent and widespread threat to human physical health, with no specific treatments available. Small molecule capsid binders have the potential...
Enteroviruses pose a persistent and widespread threat to human physical health, with no specific treatments available. Small molecule capsid binders have the potential to be developed as antivirals that prevent virus attachment and entry into host cells. To aid with broad-range drug development, we report here structures of coxsackieviruses B3 and B4 bound to different interprotomer-targeting capsid binders using single-particle cryo-EM. The EM density maps are beyond 3 Å resolution, providing detailed information about interactions in the ligand-binding pocket. Comparative analysis revealed the residues that form a conserved virion-stabilizing network at the interprotomer site, and showed the small molecule properties that allow anchoring in the pocket to inhibit virus disassembly.
Topics: Animals; Antiviral Agents; Binding Sites; Capsid; Capsid Proteins; Cell Line; Chlorocebus aethiops; Cryoelectron Microscopy; Drug Development; Enterovirus B, Human; Ligands; Molecular Docking Simulation; Protein Conformation; Virus Assembly
PubMed: 33637854
DOI: 10.1038/s42003-021-01779-x -
Journal of Virology Dec 2022Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo...
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 conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.
Topics: Humans; Albumins; Capsid Proteins; Cations; Cryoelectron Microscopy; Endosomes; Enterovirus B, Human; Enterovirus Infections; RNA; Virion; Genome, Viral
PubMed: 36448797
DOI: 10.1128/jvi.01367-22 -
Proceedings of the National Academy of... Jan 2022Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and...
Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.
Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; CD55 Antigens; Coxsackievirus Infections; Enterovirus B, Human; Host-Pathogen Interactions; Humans; Models, Molecular; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Receptors, Virus; Structure-Activity Relationship; Virus Attachment
PubMed: 35046043
DOI: 10.1073/pnas.2118590119 -
Yonsei Medical Journal Nov 2014This study investigated the possible relationship between viral infection and first trimester pregnancy loss.
PURPOSE
This study investigated the possible relationship between viral infection and first trimester pregnancy loss.
MATERIALS AND METHODS
A prospective study was performed on 51 gravidas with missed abortion, fetal anomaly, pre-term delivery, and full-tem delivery at Hanyang University Hospital. Enteroviruses were detected by semi-nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in abortive tissues and placentas. Enterovirus serotypes were confirmed by genome sequencing. Herpesviruses were detected by PCR.
RESULTS
Coxsackievirus B3 (CVB3) was detected in 8 of 14 missed abortion cases, 1 of 27 full-term cases, and none of the 9 pre-term cases. Coxsackievirus B1 (CVB1) was detected in an encephalocele case. Herpes simplex virus type 1 was found in 4 full-term cases, 3 pre-term cases, and none of the missed abortion cases.
CONCLUSION
The prevalence of CVB3 was significantly higher in missed abortion cases compared to full-term or pre-term delivery cases. CVB infection may therefore be an important etiological agent of missed abortion.
Topics: Abortion, Missed; Adult; Coxsackievirus Infections; Enterovirus B, Human; Female; Humans; Immunohistochemistry; Placenta; Pregnancy; Pregnancy Complications, Infectious; Pregnancy Trimester, First; Prevalence; Prospective Studies; Republic of Korea; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Uterus
PubMed: 25323892
DOI: 10.3349/ymj.2014.55.6.1562 -
Journal of Virology Sep 2019There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and...
There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release. There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.
Topics: Albumins; Animals; Capsid Proteins; Cell Line; Chlorocebus aethiops; Cryoelectron Microscopy; Endosomes; Enterovirus B, Human; Fatty Acids; Hot Temperature; Models, Molecular
PubMed: 31189702
DOI: 10.1128/JVI.00599-19 -
MBio Mar 2016Coxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3)...
UNLABELLED
Coxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3) Nancy strain. Using HeLa cell plaque assays with agar overlays, we noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. We identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. We determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. Our data reinforce the idea that culture-adapted laboratory virus strains can have reduced fitnessin vivo N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.
IMPORTANCE
Coxsackieviruses frequently infect humans, and although many infections are mild or asymptomatic, there can be severe outcomes, including heart inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. We used a cell culture-adapted strain of CVB3, Nancy, to examine viral replication and pathogenesis in orally inoculated mice. We found that mice shed viruses distinct from input viruses because they formed extremely large plaques in cell culture. We identified a single mutation, VP3 N63Y, that was sufficient for large-plaque formation. N63Y mutant viruses have reduced glycan binding and replication in cell culture; however, they have enhanced replication and virulence in mice. We are now using N63Y mutant CVB3 as an improved system for viral pathogenesis studies.
Topics: Adaptation, Biological; Amino Acid Substitution; Animals; Capsid Proteins; Coxsackievirus Infections; Disease Models, Animal; Enterovirus B, Human; HeLa Cells; Humans; Liver; Mice; Mutation, Missense; Polysaccharides; Protein Binding; Viral Plaque Assay; Virulence
PubMed: 27025249
DOI: 10.1128/mBio.00119-16 -
Journal of Virology Mar 2014RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing...
UNLABELLED
RIG-I-like receptors (RLRs) MDA5 and RIG-I are key players in the innate antiviral response. Upon recognition of viral RNA, they interact with MAVS, eventually inducing type I interferon production. The interferon induction pathway is commonly targeted by viruses. How enteroviruses suppress interferon production is incompletely understood. MDA5 has been suggested to undergo caspase- and proteasome-mediated degradation during poliovirus infection. Additionally, MAVS is reported to be cleaved during infection with coxsackievirus B3 (CVB3) by the CVB3 proteinase 3C(pro), whereas MAVS cleavage by enterovirus 71 has been attributed to 2A(pro). As yet, a detailed examination of the RLR pathway as a whole during any enterovirus infection is lacking. We performed a comprehensive analysis of crucial factors of the RLR pathway, including MDA5, RIG-I, LGP2, MAVS, TBK1, and IRF3, during infection of CVB3, a human enterovirus B (HEV-B) species member. We show that CVB3 inhibits the RLR pathway upstream of TBK1 activation, as demonstrated by limited phosphorylation of TBK1 and a lack of IRF3 phosphorylation. Furthermore, we show that MDA5, MAVS, and RIG-I all undergo proteolytic degradation in CVB3-infected cells through a caspase- and proteasome-independent manner. We convincingly show that MDA5 and MAVS cleavages are both mediated by CVB3 2A(pro), while RIG-I is cleaved by 3C(pro). Moreover, we show that proteinases 2A(pro) and 3C(pro) of poliovirus (HEV-C) and enterovirus 71 (HEV-A) exert the same functions. This study identifies a critical role of 2A(pro) by cleaving MDA5 and MAVS and shows that enteroviruses use a common strategy to counteract the interferon response in infected cells.
IMPORTANCE
Human enteroviruses (HEVs) are important pathogens that cause a variety of diseases in humans, including poliomyelitis, hand, foot, and mouth disease, viral meningitis, cardiomyopathy, and more. Like many other viruses, enteroviruses target the host immune pathways to gain replication advantage. The MDA5/MAVS pathway is responsible for recognizing enterovirus infections in the host cell and leads to expression of type I interferons (IFN-I), crucial antiviral signaling molecules. Here we show that three species of HEVs all employ the viral proteinase 2A (2A(pro)) to proteolytically target MDA5 and MAVS, leading to an efficient blockade upstream of IFN-I transcription. These observations suggest that MDA5/MAVS antagonization is an evolutionarily conserved and beneficial mechanism of enteroviruses. Understanding the molecular mechanisms of enterovirus immune evasion strategies will help to develop countermeasures to control infections with these viruses in the future.
Topics: Adaptor Proteins, Signal Transducing; Cysteine Endopeptidases; DEAD-box RNA Helicases; Enterovirus B, Human; Enterovirus Infections; Host-Pathogen Interactions; Humans; Interferon-Induced Helicase, IFIH1; Phosphorylation; Proteolysis; Receptors, Retinoic Acid; Signal Transduction; Viral Proteins
PubMed: 24390337
DOI: 10.1128/JVI.02712-13