Porcine MicroRNA-1307 Suppresses Foot-and-Mouth Disease Virus Replication By Destabilizing The VP1 And VP3 Regions Of Viral Genome

FMDV(Foot-and-mouth disease virus)is the etiological agent of FMD(Foot-and-mouth disease),an acute,feverous and highly contagious disease that mainly infects clove-hoofed animals like pigs,cattle and sheep.Due to its multiple disseminating ways,fast spreading speed and the records of having caused tremendous economic loss worldwide,FMD has been ranked firstly in the A list of infectious animal diseases,according to the OIE(Office International des Epizooties).Currently,vaccination is the main measure to prevent and control this disease,and the traditional inactivated vaccines still dominate the market.However,traditional vaccines await further improvement in many aspects,such as security problem,long development period,lag-behind of immune response and short protection time.Hence,developing novel vaccines is greatly meaningful for enhancement of immunization effects and efficient control of this disease,and investigation of viral infection mechanism and host immune responses will provide theoretical basis for FMD control.It has been demonstrated that micro RNAs(mi RNAs)play important regulatory roles during the interaction between virus and host cells,and manipulation of mi RNAs action to inhibit virus replication has become a novel strategy to control viral diseases.However,less is understood about the roles and mechanisms of host mi RNAs in regulating FMDV infection by now.Hence,we used the FMDV strain FMDV/O/BY/2010 currently epidemic in China,and its natural host cell line PK-15(porcine kidney cell line)as our main research objects,and performed the following studies.1.Differential expression analysis of host mi RNAs in PK-15 after FMDV infection.We collected the PK-15 cell samples after infection with FMDV for 2 h,and performed mi RNAs profiling with microarray to identify the differentially expressed mi RNAs.The results showed that mi R-1307 is significantly induced in PK-15 after FMDV infection.We obtained the similar result with RT-q PCR,further confirming the conclusion that mi R-1307 is a host FMDV-responsive gene.2.Mi R-1307 shows inhibitory effects on FMDV replication.To evaluate the possible effects of mi R-1307 on FMDV,we transfected PK-15 cells with mi R-1307 mimics before FMDV challenge,RT-q PCR was then performed to quantify the viral RNA.The results showed that transfection of mi R-1307 mimics significantly inhibits FMDV replication.Moreover,we obtained the stable cell lines overexpressing mi R-1307,and the viral replication in these cell lines were analyzed with different methods after FMDV infection.Results from RT-q PCR,Western blot and virus titer measurement all support that overexpression of mi R-1307 strongly suppresses FMDV replication.Together,these results demonstrate that mi R-1307 inhibits FMDV replication.3.Mechanisms investigation of mi R-1307 to inhibit FMDV replication.mi RNAs can either directly target viral genome or regulate host factors to exert their antiviral effects.To test the possible effects of mi R-1307 on FMDV proteins expression,we constructed the plasmids expressing FLAG-tagged different viral proteins.These plasmids were co-transfected together with mi R-1307-overexpressing plasmid into BHK-21 cells.Western blot results showed that overexpression of mi R-1307 specifically reduces the expression of FMDV structural proteins VP1 and VP3.Similar result was obtained from the co-transfection of mi R-1307 mimics with FLAG-VP1 or FLAG-VP3 plasmid.It was further demonstrated with RT-q PCR that the RNA abundance of VP1 or VP3 was already obvious reduced in those samples.We then cloned the VP1 or VP3-coding sequences after the stop codon of firefly luciferase reporter gene in pmir GLO vector.The resulting LUC-VP1 or LUC-VP3 plasmid was co-transfected with mi R-1307 mimics into BHK-21 cells,and the relative luciferase activities were measured.The results showed that transfection of mi R-1307 mimics significantly reduces the firefly luciferase activity,further confirming that mi R-1307 exerts its effects on VP1 and VP3 at RNA level.Together,these data demonstrate that mi R-1307 specifically destabilizes the VP1 and VP3 RNA sequence.Using bioinformatics tools,we predicted 4 potential mi R-1307 target sites within VP1-coding sequences and 3 within VP3.However,mutation of each of those target sites can't abolish the reductive effects of mi R-1307 on VP1 or VP3,indicating the direct target sites of mi R-1307 are outside the present prediction.The exact mechanism of how mi R-1307 reduces the RNA stability of VP1 and VP3 remains further study.4.Therapeutic potential of mi R-1307.The obvious inhibitory effects of mi R-1307 on FMDV replication in cellular assays prompted us to test its effects in animal models.Three-day-old suckling mice(Balb/c)were subcutaneously injected in the neck with mi R-1307 agomir or NC(negative control)before challenged with FMDV.The results showed that pre-injection with mi R-1307 agomir significantly delayed the FMDV-induced lethality in suckling mice,demonstrating its therapeutic potential for FMDV control.In summary,we successfully identified a porcine mi RNA mi R-1307 whose expression was significantly induced by FMDV infection.Our research demonstrated that mi R-1307 suppresses FMDV replication by specifically reducing the RNA stability of VP1 and VP3 regions of viral genome.Moreover,injection of mi R-1307 agomir significantly delays the FMDV-induced lethality in suckling mice,indicating it has therapeutic potential.