| Muscovy duck reovirus(MDRV)causes an acute infectious disease with high morbidity and mortality in Muscovy ducks and semi-Muscovy ducks,which brings serious economic losses to Muscovy duck breeding.Both MDRV and avian reovirus(ARV)are members of the Orthoreovirus genus subgroup ?,but the genomic composition and pathogenicity are different,especially in the S-group proteins closely related to the pathogenicity of orthoreoviruses.It has been reported that MDRV and ARV can stimulate the production of interferon,while ARV ?A protein has a function of resistance to host interferon(IFN)response,and the nucleotide homology between MDRV and ARV ?A protein is about 76.5%,so it can be inferred that MDRV ?A protein possibly plays an important role in the regulation of interferon expression.Therefore,in this study we explored the regulatory functions of the interferon signaling pathway by MDRV ?A protein,the main contents and results are as follows:1.In order to determine whether MDRV ?A protein could inhibit the expression of IFN-? and IFN-? like ARV ?A protein,the recombinant expression plasmid containing MDRV ?A gene was transfected into DF-1 cells,the cells were cultured for 24 h and then infected with MDRV,and ELISA method was used to detect the effect of ?A protein on interferon expression.The results showed that the expression levels of IFN-a and IFN-? in MDRV-infected DF-1 cells(MDRV group)were extremely significantly higher than those in blank control group(P<0.01),indicating that MDRV could stimulate the expression of interferon;there was no significant difference in the content of interferons between the empty vector(EV)group and ?A group(P>0.05),which indicated that ?A transfection alone did not stimulate the expression of interferons;the expression of interferon in EV+ MDRV group was significantly lower than that in both MDRV group and EV group(P<0.05),which may be due to the stimulation of empty vector transfection to produce a suitable amount of interferons,on this occasion MDRV down-regulated the expression of interferon,which is consistent with the two-way regulatory function of ARV in IFN expression;the expression of interferon of ?A+MDRV group was extremely significantly higher than that of EV+MDRV group(P<0.01),indicating that ?A probably could upregulate the expression of interferon induced by MDRV,which is inconsistent with the previous report of ARV ?A protein.To confirm above results,we further detected the effect of aA protein on the mRNA levels of interferon signaling pathway-related factors induced by MDRV with Real-time PCR method.The results showed that:(1)the mRNA levels of IFN-? and IFN-? in the MDRV group were significantly(P<0.05)or extremely significantly(P<0.01)higher than those in the blank control group;there was no significant difference in the content of interferons between the empty vector(EV)group and ?A group(P>0.05);the expression of interferons in EV+MDRV group was significantly higher than those in both EV group and MDRV group(P<0.05);while the expression of interferons in ?A+MDRV group was extremely significantly higher than those in EV+MDRV group(P<0.01),these results are consistent with the results of ELISA.(2)The detection of associated factors of interferon signaling pathway showed that:the mRNA expression of transcription factor IRF7 in MDRV group significantly increased in comparison with that in blank control group(P<0.05),and the mRNA expressions of model receptors(MDA5,TLR3 and TLR7)and antiviral factors(Mx,ISG12 and IFIT5)in MDRV group were extremely significantly higher than those in blank control group(P<0.01).The mRNA levels of the above factors in the EV+MDRV group were significantly(P<0.05)or extremely significantly(P<0.01)lower than those in the MDRV group and EV group.The mRNA level of all above factors in ?A+MDRV group was significantly higher than those in EV+MDRV group(P<0.01).These results were also consistent with the results of interferon expression.Collectively,the results showed that MDRV ?A protein up-regulated the expression of interferon signaling pathway-related factors induced by MDRV.2.The previous results showed that the expression of interferons and IFN signaling pathway-related factors in EV+MDRV group was significantly lower than those in EV group and MDRV group,suggesting that MDRV might regulate the expression of interferons in a two-way manner.To explain this phenomenon,we chose the activator poly(I:C)with positive regulatory effect instead of MDRV,using the same experimental methods to investigate the effect of ?A protein on expression of interferon and interferon-associated signaling factors activated by poly(I:C).(1)Real-time PCR results showed that the expression levels of IFN-?,MDA5,TLR3,IRF7 and Mx in the poly(I:C)group were extremely significantly(P<0.01)or significantly(IFIT5 and ISG12)higher(P<0.05)than those in the blank control group,indicating that poly(I:C)can effectively activate the mRNA expression of the above factors in the interferon signaling pathway.EV+ poly(I:C)group was significantly(P<0.05)or extremely significantly(P<0.01)higher than those in poly(I:C)group and EV group,compared with the previous experimental results,it was suggested that MDRV served as a two-way modulator in the expression of interferon.The mRNA expression levels of the above factors in ?A+poly(I:C)group was extremely significantly(P<0.01)higher than those in EV+poly(I:C)group and other groups.(2)The results of ELISA showed that the expression of those factors were consistent with those in Real-time PCR detection.Collectively,these results suggested that ?A protein enhanced the increasing expression of interferon and interferon signaling pathway-related factors induced by poly(I:C).3.In order to determine whether the amino acid residues of 155R and 273R of MDRV ?A protein are the key sites for the regulation of interferons,three mutant vectors of MDRV-YB ?A gene(155A1a mutation,273A1a mutation and 155A1a+273A1a double mutations)were constructed and then successfully expressed in DF-1 and Vero cell lines.Next,the wild-type ?A and mutants were respectively transfected into DF-1 cells for 24 h,followed by transfection with poly(I:C),and then the expressions of interferon and IFN signaling pathway-related factors were detected by Real-time PCR and ELISA.(2)Real-time PCR results showed that the mRNA expression of IFN-? and interferon signaling-related factors MDA5,TLR3,IRF7,IFIT5,ISG12 and Mx in?A155 mutant group and ?A273 mutant group were significantly(P<0.05)or extremely significantly(P<0.01)higher than those in EV group,but the mRNA expression of those factors(except TLR3)was significantly(P<0.05)or extremely significantly(P<0.01)lower than those in wild-type?A group,indicating that the single mutant could significantly(P<0.05)or extremely significantly(P<0.01)inhibited but not deprived the up-regulation of interferon and IFN signal pathway-related factors induced by poly(I:C).The mRNA expressions of above factors of?A155,273 mutant group were extremely significantly(P<0.01)lower than those of wild-type ?A group as well as single mutant groups,and simultaneously were not significantly different from EV group(P>0.05),with exception for TLR3 and MDA5,indicating that the double mutants could extremely significantly(P<0.01)inhibited,even deprived the up-regulation of interferon and IFN signal pathway-related factors induced by poly(I:C).Additionally,the mRNA expressions of those factors(except ISG12)between ?A155 mutant group and aA273 mutant group were not significant(P>0.05),even with exception for TLR3,Mx and MDA5,all factors were extremely significantly(P<0.01)higher than those of ?155,273 mutant group.(2)The ELISA results are consistent with results obtained from Real-time PCR detection.Collectively,the above results suggested that 155Arg and 273Arg were the key sites of ?A protein,and either 155Arg or 273Arg mutation significantly(P<0.05)or extremely significantly(P<0.01)inhibited the enhanced expression of interferon and its pathway-related factors induced by poly(I:C),and the double sites mutation were more effective than single site mutation during this process,and even deprived the function of?A protein in the regulation of interferon signaling pathway.In conclusion,our findings reveal that MDRV ?A protein enhances the up-regulative expressions of interferon and IFN signaling pathway-related factors induced by MDRV as well as poly(I:C),and 155Arg and 273Arg of ?A protein play a great role in this progress.This study will provide foundations for elucidation of the molecular mechanism of of MDRV ?A in influencing the natural immunity of host,and provide new insight into the control of MDRV in future. |
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