Study Of Antagonistic Drugs Against Grass Carp Reovirus Based On Host HSP70 And TLR2 Targets

Grass carp(Ctenopharyngodon Idella)is an important economic fish widely cultured in China,accounting for more than 20% of the total freshwater aquaculture in China.Grass carp hemorrhagic disease caused by Grass carp reovirus(GCRV)brings great difficulties and risks to grass carp culture every year,and seriously endangers the grass carp culture industry in China.In recent years,China has made great achievements in GCRV prevention and control,and several GCRV vaccines have been released,but due to the limited vaccine coverage,there is still a long way to go to completely solve the threat of GCRV to the grass carp culture industry in China.There are two main strategies generally recognized as more effective against the virus,one is to design drugs that target viral components(including viral proteins such as DNA or RNA polymerase)and the other is to design drugs that target host cell components,including various signal transduction pathways involved in virus-host interactions.In the case of GCRV,due to the variety of strains,the development of antiviral drugs targeting one strain alone is usually very limited and costly.Therefore,the development of preventive and control drugs against GCRV should be based on the screening of broad-spectrum antiviral drugs or the study of antiviral mechanisms.In this study,we evaluated the effectiveness of quercetin against GCRV based on two new antiviral targets,Heat Shock Protein 70(HSP70)and Toll Like Receptor 2(TLR2),in grass carp,and also screened and characterized the effect of quercetin on GCRV based on VP55-TLR2.We also screened and identified a new antiviral target,TLR2,based on VP55-TLR2 interaction,and investigated the mechanism of action of TLR2 target against virus.The details are as follows:1.Quercetin,a plant-derived flavonoid compound,can inhibit the expression of grass carp heat shock proteins in response to grass carp heat shock and effectively inhibit the proliferation of type I and type III GCRV in vitro.Quercetin is a major dietary flavonoid and a known transcriptional repressor of Heat Shock Factor 1(HSF1).Our study suggests that HSP70 plays an active role in GCRV infection through an unknown mechanism.Therefore,we predict that HSP70 may be a potential broad-spectrum antiviral target.We down-regulated the expression of HSP70 in grass carp by plant-derived quercetin to assess the potential of HSP70 as a new antiviral target in fish.The results showed that the expression of HSP70 in grass carp was significantly inhibited by either GCRV-104 infection or heat shock treatment in the presence of 50 μM quercetin.The reduction of viral protein expression and daughter viruses after GCRV-104 infection of grass carp cells was correlated with the downregulation of HSP70 by quercetin,and the pro-viral effect of HSP70 could be attenuated by quercetin,suggesting that HSP70 is involved in quercetin-mediated antiviral targets.The same dose-dependent antiviral effect of quercetin on different genotypes of GCRV JX-01 suggests that quercetin,as a specific inhibitor of HSP70 expression and GCRV replication in grass carp,has the potential to be developed into a broad-spectrum antiviral drug by targeting the proviral target of HSP70 in fish.2.Using phage display technology to screen for grass carp target proteins that may interact with VP55,TLR2 and VP55 interactions were verified using GST-Pull down,Dot blot,and co-localization,and TLR2 was identified as a novel anti-GCRV target using sh RNA interference technology.The interaction between virus and host is an important reference for us to find antiviral targets and select appropriate antiviral strategies.VP55 protein is the outer shell fibronectin of GCRV-104 and plays a key role in the first step(adsorption process)of GCRV-104 infection.We screened the host protein interacting with VP55 as a new antiviral target,performed prokaryotic expression and purification of VP55,and obtained purified GST-VP55 protein;encapsulated GST-VP55 protein on 6-well plates,and screened the peptide interacting with GST-VP55 using phage display technology;then used the protein library of grass carp to compare the screened peptide The interaction between VP55 and TLR2 was identified and verified using GST-Pull down,Dot blot,and co-localization,and the interaction between TLR2 and VP55 was confirmed.sh RNA interference was then used to knock down the TLR2 expression abundance in GCO cells,and TLR2 was detected.expression abundance in GCO cells using sh RNA interference to detect the effect of TLR2 target on GCRV replication,and validated TLR2 as a novel anti-GCRV target.3.A preliminary investigation of the antiviral mechanism of TLR2-based target drugs showed that VP55 could inhibit the expression of TLR2 and its downstream genes and attenuate the immune response of TLR2-TNFα pathway;overexpression of TLR2 could upregulate the expression of TLR2 and its downstream genes,enhance the immune response of TLR2-TNFα pathway,and inhibit the expression of viral genes in infected cells and the supracellular The overexpression of TLR2 could enhance the immune response of TLR2-TNFα pathway,inhibit the expression of viral genes in infected cells and the generation of daughter viruses in cell supernatants.To evaluate the potential of developing antiviral drugs against TLR2 targets,we explored the role of TLR2-TNFα pathway in the process of GCRV infection.We identified the tissue expression profiles of TLR2,NFk B,and TNFα genes on grass carp and correlated them with the histophilicity of GCRV.Then we examined the expression changes of these genes during GCRV infection and verified whether these changes correlated with the interactions between TLR2 and VP55,and then verified the regulatory role of TLR2 on its downstream gene expression and its effect on viral replication by knocking down and overexpressing TLR2.It was found that the expression distribution of TLR2,NFk B,TNFα and other genes were negatively correlated with the histophilicity of GCRV;VP55 could inhibit the expression of TLR2 and its downstream genes and weaken the immune response of TLR2-TNFα pathway;while adjusting the expression abundance of TLR2 could regulate the expression of TLR2 downstream genes and change the strength of the immune response of TLR2-TNFα pathway This affects the expression of viral genes in infected cells and the production of daughter viruses in cell supernatants.Based on these data,we believe that TLR2 targets are very promising targets for antiviral drug development,suggesting that some of our TLR2 agonists may have antagonistic functions against GCRV.In this study,we evaluated the inhibitory effect of a plant-derived small molecule drug quercetin based on the HSP70 target on GCRV,and also screened and identified the grass carp TLR2 protein that interacts with the type III GCRV coat protein VP55 using phage display technology,identified TLR2 as a new antiviral target using sh RNA interference technology,and conducted a preliminary investigation on the development of antiviral drugs based on the TLR2 target.A preliminary investigation of the mechanism of action of TLR2-based antiviral drugs was carried out.From the perspectives of antiviral drugs targeting HSP70 in grass carp and antiviral drugs based on TLR2 targets,we provide reliable strategies and new ideas for the development of GCRV prevention and control drugs.