Understanding The Origin Of The Extra Dinucleotide AC At The 5' Termini Of Rice Stripe Tenuivirus MRNAs

Segmented negative sense RNA viruses?sNSVs?commonly use a mechanism called cap snatching to synthesize capped mRNAs.During this mechanism,sNSVs snatch a capped-RNA leader m⁷GpppN-N_X?X=10-20?from host cellular mRNAs and uses the snatched leaders as a primer for the transcription of viral template RNAs,giving rise to viral mRNAs with a host-derived capped-RNA leader at their 5^'ends.Rice stripe virus?RSV?is one of the most important plant sNSVs.Recently,our lab determined the 5^'ends of 6 RSV mRNAs using total RNA extracted from systemically infected rice as the staring material.A large number of RSV mRNA were found to contain one or more extra dinucleotide AC between between the host-derived capped-RNA leader and transcribed sequence(m⁷GpppN-N_X?AC?_n?n?1?A₁C₂A₃C₄AAAGUC-,transcribed sequence is underlined).These mRNAs account for about 42%of the transcripts from genomic RNAs?vRNA,such as NP?,and about 19%of the transcripts from antigenomic RNAs?vcRNA,such as NCP?.According to the relevant models reported in the literature,our lab proposed that RSV uses the prime-and-realign mechanism during cap-snatching,and RSV has a greater tendency to use this mechanism in transcribing genomic mRNAs than that of antigenomic RNAs.However,another explanation for extra dinucleotide AC is that RSV frequently snatches RNA leaders from its own mRNAs.For example,RSV will obtain m⁷GpppN-N_x?AC?A₁C₂A₃C₄AAAGUC-when it cleaves one of its mRNAs,m⁷GpppN-N_xA₁C₂A₃C₄AAAGUC-,at the position C₂,and RSV snatchedthecapfromtheC₂ofthem⁷GpppN-N_x?AC?A₁C₂A₃C₄AAAGUC-,RSVwillobtainthem⁷GpppN-N_x?AC?2A₁C₂A₃C₄AAAGUC-.The following two experiments are used to study whether this explanation is valid:???To see the frequency of the extra dinucleotide AC in the mRNAs produced in early stages of RSV infection:The total RNA was extracted from the inoculated leaves at 4,8,12,24 and 48 hours after RSV friction inoculation of Nicotiana benthamiana,We obtained 5^'sequences of RSV NP and NCP mRNA at different time by the high throughput sequencing method.If the extra dinucleotide AC derived from the RSV own RNA,we can foresee:???in the early stage of RSV infection,m⁷GpppN-N_X?AC?_nA₁C₂A₃C₄AAAGUC-was rare in mRNA.???The proportion of m⁷GpppN-N_X?AC?_nA₁C₂A₃C₄AAAGUC-in NP and NCP mRNA in the early stage of RSV infection was not significantly different.???To track RSV mRNA primed by a defined and exogenously introduced capped RNA leader:Recently,our lab found that RSV can snatch capped-RNA leaders from the heterologously expressed mRNA in Nicotiana benthamiana.Based on this finding,this study expressed a GFP mRNA through agroinfiltration in RSV infected Nicotiana benthamiana.To present a capped-RNA leader,m⁷GpppN-ACGGGGGACTTC₁₂,to RSV.Then This study obtained RSV NP and NCP mRNAs primed by m⁷GpppN-ACGGGGACTTC₁₂ at 1,2,3,5,9,and 12 days,and calculated theproportionofm⁷GpppN-ACGGGGGACTTC₁₂?AC?_nA₁C₂A₃C₄AAAGUC-at each time point.If the extra dinucleotide AC is mainly derived from self-snatching,we can foresee:???Theproportionofm⁷GpppN-ACGGGGGACTTC₁₂?AC?_nA₁C₂A₃C₄AAAGUC-is lower in the early stage and higher in the later stage.???The different proportions of m⁷GpppN-ACGGGGGACTTC₁₂?AC?_nA₁C₂A₃C₄AAAGUC-in NP and NCP mRNA was more obvious in the later periodThe results of experiment???showed that m⁷GpppN-N_X?AC?_n A₁C₂A₃C₄AAAGUC-is common in the early stage of RSV infection,and NP mRNA?28.17-37.86%?has a greater tendency to contain m⁷GpppN-N_X?AC?_nA₁C₂A₃C₄AAAGUC-than NCP?17.34-20.53%?early in RSV infection.The results of experiment.???showed that the proportion of m⁷GpppN-ACGGGGGACTTC₁₂?AC?_nA₁C₂A₃C₄AAAGUC-didnot change significantly with time,and NP?38.97-57.58%?has morem⁷GpppN-ACGGGGGACTTC₁₂?AC?_n A₁C₂A₃C₄AAAGUC-thanNCP?0-16.30%?at all time points.Based on the results of the two experiments,we concluded that the extra dinucleotide AC in RSV mRNA was derived from the prime-and-realign mechanism,not from self-snatching.In addition,the following two observations were obtained in this study:???RSV has already begun to transcribe its genomes 4 hours after it was inoculated into leaves of N.benthamiana;?ii?RSV produces much more NCP mRNAs than NP mRNAs early in its infection.