Functional Characterization Of Cyclophilin A During Red Spotted Grouper Nervous Necrosis Virus (RGNNV) Infection

Cyclophilin A(Cyp A)is a ubiquitously expressed cellular protein that plays an important role in several pathological conditions,as well as inflammation and infection.Cyp A acts as a key factor in the replication of several viruses.However,little is known about the role of Cyp A in the replication of the nervous necrosis virus.NNV was initially isolated from diseased marine fish species and has been reported in more than 120 cultured and wild range fish species;thus caused a huge economic loss to the aquaculture industry.Recently it also reported during experimental conditions that some freshwater aquaculture species are also susceptible to NNV.Though,prevailing efforts have been made to improve therapeutic and prophylactic approaches against viral infection.In this study,functions of Cyclophilin A during Red spotted Grouper Nervous Necrosis Virus(RGNNV)infection were characterized.The main results are as follows:1.Cloning and characterization of Cyp A from orange-spotted grouper(Epinephelus coioides).Grouper Cyp A(GF-Cyp A)was cloned from the grouper fin cell line(GF-1)derived from orange-spotted grouper(E.coioides).Sequence analysis found that the full length of gene consists of 743 bp.GF-Cyp A open reading frame(ORF)of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4 k Da.Through bioinformatics analysis,the secondary and 3D structure of GF-Cyp A was predicted.Loops,helix and ?-strands were predicted using online software Phyre2.Binding sites,solvent accebality and percentage of loops,helix and strands were also predicted.Phylogenetic analysis and multiple sequence alignment and homology analysis were also carried out using several bioinformatics related softwares.The deduced amino acid sequence shared highly conserved regions including PPIase domain with Cyp A of other animal species,showing that GF-Cyp A is a new member of Cyclophilin A family.2.The inhibition of RGNNV replication by GF-Cyp A.To investigate the role of GFCyp A during RGNNV infection,a series of experiments were conducted.We observed that GF-Cyp A was up-regulated in the GF-1 cells infected with RGNNV.Additionally,overexpression of Cyp A could significantly inhibit the replication of RGNNV in GF-1 cells at both transcriptional and translational levels.Similarly the RGNNV inhibition was also observed by viral titration assay.By contrast,when the GF-Cyp A was knock-downed by si RNA in GF-1 cells,the replication of RGNNV was enhanced.Furthermore,the expressions of pro-inflammatory factors,such as TNF-2,TNF-?,IL-1b,and ISG-15 were increased in GF-Cyp A transfected GF-1 cells challenged with RGNNV,indicating that GF-Cyp A might be involved in the regulation of the host pro-inflammatory factors.Altogether,we conclude that GFCyp A plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1 cells during RGNNV infection.3.Cyclosporin A and RGNNV replication.Cyclosporin A(Cs A)is an immunosuppressive drug which binds to Cyp A and form Cs-Cyp A complex.It inhibits calciniurin phosphatase activity and leads ti immune suppressive activity by preventing T-cells activation.Due to this relationship of Cs A with Cyp A,the role of Cs A was also investigated during RGNNV infection in GF-1 cells.Firstly,GF-1 cells proliferation was observed after several concentrations(0,2,4,8,16,& 32 ?M)at different time points(12,24,48,72 & 96)hours post Cs A treatment.The results revealed that GF-1 cells viability,treated with 4?M was more than 80 % upto 48 hours post Cs A treatment,so this amount was set as nontoxic for GF-1 cells used for further RGNNV replication studies.Eventually,it was found Cs A doesn't effect RGNNV replication during the early stages(0,1,2 & 3 hpi).However,during the later stages(6,12,24 & 48 hpi)of viral life cycle,Cs A was found to negatively effect RGNNV replication.Eventually,when the GF-Cyp A was knock-downed by si RNA in GF-1 cells treated with Cs A and infected with RGNNV,the replication of RGNNV was still not enhanced and inhibited at either time points i.e.early and late steps of infection,indicating that inhibition of RGNNV by Cs A is not Cyp A-dependent.To examine the impact of Cs A on cytokines expression during RGNNV infection after Cs A treatment,several cytokines expressions i.e.ISG-15,IL-1?,TNF-2 and TNF-? were analyzed.Our findings indicated that,these cytokines were inhibited at either time points during RGNNV infection after Cs A treatment as compared with pc DNA3.1-Cyp A and mock treatmentTaken together,these results will shed new light on the function of Cyp A in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish.