Regulatory Mechanisms Of CyHV-2 Replication Via Interaction Of CyHV-2 Immediate Early Protein ORF121 With Crucian Carp C-Myc Transcriptional Regulator

Cyprinid Herpesvirus 2(CyHV-2)infecting crucian carp and goldfish causes herpes viral haematopoietic necrosis(HVHN),which causes severe economic losses to crucian carp aquaculture.DNA viruses typically carry hundreds of genes,including immediate early genes(IE),early genes(E)and late genes(L).The expression of immediate early genes in the early stage of viral infection affects the transcription and translation of early and late genes of the virus and associated host genes.In our previous study,cycloheximide(CHX)and cytarabine(Ara C)were used to establish an immediate early gene screening model and five immediate early genes(ORF54,ORF121,ORF141,ORF147,ORF155)were obtained.Among them,ORF121 is highly expressed in all tissues of dying crucian carp of CyHV-2 infection,however,the function of ORF121 in virus replication is still unknown.Herein our study mainly focuses on the interaction analyses with ORF121 and the host factors.Our results show the following:1 Yeast two-hybrid system was used to screen crucian carp proteins interacting with ORF121,and the structure of C-Myc was analyzed.In order to investigate the function of ORF121,we screened crucian carp proteins interacting with ORF121 by Mating method.The Y2 H Gold yeast strain transformed into pGBKT7-ORF121 was co-cultured with the Y187 yeast strain transformed into the crucian carp cDNA library,and cultured in auxotrophic medium.Five proteins interacting with ORF121 were obtained: C-Myc transcriptional regulator,centrosome and spindle pole-associated protein 1-like isoform X4,zinc finger protein 518B-like,pre-m RNAprocessing factor 40 homolog A-like,galectin-3-like.Bioinformatics analysis showed that there was a BHLH domain binding DNA sequence in crucian carp C-Myc.Tyr 68 of ORF121 binding with Glu 349 of C-Myc,and Gln 127 of ORF121 binding with Lys 332 of C-Myc.2 Dot-blot overlay and subcellular co-localization were used to verify the interaction between ORF121 and C-Myc.Using siRNA to interfere with C-Myc expression,we found that C-Myc is closely related to CyHV-2 replication.Virus replication affected by the interaction between virus and host.Based on the clear interaction between ORF121 and C-Myc transcriptional regulator in yeast,dot-blot overlay experiment was used to confirm the interaction between them,and subcellular co-localization experiment was used to found that ORF121 and C-Myc co-located in the cell nucleus.In order to explore the specific role of C-Myc in viral infection,we studied the function of C-Myc.In CyHV-2 infection,ORF121 and C-Myc were up-regulated synchronously.With the interference of C-Myc expression by siRNA,the expression of ORF121 was decreased,and the copies of CyHV-2 was also significantly decreased.These results indicated that C-Myc protein not only interacts with ORF121 protein,but also is closely involved in the replication of CyHV-2.3 Transcriptome analysis showed that PI3K/AKT/mTOR signaling pathway was significantly upregulated in CyHV-2 infection.10058-F4 inhibition experiment indicated that the expression of C-Myc not only affected the transcription of the whole CyHV-2genome,but also affected the expression of PI3K/AKT/mTOR signaling pathway genes.Rapamycin inhibition experiment indicated that ORF121 interacts with C-Myc to affect the PI3K/AKT/mTOR signaling pathway by regulating the phosphorylation of mTOR.In order to explore which pathways are regulated by the interaction between ORF121 and C-Myc to affect the replication of CyHV-2,we first detected the distribution of CMyc in tissues of crucian carp and goldfish,and the results showed that the expression of C-Myc was the highest in gill tissues,which was nearly 4.5 times that of other tissues.Then the CyHV-2 SH-01 strain was artificially injected into crucian carp and goldfish,and the transcriptome analysis was performed together with healthy ones.The results showed that PI3K/AKT/mTOR signaling pathway was significantly up-regulated,which was confirmed by RT-qPCR assay of GiCF cells infected with CyHV-2.Next,C-Myc inhibitor 10058-F4 was selected for the experiment,and the 10058-F4 inhibition experiment results showed that ORF121 was inhibited at both transcription and translation levels,as well as the expression of the other four immediate early genes(ORF54,ORF141,ORF147,ORF155),one early gene(ORF24)and one late gene(ORF7)was suppressed at the transcriptional level,and the copies of CyHV-2 was decreased.At same time,PI3K/AKT/mTOR signaling pathway genes were inhibited.The results of rapamycin inhibition experiment on mTOR inhibitor showed that ORF121 and C-Myc were inhibited at the transcriptional level after mTOR phosphorylation was inhibited,and the expression of the other four immediate early genes(ORF54,ORF141,ORF147,ORF155),one early gene(ORF24)and one late gene(ORF7)were inhibited,and the copies of CyHV-2 was decreased,as well as some genes of PI3K/AKT/mTOR signaling pathway were inhibited.In conclusion,this study revealed a novel host protein,C-Myc transcriptional regulator,which interacts with the CyHV-2 immediate early gene ORF121.The expression of C-Myc transcriptional regulator is closely related to the replication of CyHV-2.Furthermore,ORF121 interacts with C-Myc to regulate the PI3K/AKT/mTOR signaling pathway by affecting the phosphorylation of mTOR,and indirectly regulates the replication of CyHV-2.Our study found that C-Myc and mTOR may be two effective drug targets for inhibiting CyHV-2,providing a new strategy for the prevention and control of CyHV-2 and the development of new environmental and green fishery drugs.