Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design.

Reply to Alberts, P. Comment on "Hietanen et al. Cytolytic Properties and Genome Analysis of Rigvir® Oncolytic Virotherapy Virus and Other Echovirus 7 Isolates. Viruses 2022, 14, 525".

Our work focused on comparing the cellular effects of Rigvir to other echovirus 7 isolates, because originally Rigvir is also an echovirus 7 isolate [...].

Cytolytic Properties and Genome Analysis of Rigvir® Oncolytic Virotherapy Virus and Other Echovirus 7 Isolates.

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.

Isolation and characterization of phage display-derived scFv antibodies against human parechovirus 1 VP0 protein.

Human parechoviruses (PeVs) are common viruses that are associated with a variety of diseases from mild gastrointestinal and respiratory symptoms to severe central nervous system infections. Until now there has not been antibodies for visualizing parechovirus infection. We used E. coli recombinant PeV-A1-VP0 protein as a target in phage display single chain variable fragment (scFv) antibody library panning. Three rounds of panning allowed identification and isolation of several candidate scFv clones, which tested positive in enzyme-linked immunosorbent assay (ELISA) against VP0. Three scFv clones (scFv-55, -59 and -71) with different CDR-3 sequences were further purified and tested in ELISA, Western blot and immunofluorescence microscopy (IFA) against a set of PeV-A1 isolates and a few isolates representing PeV types 2-6. In IFA, all three scFv binders recognized twenty PeV-A1 isolates. ScFv-55 and -71 also recognized clinical representatives of PeV types 1-6 both in IFA and in capture ELISA, while scFv-59 only recognized PeV-A1, -A2 and -A6. PeV-A1-VP0 (Harris strain) sequence was used to generate a peptide library, which allowed identification of a putative unique conformational antibody epitope with fully conserved flanking regions and a more variable core VVTYDSKL, shared between the scFv antibodies. Sequencing of the VP0 region of virus samples and sequence comparisons against parechoviral sequences in GenBank revealed 107 PeV-A1, -A3, -A8, -A17, -A (untyped) sequences with this exact epitope core sequence, which was most dominant among PeV-A1 isolates. These data suggest the first-time isolation of broad range phage display antibodies against human parechoviruses that may be used in diagnostic antibody development.

Monoclonal antibody against VP0 recognizes a broad range of human parechoviruses.

Parechoviruses (PeVs) are common viruses that cause mild gastrointestinal or respiratory symptoms to severe central nervous system infections. In infants, parechovirus infection is one of the leading causes of life-threatening viral disease. High-quality antibodies with broad binding specificities are essential to improve accurate parechovirus diagnosis in diagnostic laboratories. Such antibodies have potential in the development of rapid antigen detection assay against PeVs. In the present study, VP4 and VP2 genes from human parechovirus A1 (PeV-A1) were cloned and VP0 fusion protein produced to develop monoclonal antibodies against PeVs. Two pan-parechovirus antibodies, one IgG and one IgM isotype, were isolated. The properties of IgG1/κ monoclonal (designated as Mab-PAR-1) was studied further. Mab-PAR-1 was shown to be functional in western blot against denatured recombinant protein and viral particles. In immunofluorescence assay, the antibody tested positive for nineteen PeV-A1 isolates while showing no cross-reactivity to fourteen entero- and rhinovirus types. In addition, Mab-PAR-1 showed positive reactivity against five other cultivable parechovirus types 2-6. A unique Mab-PAR-1 epitope located in the junction of the three capsid proteins VP0, VP1, and VP3 was identified using a peptide library screen. This study demonstrates that PeV-A1-VP0 protein is functional antigen for developing monoclonal antibody for diagnosis of broad range of parechovirus infections.

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

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.

Genome Sequences of RIGVIR Oncolytic Virotherapy Virus and Five Other Echovirus 7 Isolates.

We report here the nearly complete Illumina-sequenced consensus genome sequences of six isolates of echovirus 7 (E7), including oncolytic virotherapy virus RIGVIR and the Wallace prototype. Amino acid identities within the coding region were highly conserved across all isolates, ranging from 95.31% to 99.73%.