Screening Of Deubiquitinase UL36^USP Inhibitor And Its Suppression Effect On MDV Replication

Marek's Disease(MD)of chickens is a lymphoproliferative disease caused by the pathogenic Marek's Disease Virus(MDV).It is highly contagious and highly pathogenic,and can cause serious economic loss in the poultry industry.MDV belongs to alpha-herpesvirus,and is divided into three serotypes.Pathogenic MDV belongs to serotype I,serotype II and III are widely used as anti-MD vaccines in the control of Marek's disease.Along with the usage of MD vaccines,more virulent MDVs(vv)and even MDV(vv+)have emerged,and the virulence of MDV has been continuously enhanced.It is still uncertain whether the more virulent MDV will appear.Deubiquitinating enzymes are a class of isopeptidases that eliminate ubiquitin from ubiquitinated substrates or polyubiquitinated chains.Deubiquitinases are usually involved in various cellular processes,such as cell proliferation,apoptosis,and viral pathogenecity.Therefore,some deubiquitinases have been used as targets for the screening of anticancer drugs.MDV encodes a large tegument protein(UL36)which has deubiquitination activity.The UL36-480 protein is the N-terminal fragment of UL36 that containing a catalytic core.Destruction of its deubiquitinase activity can inhibit the replication and packaging of MDV in chicken cells,as well as the shedding of MDV,thereby reduce the occurrence of MD tumor.In the current study,UL36 was used as a target to screen the compounds that effectively anti-MDV and were validated in chicken cells.The results showed that two compounds,b-AP15 and LDN-57444,could effectively inhibit UL36 activity.The inhibition kinetics of these two inhibitors were then analyzed.The kinetic curves were fitted and the Km value and Vmax were calculated.Both showed non-competitive inhibition on UL-36 activity.CCK-8 was used to determine the maximum safe concentration of these two inhibitors on CEF cells.The plaque reduction and real-time PCR were utilized to determine the antiviral effect of the inhibitor on MDV.The results showed that b-AP15 and LDN-57444 can inhibit the replication of MDV in CEF cells,and also inhibit the horizontal transmission of MDV between the cells.To conclude,the current study determined that b-AP15 and LDN-57444effectively inhibit the deubiquitination activity of UL36.Both inhibitors could suppress the replication of MDV in CEF cells,reduced transmission of MDV between the cells.The in vitro results of this study provide a new way to effectively inhibit the replication and packaging of MDV in chicken cells and effectively prevent and control the shedding of MDV virus.