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Recombination Events and Conserved Nature of Receptor Binding Motifs in Coxsackievirus A9 Isolates.

ABSTRACT: 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.

SUBMITTER: Hietanen E

PROVIDER: S-EPMC7019539 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Recombination Events and Conserved Nature of Receptor Binding Motifs in Coxsackievirus A9 Isolates.

Hietanen Eero E Susi Petri P

Viruses 20200106 1

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 seque ...[more]

PMID: 31935831

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Project description:Coxsackievirus A9 (CV-A9) is a pathogenic enterovirus type within the family Picornaviridae. CV-A9 infects A549 human epithelial lung carcinoma cells by attaching to the ?V?6 integrin receptor through a highly conserved Arg-Gly-Asp (RGD) motif, which is located at the exposed carboxy-terminus of the capsid protein VP1 detected in all studied clinical isolates. However, genetically-modified CV-A9 that lacks the RGD motif (CV-A9-RGDdel) has been shown to be infectious in some cell lines but not in A549, suggesting that RGD-mediated integrin binding is not always essential for efficient entry of CV-A9.Two cell lines, A549 and SW480, were used in the study. SW480 was the study object for the integrin-independent entry and A549 was used as the control for integrin-dependent entry. Receptor levels were quantitated by cell sorting and quantitative PCR. Antibody blocking assay and siRNA silencing of receptor-encoding genes were used to block virus infection. Peptide phage display library was used to identify peptide binders to CV-A9. Immunofluorescence and confocal microscopy were used to visualize the virus infection in the cells.We investigated the receptor use and early stages of CV-A9 internalization to SW480 human epithelial colon adenocarcinoma cells. Contrary to A549 infection, we showed that both CV-A9 and CV-A9-RGDdel internalized into SW480 cells and that function-blocking anti-?V integrin antibodies had no effect on the binding and entry of CV-A9. Whereas siRNA silencing of ?6 integrin subunit had no influence on virus infection in SW480, silencing of ?2-microglobulin (?2M) inhibited the virus infection in both cell lines. By using a peptide phage display screening, the virus-binding peptide identical to the N-terminal sequence of HSPA5 protein was identified and shown to block the virus infection in both A549 and SW480 cell lines. HSPA5 was also found to co-localize with CV-A9 at the SW480 cell periphery during the early stages of infection by confocal microscopy.The data suggest that while ?V?6 integrin is essential for CV-A9 in A549 cell line, it is not required in SW480 cell line in which ?2M and HSPA5 alone are sufficient for CV-A9 infection. This suggests that the choice of CV-A9 receptor(s) is dependent on the tissue/cellular environment.

Dataset Information

Recombination Events and Conserved Nature of Receptor Binding Motifs in Coxsackievirus A9 Isolates.

Publications

Recombination Events and Conserved Nature of Receptor Binding Motifs in Coxsackievirus A9 Isolates.

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