Mechanisms Of A77 1726-meidated Restriction Of Canine Distemper Virus Replication

Canine distemper(CD)is a highly contagious and acute fever disease caused by canine distemper virus(CDV).The symptoms are mainly related to the respiratory and gastrointestinal tracts as well as the nerves system.CDV is a member of the genus measles virus of the Paramyxoviridae family.It has a wide range of hosts that can be infected by multiple routes.Different species of animal hosts have distinct incubation periods.CD poses great challenges in treatment and prevention.In particular,currently there is no effective drugs for treating it.It is essential to develop novel antiviral drugs for treating CD.Leflunomide is an anti-inflammatory drug used clinically to treat human rheumatoid arthritis(RA),systemic immune disease,and organ transplantation.Its active metabolite A77 1726 inhibits the activity of protein tyrosine kinases and S6K1 kinases.In addition,A771726 inhibits the biosynthesis of pyrimidine nucleotides by targeting DHO-DHase.Prior studies have shown that A77 1725 possesses a wide range of antiviral activity.For example,it inhibits the replication of polyoma virus and rotavirus by inhibiting pyrimidine nucleotide synthesis but inhibits the replication of cytomegalovirus(CMV)and BK virus by other mechanisms.Whether A77 1726 inhibits CD V replication remains unknown.The purpose of this study is to investigate the ability of A77 1726 its underlying mechanisms.Vero cells infected with the CDV vaccine strain CDV-Rockborn or virulent isolate CDV-1 were incubated in the absence or presence of A77 1726 were analzyed for the expression of N and M proteins by Western blot and for virus titers in the conditioned media by measuring the TCID50 values.We found that A77 1726 reduced the levels of viral N and M proteins and viral titer in a concentration-dependent manner.Addition of exogenous uridine in cell culture blocked the inhibitory effect of A77 1726.Brequinar sodium(BQR),a specific inhibitor of DHO-DHase,also decreased the levels of viral M and N proteins,which could be reversed by uridine.These results suggest that A77 1726 inhibits CDV replication by inhibiting pyrimidine nucleotide synthesis.Prior studies have shown that S6K1 can promote pyrimidine nucleotide synthesis by phosphorylating and activating CAD(caspase activated deoxyribonuclease),a rate-limiting enzyme in pyrimidine nucleotide synthesis.We further investigated if A77 1726 also inhibited CDV replication by inhibiting S6K1 enzyme activity.We first analyzed S6K1 kinase activity in Vero cells and induced feedback activation of PI-3 kinase signaling pathway.We found that A77 1726 resulted in concentration-dependent up-regulation of AKT and S6K1 phosphorylation levels,and down-regulation of 4E-BP and S6 phosphorylation levels.S6K1 gene silencing were significantly decreased the virus titers and viral protein levels and enhanced the inhibitory effect of A77 1726 on CDV replication.The S6K1 specific inhibitor PF-4708671 and its upstream mTOR inhibitor rapamycin both effectively inhibited CDV replication.Both PF-4708671 and rapamycin suppressed S6 and 4E-BP phosphorylation but increasing AKT phosphorylation.PF-4708671 increased S6K1 phosphorylation,whereas rapamycin decreases S6K1 phosphorylation.In addition to inhibit DHO-DHase and S6K1,A77 1726 also inhibits the acti ity of various tyrosine kinases.Therefore,we investigated whether A77 1726 exerted antiviral effects by the JAK and Src family tyrosine kinases.SU6656,a Src inhibitor,and Ruxolitinib(Rux),a JAK inhibitor,had no effect on viral protein expression.In summary,our study provides evidence that A77 1726 inhibits CDV replication by inhibiting pyrimidine nucleotide synthesis and by inhibiting S6K1 activity.Its antiviral activity was not related to its inhibitory effect on protein tyrosine kinase activity.Our study warrants further investigation on its antiviral activity in vivo.