Screening Of Anti-SVCV Active Molecules Against The Interaction Interface Between Glycoprotein And TCP1

Spring viraemia of carp(SVC)has been recognized as one of the class I diseases by animal epidemic prevention law of the people’s republic of China.Once viral infection is established,all aquatic life should be slaughtered,destructed or processed by other mandatory measures,which causes significant losses to fishery economy.At present,preventive medication in aquaculture is particularly important due to the weakness and limitation of vaccine immunization research and application.In the process of drug development,target-based precision drug design could avoid blindness.Since protein-protein interactions(PPIs)are highly specific and may cause changes in biological functions in organisms once they are disturbed or disrupted by external factors,targeting PPIs has become a hot spot in drug development.The pathogen that induces SVC is spring viraemia of carp virus(SVCV)of the genus Sprivivirus in the family Rhabdoviridae,whose glycoprotein(G)on the envelope mediates viral entry,assembly and release by binding to different host factors.In this study,we use protein-protein interaction techniques to identify potential binding receptors for glycoprotein.Based on the molecular mechanism of glycoprotein-receptor interaction,we search for inhibitors targeting key amino acid residues at the interaction interface to further discover drug molecules against SVCV.The following main results are achieved.1.Identification of glycoprotein binding receptorsIn vitro,a comparative analysis of differentially expressed genes(DEGs)in EPC cells before and after SVCV infection using transcriptome sequencing reveals that most DEGs are mapped to GO terms such as protein binding,DNA binding,receptor binding,protein kinase activity,receptor activity,and are involved in antiviral innate immune responses and cytokine-mediated signaling pathways.In vivo,the screening of receptor proteins interacting with SVCV glycoprotein in zebrafish embryo libraries using yeast two-hybrid technique reveals positive candidate proteins with binding,molecular functional regulator and transcriptional regulatory activities,and involved in translation,folding,and transport pathways.A total of 9 positive clones related to the viral infection process are obtained by rotary verification,including TCP1,GFAP,ACTB2,RPL24,RPS3,etc.The gene expression levels of receptor proteins(except GFAP)are all down-regulated in EPC cells infected with SVCV for 48 h.CO-IP technique further corroborates the interaction between glycoprotein and TCP1 protein.Functional studies of the TCP1 protein using overexpression technique reveals that overexpressed TCP1 is able to promote SVCV replication,indicating that TCP1 protein is required for virus replication during SVCV infection.2.Exploration of the interaction mode between glycoprotein and TCP1Exogenous expression of glycoprotein is carried out by cell-free synthesis system,Escherichia coli,yeast,insect cells and mammalian cells.It is found that the introduction of Trigger factor tag could well solve the solubility problem of glycoprotein in E.coli,and10 mg/m L soluble fusion protein with a purity of>95%is obtained after purification.The secondary structure of glycoprotein is identified using UV-Vis absorption spectroscopy,fluorescence spectroscopy and circular dichroism.The results show that there is an absorption peak at around 280 nm,indicating that glycoprotein sequence contains aromatic amino acid residues.A strong fluorescence intensity at about 338 nm indicates that the fluorescence of glycoprotein is mainly generated by tryptophan residues.Glycoprotein has one peak at 215 nm representing theα-helix structure.The secondary structure of glycoprotein is mainly dominated by theα-helix(97.3%),with theβ-turn and random coil contents of 2.5%and 0.2%,respectively.The three-dimensional structure of glycoprotein is predicted using SWISS-MODEL,I-TASSER,Phyre~2 and Alpha Fold2.Among these models,the Alpha Fold2 model represents the best prediction.By comparison with the homologous protein VSV-G,it is found that the SVCV glycoprotein ectodomain folds into 4 distinct domains,a topβsheet-rich lateral domain(residues 19 to 35 and 328 to 401),a centralα-helical domain(36 to 53,277 to 327 and 402 to 423),a pleckstrin homology domains(54to 68 and 199 to 276)in the neck,and the elongated fusion domain(69 to 198),respectively.Based on the above structural information,molecular dynamics simulations reveal that the interaction between glycoprotein and TCP1 is mainly through hydrogen bonding and hydrophobic interactions,and binding sites are located at the ARG71～HIS80 position of fusion domain of glycoprotein.The G-TCP1 complex forms 4 pairs of hydrogen bonds,including ARG133-GLN634(bond length 2.92(?)),ARG133-GLN634(3.00(?)),ALA477-TYR631(2.85(?))and GLN478-TYR635(2.70(?)),indicating that hydrogen bonds play an important role in the binding of glycoprotein to TCP1.3.Discovery of active molecules targeting the G-TCP1 interaction interfaceUsing EPC cells as a drug screening model,32 arctigenin derivatives are found to significantly inhibit the gene expression of glycoprotein by real-time quantitative PCR(RT-q PCR)assay,indicating that the derivatives have anti-SVCV activity.Combined with the anti-SVCV drugs previously identified in our laboratory,we establish a library of active molecules against SVCV covering 63 arctigenin derivatives and 49 coumarin derivatives.Using the key residues(ARG71～HIS80)at the interaction interface between glycoprotein and TCP1 as the docking pocket,candidate molecules 12,36,63,92,T1609,T5725(SAB),T1716 and T2727 are screened from the active and commercial molecular libraries,respectively,using virtual screening technique,and are verified to effectively inhibit SVCV replication(>80%inhibition rate)by RT-q PCR.By means of spectroscopy,it is found that the candidate molecules could lead to regular quenching of the intrinsic fluorescence of glycoprotein by causing conformational changes in glycoprotein.Among them,the active molecule SAB has the best inhibition effect on glycoprotein.The spontaneous exothermic reaction between SAB and glycoprotein is confirmed by isothermal titration calorimetry method.Molecular dynamics simulations reveal that SAB interacts with glycoprotein through hydrogen bonding,hydrophobic interactions,salt bridges andπ-πstacking.Among them,the hydrogen bonds contribute the most.SAB forms 8 hydrogen bonds with LYS65(bond length 3.10(?)),THR67(2.90(?)),THR67(3.22(?)),TYR73(2.95(?)),THR79(2.88(?)),HIE80(2.84(?)),SER81(2.62(?))and HIE83(2.80(?)).In summary,this study identifies the TCP1 protein,a key host interacting factor of SVCV glycoprotein,establishes a screening platform for targeting G-TCP1 interaction interface inhibitors,and uses this platform to discovery anti-SVCV active molecules such as SAB,T1716 and T2727.This study advances the process of drug development targeting the interaction interface between glycoprotein and receptor proteins,with a view to providing drug guarantee for the prevention and control of SVC in aquaculture.