Preliminary Study On The Effect Of Heat Shock Protein HSP90 On The Replication Of Spring Viremia Of Carp Virus

Spring viremia of carp(SVCV)is the pathogen of spring viremia of carp(SVCV).It can cause acute infectious diseases with high mortality among many Cyprinidae fishes,including carp,black carp and zebrafish,and the mortality rate in young fish is as high as 90%,causing huge economic losses to aquaculture.HSP90 is a highly conserved ATP dependent chaperone,accounting for 1% of all proteins in eukaryotes.They remodel hundreds of substrate proteins and participate in many cellular functions,such as protein transport,signal transduction and receptor maturation.Proper folding of viral and host proteins is a prerequisite for protein function and protein stability.As an important chaperone protein for stable protein folding,HSP90 plays a crucial role in the replication of many viruses with different genomic structures and different replication strategies.In this study,the HSP90 gene was cloned from EPC(Epithelioma papulosum cyprini)and its biological characteristics were analyzed.In addition,its influence on SVCV virus infection was studied through overexpression of HSP90 and inhibition of HSP90,so as to provide certain theoretical basis for the study of pathogenic mechanism of SVCV and anti-SVCV drugs.1.Cloning and biological characteristics analysis of HSP90In this study,HSP90 gene was cloned from EPC cells.The full length of its CDS region was 2175 bp,encoding 725 amino acids.Through protein sequence homology alignment,it was found that it had the closest genetic relationship with carp and crucian carp,and the sequence homology with Homo＿sapiens,Mus＿musculus,Gallus＿gallus and Danio＿rerio were 90.38%,90.66%,89.7% and 97.25%,respectively.The expression of HSP90 in various tissues(heart,brain,eye,muscle,intestine,gill,liver,kidney and spleen)was studied by fluorescence quantitative analysis and WB.The results showed that HSP90 was distributed most in liver,intestine and gill,and least in spleen,eye and muscle.The results of immunofluorescence experiment showed that HSP90 was distributed in the cytoplasm of EPC cells.After SVCV infection,the expression of SVCV-G and HSP90 in kidney,spleen,liver and gill tissue increased gradually with the increase of time,and reached the peak on the third day.The expression of SVCV-G gene was significantly increased after EPC cells were infected with SVCV virus and reached the peak at 24 h.With the increase of time,the titer of the virus increased gradually,and the virulence of the virus reached the highest level at48 h.The level of HSP90 also increased with the duration of infection.Microscopic observation of the cells infected with SVCV showed that the cells gradually lost their vitality until a large number of cells died,and empty spots appeared.2.The effect of overexpression of HSP90 on replication of SVCV virusAfter transfection with recombinant plasmid Pcs2+8Cm Cherry-Flag-HSP90,the cells were infected with the virus.The apoptosis rate was detected by flow cytometry.The results showed that the apoptosis rate and necrosis rate of the cells were significantly increased after infection with SVCV after overexpression of HSP90.The results showed that over expression of HSP90 could significantly up regulate the transcription and viral titer of SVCV-G,and up regulate the expression of IRF3 and IRF7,and the expression of TLR3,TBK1 and IFN-β and TRAF6 were not significantly regulated.3.Inhibit the effect of HSP90 on the replication of SVCV virusThe specific inhibitor 17-AAG(17-acrylamino-17-demethoxygeldanamycin)inhibited Hsp90,which could significantly reduce the viral titer of SVCV and the expression of SVCV-G,as well as the rate of apoptosis and necrosis.The results showed that 17-AAG could significantly down regulate TBK1,IRF3/7,IFN-β and TRAF6.In conclusion,HSP90 can promote viral replication and affect the expression of immune genes in cells to a certain extent.