Study On The Regulation Of Helicase Proteins,DDX23 And DDX17 On FMDV Replication

The unprecedented contributions of DEAD-box helicases in cellular life are of utmost importance,without which cellular life is inevitable.DEAD-box helicase 23(DDX23)is a host nuclear helicase,which is a part of the spliceosomal complex and involved in pre-m RNA splicing.To investigate whether DDX23,an internal ribosomal entry sites transacting factor(ITAF)affects foot-and-mouth disease virus(FMDV)replication through internal ribosome entry site(IRES)-dependent manner.For this,we utilized a pull-down assay,Western blotting,quantitative real-time PCR,confocal microscopy,overexpression,and small interfering RNA knockdown,as well as the median tissue culture infective dose.Our findings showed that FMDV infection inhibited DDX23 protein expression,and a cleavage of approximately 55 k Da was observed.A significant increase in the m RNA level of DDX23 was observed during foot-and-mouth disease virus infection.In a counter-attack,the overexpression of DDX23 reduced FMDV protein expression,m RNA level,and viral titer.In contrast,CRISPR Cas9knockout/small interfering RNA knockdown increased FMDV replication.The pull-down and massspectrometry results showed the interaction between DDX23 and FMDV IRES.Insight investigation revealed that DDX23 interacted with FMDV IRES domain III and IV.Foot-and-mouth disease virus 3C proteinase inhibited DDX23 protein expression.In addition,the inhibition of DDX23 was 3C proteinase dosage-dependent,which was due to direct interaction between DDX23 and FMDV 3C proteinase.The enzymatic activity of FMDV 3C proteinase degraded DDX23,whereas FMDV primarily degraded DDX23 via the lysosomal pathway.Additionally,the FMDV IRES activity was significantly suppressed in DDX23-overexpressed cells,and an upsurge in the activity was observed with DDX23 knocked down.Collectively,our results demonstrated that DDX23 negatively affects FMDV replication,which could be a useful target for the design of antiviral drugs.Moreover,this study also investigated the association between DDX17 and FMDV IRES.DDX17 is involved in gene regulation,alternative splicing,ribosomal biogenesis,binding to double-stranded and single-stranded RNA and affecting virus replication.The interaction between DDX17 and FMDV IRES was confirmed through pull-down assay and RNA immunoprecipitation assay.During FMDV infection,DDX17 was degraded and cleaved into approximately a 50 k Da particle.In addition,the DDX17 m RNA level was significantly increased with the progression of FMDV infection.Knockdown of DDX17 showed no change in the FMDV protein expression,although,a significant increase in m RNA and viral titer was observed.As expected,the overexpression of DDX17 significantly decreased the m RNA level and viral titer,but no significant change was observed in FMDV protein expression.Interestingly,the FMDV,Classical swine fever virus(CSFV),Seneca valley virus(SVA)IRES activity was significantly increased with the knockdown of DDX17 and decreased during the overexpression of DDX17.Furthermore,DDX17 was degraded during FMDV infection via lysosomal and proteasomal pathways.This novel ITAF,which negatively regulates the FMDV IRES activity,could be used as an antiviral to restrict viral replication.