Studies On The Interaction Between Rice Stripe Virus And The Nonsense-mediated Decay Of The Host During Its Cap-snatching

Rice stipe virus(RSV),an important plant-infecting bunyavirus,has caused great economic losses in China in the past decades.Like all other bunyaviruses,RSV uses a mechanism called cap-snatching to acquire a cap structure for its own m RNAs.How RSV interacts with the host during the process of cap-snatching,however,remains poorly understood.Eukaryotic cells often produce nonsense m RNAs(ns RNAs)with premature termination codons(PTC).The ns RNAs are recognized by a multi-step pathway named nonsense-mediated decay(NMD),which transports ns RNAs to cellular processing bodies(P-bodies)for decapping and degradation.Some studies suggest that bunyaviruses may preferentially target ns RNAs during cap-snatching and consequently bunyaviruses may benefit from NMD.The major aim of this study is to investigate whether this idea is true for RSV.As the first step for this aim,two experiments were carried out to test whether RSV preferentially targets ns RNAs during cap-snatching.In the first experiment,a competition was done between a normal GFP m RNA(GFP-n)and a PTC-containing GFP m RNA(GFP-m).In the second experiment,two competitions were done,one between a normal phytohemagglutinin(PHA)m RNA and a normal GFP,the other between a PTC-containing PHA(PHA-m)and a normal GFP.The results showed that RSV prefers neither GFP-m nor PHA-m.In addition,the cap-snatching of RSV from m RNAs with intrinsic instability elements was investigated.The results showed that,like ns RNAs,RSV did not prefer these m RNAs during cap-snatching.As the second step,virus induced gene silencing was used to silence UPF1 or SMG7,each encoding a key protein involved in early NMD,to investigate the effects of NMD on the cap-snatching of RSV from ns RNAs.The cap-snatching frequency of RSV from PHA-m was increased in UPF1-silenced plants,although it was unchanged in SMG7-silenced plants.In contrast,the cap-snatching frequency of RSV from GFP-m was increased in SMG7-silenced plants,although it was unchanged in UPF1-silenced plants.As the third step,the infection efficiency of RSV in UPF1 or SMG7-silenced plants was investigated.Although the symptoms developed in UPF1 or SMG7-silenced plants were comparable to those developed in wildtype plants,the viral accumulation level was increased in these plants.This suggested that the infection efficiency of RSV was enhanced by silencing of UPF1 or SMG7.Taken together,the observations obtained above indicate that RSV does not show preference to ns RNAs during cap-snatching.The idea that RSV benefits from NMD seems to be very unlikely to be true.In contrast,it seems that,by degrading ns RNAs,NMD restricts the cap-snatching of RSV and may thus impose a constriction to the infection of RSV.Consistent with this,this study found that RSV NP co-localizes with SMG7 when expressed together in plant cells.Given these ideas,it is interesting to know whether plants deliberately enhance NMD in response to RSV infection.As an attempt to answer this question,the expression level of several NMD-related genes in RSV-infected plants was investigated.It was found that UPF1,SMG7,DCP2 and XRN4 were all upregulated.Is it possible that plants recognize specific RSV proteins to upregulate NMD-related genes? To answer this question,transgenic plants overexpressing the NP of RSV were obtained and the expression of NMD-related genes in these plants was investigated.Like the situation observed from RSV-infected plants,UPF1,DCP2 and XRN4 showed significant increase in expression.However,SMG7 was downregulated in the transgenic plants.In summary,preliminary investigations were carried out to probe the interaction between RSV and host NMD during its cap-snatching.The data may be useful in understanding the cap-snatching of RSV.