Dataset Information

Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

ABSTRACT: Senecavirus A (SVA) is a member of the genus Senecavirus of the family Picornaviridae. SVA-associated vesicular disease (SAVD) outbreaks have been extensively reported since 2014-2015. Characteristic symptoms include vesicular lesions on the snout and feet as well as lameness in adult pigs and even death in piglets. The capsid protein VP3, a structural protein of SVA, is involved in viral replication and genome packaging. Here, we developed and characterized a mouse monoclonal antibody (mAb) 3E9 against VP3. A motif 192GWFSLHKLTK201 was identified as the linear B-cell epitope recognized by mAb 3E9 by using a panel of GFP-tagged epitope polypeptides. Sequence alignments show that 192GWFSLHKLTK201 was highly conserved in all SVA strains. Subsequently, alanine (A)-scanning mutagenesis indicated that W193, F194, L196, and H197 were the critical residues recognized by mAb 3E9. Further investigation with indirect immunofluorescence assay indicated that the VP3 protein was present in the cytoplasm during SVA replication. In addition, the mAb 3E9 specifically immunoprecipitated the VP3 protein from SVA-infected cells. Taken together, our results indicate that mAb 3E9 could be a powerful tool to work on the function of the VP3 protein during virus infection.

SUBMITTER: Chen M

PROVIDER: S-EPMC8621820 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

Chen Mi M Chen Lulu L Wang Jing J Mou Chunxiao C Chen Zhenhai Z

Viruses 20211118 11

Senecavirus A (SVA) is a member of the genus Senecavirus of the family Picornaviridae. SVA-associated vesicular disease (SAVD) outbreaks have been extensively reported since 2014-2015. Characteristic symptoms include vesicular lesions on the snout and feet as well as lameness in adult pigs and even death in piglets. The capsid protein VP3, a structural protein of SVA, is involved in viral replication and genome packaging. Here, we developed and characterized a mouse m ...[more]

PMID: 34835106

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Project description:Viruses deploy sophisticated strategies to hijack the host's translation machinery to favor viral protein synthesis and counteract innate cellular defenses. However, little is known about the mechanisms by which Senecavirus A (SVA) controls the host's translation. Using a series of sophisticated molecular cell manipulation techniques, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as an essential host factor involved in translation control in SVA-infected cells. It was also determined that the SVA structural protein, VP3, binds to and relocalizes hnRNPA2B1, which interferes with the host's protein synthesis machinery to establish a cellular environment that facilitates viral propagation via a two-pronged strategy: first, hnRNPA2B1 serves as a potent internal ribosome entry site (IRES) trans-acting factor, which is selectively co-opted to promote viral IRES-driven translation by supporting the assembly of translation initiation complexes. Second, a strong repression of host cell translation occurs in the context of the VP3-hnRNPA2B1 interaction, resulting in attenuation of the interferons response. This is the first study to demonstrate the interaction between SVA VP3 and hnRNPA2B1, and to characterize their key roles in manipulating translation. This novel dual mechanism, which regulates selective mRNA translation and immune evasion of virus-infected cells, highlights the VP3-hnRNPA2B1 complex as a potential target for the development of modified antiviral or oncolytic reagents.ImportanceViral reproduction is contingent on viral protein synthesis, which relies entirely on the host's translation machinery. As such, viruses often need to control the cellular translational apparatus to favor viral protein production and avoid host innate defenses. Senecavirus A (SVA) is an important virus, both as an emerging pathogen in the pork industry and as a potential oncolytic virus for neuroendocrine cancers. Here, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) was identified as a critical regulator of the translational landscape during SVA infection. This study supports a model whereby the VP3 protein of SVA efficiently subverts the host's protein synthesis machinery through its ability to bind to and relocalize hnRNPA2B1, not only selectively promoting viral internal ribosome entry site-driven translation but also resulting in global translation shutdown and immune evasion. Together, these data provide new insights into how the complex interactions between translation machinery, SVA, and innate immunity contribute to the pathogenicity of the SVA.

Project description:Senecavirus A (SVA) is a type of nonenveloped single-stranded, positive-sense RNA virus. The VP2 protein is a structural protein that plays an important role in inducing early and late immune responses of the host. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the B epitopes of the VP2 protein is of great importance to revealing its antigenic characterization. In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the VP2 protein from the SVA strain CH/FJ/2017 using the Pepscan approach and a bioinformatics-based computational prediction method. The following four novel IDEs of VP2 were identified: IDE1, 41TKSDPPSSSTDQPTTT56; IDE2, 145PDGKAKSLQELNEEQW160; IDE3, 161VEMSDDYRTGKNMPF175; and IDE4, 267PYFNGLRNRFTTGT280. Most of the IDEs were highly conserved among the different strains. To our knowledge, the VP2 protein is a major protective antigen of SVA that can induce neutralizing antibodies in animals. Here, we analyzed the immunogenicity and neutralization activity of four IDEs of VP2. Consequently, all four IDEs showed good immunogenicity that could elicit specific antibodies in guinea pigs. A neutralization test in vitro showed that the peptide-specific guinea pig antisera of IDE2 could neutralize SVA strain CH/FJ/2017, and IDE2 was identified as a novel potential neutralizing linear epitope. This is the first time VP2 IDEs have been identified by using the Pepscan method and a bioinformatics-based computational prediction method. These results will help elucidate the antigenic epitopes of VP2 and clarify the basis for immune responses against SVA. IMPORTANCE The clinical symptoms and lesions caused by SVA are indistinguishable from those of other vesicular diseases in pigs. SVA has been associated with recent outbreaks of vesicular disease and epidemic transient neonatal losses in several swine-producing countries. Due to the continuing spread of SVA and the lack of commercial vaccines, the development of improved control strategies is urgently needed. The VP2 protein is a crucial antigen on the capsids of SVA particles. Furthermore, the latest research showed that VP2 could be a promising candidate for the development of novel vaccines and diagnostic tools. Hence, a detailed exploration of epitopes in the VP2 protein is necessary. In this study, four novel B-cell IDEs were identified using two different antisera with two different methods. IDE2 was identified as a new neutralizing linear epitope. Our findings will help in the rational design of epitope vaccines and further understanding of the antigenic structure of VP2.

Project description:Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

Dataset Information

Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

Publications

Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

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