Screening And Identification Of Small Molecular Compounds Inhibiting PRRSV Replication

Porcine reproductive and respiratory syndrome virus(PRRSV)is a single stranded positive RNA virus,the virus appeared in the late 1980s and spread widely in the world,causing huge economic losses to the global pig industry.Since the birth of the first commercial PRRSV vaccine in 1994,no vaccine is safe,effective and has broad-spectrum protective effect.The conventional inactivated vaccine could not induce the protection of heterologous virus;the attenuated vaccine will make the virulence of the virus return to strong and increase the risk of infection;The anti PRRSV drugs developed in the market are not effective in preventing and treating the spread and outbreak of porcine reproductive and respiratory syndrome(PRRS).Therefore,the research and development of new vaccines and drugs to effectively prevent and control PRRSV has become a key problem to be solved.ML323,a specific inhibitor of USP1-UAF1 complex,could significantly inhibit the proliferation of PRRSV.It is suggested that m1323 may be an important candidate for anti PRRSV drugs.Therefore,it is necessary to verify the inhibitory effect of ML323 on PRRSV replication and proliferation,and explore its key target.Ubiquitin specific protease 1(USP1),as a member of the ubiquitin specific protease family,forms a close complex with the WD repeat domain 48 protein uafl(USP1 associated factor,UAF1)to play its function.A large number of studies have shown that the replication of PRRSV requires an active ubiquitin proteasome system,and it is speculated that USP1 may play an important regulatory role in the process of PRRSV infection.In this study,PRRSV-BJ4 and PRRSV-GFP were used as model viruses,and the small molecular compound library constructed by our team was used to screen the small molecular compounds that significantly regulate PRRSV infection and are related to ubiquitination.ML323,a specific inhibitor of USP1-UAF1 complex,was used as the breakthrough point to verify the regulatory effect of ML323 on PRRSV proliferation in MARC-145 cells in a dose-dependent and time-dependent manner by RT-qPCR.Small interfering RNA(siRNA)was used to interfere the expression of USP1 and UAF1 in cells,and the overexpression cell line was constructed to verify its role in the process of PRRSV infection.The viral protein encoded by PRRSV-BJ4 and its main binding ubiquitination sites were explored by Co-IP method.Finally,the viral protein encoded by PRRSV-BJ4 and its regulatory mechanism on ubiquitination were explored.Western blot,RT-qPCR and TCID50 showed that ML323 could inhibit PRRSV infection in MARC-145 and PAM Tang cells,and the infection of PRRSV also promoted the expression of USP1 and UAF1,The expression of UAF1 and USP1 was inhibited by siRNA the results of Western blot,RT-qPCR and TCID50 showed that the infection of PRRSV was significantly inhibited,and overexpression of USP1 could significantly promote the proliferation of PRRSV,Co-IP test showed that the nonstructural protein Nsp1? of PRRSV-BJ4 mainly combined with K48 ubiquitin,and ML323 inhibited the de ubiquitination of USP1 and promoted the formation of ubiquitination chain of Nsp1? at K48.It was found that PRRSV infection could promote the expression of USP1 and UAF1,and ML323,a specific inhibitor of USP1-UAF1 complex,could inhibit virus infection,ML323 could inhibit the expression of Nsp1? and promote the formation of its K48 ubiquitin chain,thus inhibiting virus infection.In this study,we screened a small molecule compound ML323 that can inhibit PRRSV,and verified its regulatory effect by cell biology and molecular biology methods.We explored the role of ML323 in virus infection,which provided theoretical support for the development of drugs for the treatment of PRRS.