Screening and identification of cyprinid herpesvirus 2 (CyHV-2) ORF55-interacting proteins by phage display

Abstract

Background Cyprinid herpesvirus 2 (CyHV-2) is a pathogenic fish virus belonging to family Alloherpesviridae. The CyHV-2 gene encoding thymidine kinase (TK) is an important virulence-associated factor. Therefore, we aimed to investigate the biological function of open reading frame 55 (ORF55) in viral replication. Methods Purified CyHV-2 ORF55 protein was obtained by prokaryotic expression, and the interacting peptide was screened out using phage display. Host interacting proteins were then predicted and validated. Results ORF55 was efficiently expressed in the prokaryotic expression system. Protein and peptide interaction prediction and dot-blot overlay assay confirmed that peptides identified by phage display could interact with the ORF55 protein. Comparing the peptides to the National Center for Biotechnology Information database revealed four potential interacting proteins. Reverse transcription quantitative PCR results demonstrated high expression of an actin-binding Rho-activating protein in the latter stages of virus-infected cells, and molecular docking, cell transfection and coimmunoprecipitation experiments confirmed that it interacted with the ORF55 protein. Conclusion During viral infection, the ORF55 protein exerts its biological function through interactions with host proteins. The specific mechanisms remain to be further explored.

Generation and Characterization of ORF55/ORF57-Deleted Recombinant Cyprinid herpesvirus 2 Mutants with Chimeric Capsid Protein Gene of Grouper Nervous Necrosis Virus

“…The findings suggest that the ORF57 and TK genes are conserved in CyHVs and may have an impact on the virulence of CyHV-2. Furthermore, the TK gene was revealed to be unnecessary for replication in vitro but pertinent to virulence in vivo as shown in numerous herpesviruses including CyHV-3 [ 28 , 29 , 30 , 31 ]. Therefore, this study selected the ORF55 (TK) and ORF57 genes as targets for constructing CyHV-2 recombinant mutants.…”