Comparative Study On The Cap-snatching Mechanisms Of Rice Stripe Virus And Rice Grassy Stunt Virus

Segmented negative-sense RNA viruses are an important group of viruses,several of which pose a serious threat to food safety or human living and animal husbandry.This group of viruses use a conserved mechanism termed cap-snatching during genome transcription.In cap-snatching,the viruses use a capped RNA fragment of the host as a primer to initiate mRNA production.Cap-snatching results in a chimeric virus mRNA containing a heterogeneous 5’ teminus derived from the host.Understanding cap-snatching is important to clarify the infection mechanisms of segmented negative-sense RNA viruses and may provide novel targets for the development of antivirals.Normally,the snatched capped RNAs of a virus are 10-15 nucleotide(nt)in size and have a high sequence diversity.These,plus the difficulty in obtaining 5’ terminal sequence of a mRNA,results in a fact that many of the important issues regarding cap-snatching are unclear at present.These include:(1)whether or not and how a virus select a specific set of host RNAs;(2)the diversity of the snatched capped RNAs;(3)if there are some rules according to which a virus selects cap donors;(4)Do viruses cleave and use a specific subset of host mRNAs?In addition,it has been noted that viruses use a mechanism called prime-and-realign with varying frequencies.However,the mechanism underlying this variation is poorly understood.In this study,with an attempt to solve these problems,we studied the cap-snatching mechanisms of the two tenuiviruses Rice stripe virus(RSV)and Rice grassy stunt virus(RGSV).Using artificial inoculation with viruliferous planthoppers,rice plants co-infected by RSV and Rice ragged stunt virus(RRSV),a plant-infecting reovirus,or RGSV and RRSV were obtained.The results of nested RT-PCR showed that both RSV and RGSV snatched capped RNAs from RRSV in the co-infected rice.A total of 347 chimeric mRNA clones of RSV/RGSV NP/NCP were sequenced.An analysis of the sequences suggested a scenario consistent with previously reported data on other related viruses,in which both tenuiviruses prefer leaders having a 3’-terminal A,C,AC or CA that can base pair with the template at the 3’ termini and upon base pairing the RRSV leaders prime processive transcription directly or after one to several cycles of priming and realignment(repetitive prime-and-realign).A comparison of the two viruses revealed that RSV has a higher tendency to use repetitive prime-and-realign than RGSV even with the same leader derived from the same RRSV mRNA.Combining with relevant data reported previously,this points towards an intrinsic feature of RSV.To further study the cap snatching mechanisms of the two viruses,a method based on specific enzyme digestion reactions was developed to enrich RSV/RGSV mRNAs and sequence them with the next-generation sequencing technologies.With this method,the 5’ termini of RSV/RGSV NP/N CP mRNAs from 12 different rice samples and one wheat sample were sequenced and a total of 22,551,079 sequences were obtained.An analysis of these sequences revealed that:(1)both viruses tend to cleave the cap donor RNA 10-13 nt downstream of the cap after an A,C,AC or CA.(2)RSV uses the mechanism "prime-and-realign" more frequently that RGSV.This is not affected by the co-infecting RRSV.In addition,the high tendency of RSV to use"prime-and-realign" can also be found in wheat.(3)RSV uses the mechanism"prime-and-realign" more frequently when producing the NP mRNAs than when producing the NCP mRNAs.(4)A very high diversity of the capped RNAs were found for both viruses.However,the two viruses showed some detectable differences in their "spectrums" of the snatched capped RNAs,indicating that viruses may select their capped donors according to some rules.To identify the cap donor RNAs,a modified Capseq method was developed.With this method,more than one million capped RNAs were sequenced from RS V or RGSV infected rice,creating a database for rice 5’ un-translated regions,A search of this database using the abovementioned capped RNAs of RSV/RGSV identified the source RNAs for more than 40 thousand capped fragments.An analysis of these source RNAs revealed that they correspond to 4,520 distinct rice genes.An analysis of the capped fragments that were used with a high frequency by both viruses revealed a significant enrichment of the GO terms related to chloroplast function.Thus,RSV and RGSV may specifically use chloroplast related mRNAs as cap donors.This represents the first study in which the cap snatching of two segmented negative-sense RNA viruses were studied in parallel.In addition,this represents the first study on the cap-snatching mechanisms of segmented negative-sense RNA viruses replicating in the cytoplasm using high throughput sequencing.The results of this study provided new insights into the cap-snatching mechanisms of tenuiviruses and related viruses.