The Cap Snatching Of Rice Stripe Virus As A Tool To Map The Transcription Start Sites Of TYLCV

To produce mRNAs with a cap structure at their 5' ends,segmented negative-sense RNA viruses(sNSVs)commonly employ a mechanism called cap-snatching.In this mechanism,sNSVs snatch capped-RNA leaders from host cellular mRNAs and initiate genome transcription with the snatched leaders as primers,giving rise to viral mRNAs with host-derived heterogeneous sequences at their 5' ends.Rice stripe tenuivirus(RSV)is an important plant-infecting sNSV.In recent years,our lab established an approach to sequence the cap-snatching repertoire of RSV.Tomato yellow leaf curl virus(TYLCV)depends on the host transcription machinery to synthesize its mRNAs.It is known that the genome of TYLCV has six open reading frames(ORFs),but the sites on the TYLCV genome from which host RNA polymerase transcribes mRNAs containing these ORFs are unclear at present.This study was aimed to solve this issue with the cap-snatching of RSV,namely to establish a system in which RSV can snatch capped-RNA leaders from the mRNAs of TYLCV and the snatched leaders can be obtained by high throughput sequencing.Firstly,this study established a method through which Nicotiana benthamiana co-infected by RSV and TYLCV can be obtained reproducibly.In this method,crude preparations of RSV obtained from rice is used to rub-inoculate four-to-six leaf stage N.benthamiana.After a green-house growth of two weeks,N.benthamiana infected by RSV is super-infected by TYLCV by agro-infiltration of a TYLCV infectious clone.After another two weeks,total DNA or RNA is extracted from the N.benthamiana to detect TYLCV or RSV with PCR or RT-PCR,respectively.As the second step,N.benthamiana co-infected by RSV and TYLCV was collected and the capped-RNA leaders snatched by RSV was sequenced(library 1).N.benthamiana infected by RSV alone was used as a control(library 2).The results showed that 715,896 capped-RNA leaders,comprising 11,833 unique sequences,were obtained for library 1.Among these capped-RNA leaders,12,599,comprising 30 unique sequences matched the genome of TYLCV.After mapping these capped-RNA leaders to the genome of TYLCV,21 putative transcription start sites were obtained,9 of which were located to the virion-sense DNA,while 12 of which were located to the complementary-sense.The numbers of capped-RNA leaders mapped to each transcription start site ranged from 42 to 2,075,with an average of 419.Among the 715,896 TYLCV-matching capped-RNA leaders in library 1,only 49(4 unique)could be found in library 2.This indicated that most of these TYLCV-matching capped-RNA leaders were derived from mRNAs of TYLCV,rather than those of the host.To confirm the results obtained above,conventional RLM-RACE was used to identify the transcriptional start sites upstream of the ORF AV2 or AC2(AC3).The results showed that most transcriptional start sites obtained by cap-snatching could be confirmed by conventional methods,and vice versa.However,the cap-snatching approach has obvious advantages over conventional methods:(i)with the cap-snatching approach,researchers do not need to design different primers for each mRNA;(ii)with the cap-snatching approach,all the transcriptional start sites are obtained in a single high throughput sequencing reaction;(iii)the cap-snatching approach s much cheaper than conventional methods.Despite the fact that the cap-snatching mechanism of sNSVs has been studied for nearly 40 years,this study represents the first attempt to use the this mechanism as a tool for molecular studies.As a group,sNSVs have a very broad host range.In theory,the approach used here can be applied to any virus that is able to co-infect any host with any sNSV.Thus,this study established an effective method for transcriptional start site identification and this method is potentially very useful.In addition,because TYLCV is feasible to genetic manipulations,the co-infection of RSV and TYLCV provided a valuable system for further studies on the cap-snatching mechanism of RSV.