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Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

ABSTRACT: The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication.The purified BEFV G1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G1 was assayed by ELISA. Then the roles of specific G1-binding peptides in the context of BEFV infection were analyzed.The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner.Two antiviral peptide ligands binding to bovine ephemeral fever virus G1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

SUBMITTER: Hou P

PROVIDER: S-EPMC5753476 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Biopanning of polypeptides binding to bovine ephemeral fever virus G<sub>1</sub> protein from phage display peptide library.

Hou Peili P Zhao Guimin G He Chengqiang C Wang Hongmei H He Hongbin H

BMC veterinary research 20180104 1

<h4>Background</h4>The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G<sub>1</sub> protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G<sub>1</sub> protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G<sub>1</sub> protein with high-affinity and inhibit BEFV replication.<h4>Methods</h4>The purified BEFV G<sub>1< ...[more]

PMID: 29301517

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Project description:Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia, and Australia; however, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956 to 2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated to be between 56 and 65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936 and that the 1955-1956 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent and at a high evolutionary rate of ∼10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2, and G4 have been relatively stable, although variations in site G3a/b defined four antigenic subtypes. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220, and 223, which map to the tip of the spike on the prefusion form of the G protein. Finally, we propose that positive selection on residue 215 was due to cross-reacting neutralizing antibody to Kimberley virus (KIMV). This is the first study of the evolution of BEFV in Australia, showing that the virus has entered the continent only once during the past 50 to 60 years, it is evolving at a relatively constant rate as a single genetic lineage, and although the virus is relatively stable antigenically, mutations have resulted in four antigenic subtypes. Furthermore, the study shows that the evolution of BEFV in Australia appears to be driven, at least in part, by cross-reactive antibodies to KIMV which has a similar distribution and ecology but has not been associated with disease. As BEFV and KIMV are each known to be present in Africa and Asia, this interaction may occur on a broader geographic scale.

Project description:Detection of protection-associated epitopes via reverse vaccinology is the first step for development of subunit vaccines against microbial pathogens. Mapping subunit vaccine targets requires high throughput methods, which would allow delineation of epitopes recognized by protective antibodies on a large scale. Phage displayed random peptide library coupled to Next Generation Sequencing (PDRPL/NGS) is the universal platform that enables high-yield identification of peptides that mimic epitopes (mimotopes). Despite being unsurpassed as a tool for discovery of polyclonal serum mimotopes, the PDRPL/NGS is far inferior as a quantitative method of immune response. Difficult-to-control fluctuations in amounts of antibody-bound phages after rounds of selection and amplification diminish the quantitative capacity of the PDRPL/NGS. In an attempt to improve the accuracy of the PDRPL/NGS method, we compared the discriminating capacity of two approaches for PDRPL/NGS data analysis. The whole-unique-sequence-based analysis (WUSA) involved generation of 7-mer peptide profiles and comparison of the numbers of sequencing reads for unique peptide sequences between serum samples. The motif-based analysis (MA) included identification of 4-mer consensus motifs unifying unique 7-mer sequences and comparison of motifs between serum samples. The motif comparison was based not on the numbers of sequencing reads, but on the numbers of distinct 7-mers constituting the motifs. Our PDRPL/NGS datasets generated from biopanning of protective and non-protective anti-Borrelia burgdorferi sera of New Zealand rabbits were used to contrast the two approaches. As a result, the principle component analyses (PCA) showed that the discriminating powers of the WUSA and MA were similar. In contrast, the unsupervised hierarchical clustering obtained via the MA classified the preimmune, non-protective, and protective sera better than the WUSA-based clustering. Also, a total number of discriminating motifs was higher than that of discriminating 7-mers. In sum, our results indicate that MA approach improves the accuracy and quantitative capacity of the PDRPL/NGS method.

Dataset Information

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

Publications

Biopanning of polypeptides binding to bovine ephemeral fever virus G<sub>1</sub> protein from phage display peptide library.

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