RNA Structure Basis And Molecular Evolution Of Cap-independent Translation In Tobacco Bushy Top Virus

Tobacco bushy top virus（TBTV）,a member of the genus of Umbravirus in the family of Tombusviridae,contains a positive single-strand RNA with 4,152 nts.The genome of TBTV lacks 5’cap and 3’poly（A）tail and encodes 4 ORFs,including replicase components p35 and its-1 frameshift product p98（the RdRp）,and movement proteins（p26 and p27）.TBTV contains a functional BTE in its 3’UTR,which can regulate cap-independent translation of the reporter gene FLuc.In this study,we analyzed the function of BTE within TBTV full-length RNA on the translation of p35 and in vivo RNA accumulation in BY-2 protoplasts.Three new mild TBTV isolates were cloned and analyzed for in vivo accumulation and p35expression,which suggested that attenuated virulence of mild TBTV isolates was associated with low-level expression of p35 due to the structural alteration of BTE and the inhibition of long-diatance RNA-RNA interaction.In addtion,deletion mutagenesis in TBTV full-length RNA identified second type cap-independent translation element locating at the 3’terminal region.The detailed data were shown as following.The colne and molecular evolution of TBTV:Three new isolates of Tobacco bushy top virus（TBTV）that exhibit mild pathogenicity in the field were colned using 5’and 3’RACE combined with one step full-length RT-PCR.The genomes of three isolates,termed TBTV-JC（KM016224）,TBTV-MD-I（KM016225）and TBTV-MD-II（KM067277）,were all4,152 nts in length and included one distinctive difference from previously reported TBTV isolates:the first nucleotide of the genome was a guanylate,not an adenylate.Phylogenetic analysis among all 8 TBTV isolates indicated that variant ORFs and 3’UTR in TBTV evoluted separately and ORF1 coding region was the most variable（75.0%-99.2%）.Recombination analysis detected 32 potential recombination events in 7 TBTV isolates.The effect of BTE on translation and replication of TBTV:The function of BTE in TBTV full-length RNA on the cap-independent translation of p35 was analyzed using WGE.Three core characteristic of BTE regulating the translation of TBTV were identified,which is consist of 17 nt conserved sequences in SL-I,long-distance RNA-RNA interaction between5’UTR and SL-III A and the structural stability of BTE.Moreover,BTE also played essential role in in vivo RNA accumulation of TBTV in BY-2 protoplasts.It is suggested that BTE affects the virulence of TBTV through regulating the translation of viral proteins.Relative RNA accumulation of the new isolates:Three new isolates of TBTV（JC,MD-I and MD-II isolates）from samples exhibiting mild virulence were compared with TBTV-Ch（wt）on RNA accumulation in BY-2 protoplasts.Relative RNA accumulation of these new isolates was only 30 to 40%of TBTV-Ch levels.In vitro synthesis of p35 for TB-JC,TB-MD-I and TB-MD-II was 25%,38%and 20%of wt,respectively.The low-level expression of p35 may be associated with the less virulent of the new isolates of TBTV.Molecular mechanism of TBTV isolates with low-level expression of p35:Based on translation data of chimeric TBTV RNAs,low-level expression of p35 in three new isolates was determined by two regions:the 5’terminal 500 nt region（RI）and the 3’internal region（RV）including the BTE.For RV region,natural mutation in BTE did not affect the translation of p35.However,natural mutation U³⁵⁸⁰→A³580580 decreased the translation of p35 to50%of wt.Secondary structure predictions using Mfold showed that TBTV isolates with low-level expression of p35 have low ratio of BTE typical structure.SHAPE structural probing also confirmed that the structure of BTE region in TB-JC,TB-MD-I and TB-MD-II showed remarkable difference from that of TBTV-Ch（wt）.It is implied that the negative effect of RV on the translation of p35 may be associated with the structural alteration of BTE due to the outside mutation.This is the first report on structural evolution of 3’cap-independent translation elements（BTE in this study）among different isolates of a given RNA virus,which is associated with variation in virulence of TBTV.For RI region,SHAPE structural probing showed that long-distance RNA-RNA interaction between the 5’UTR and SLIII-A in BTE was destroyed although the potential RNA-RNA interaction sequences existed.This inhibiton on long-distance RNA-RNA interaction was restored by two additional 5’UTR,which suggested that mutations in RI region changed local structure of 5’500 nt resulting into the inhibition of long-distance RNA-RNA interaction between the5’UTR and SLIII-A in BTE.Second type cap-independent translation element in TBTV:In addition,deletion mutagenesis showed that deletion of 4040-4099（D9）or deletion of 4093-4152（D10）decreased the translation of p35 in TBTV.SHAPE analysis showed that D9 or D10 did not affect the strcuture of BTE,which implied that the region of 4040-4152 may contain second type cap-independent translation elelment（CITE）in TBTV.Mfold structure prediction on TBTV showed that the region of 4003-4152 froms an independent domain.Based on the data of In-line structural probing,the structure of 4003-4152 includes 5 stem-loops and 3pseudonodules.The structure of 4003-4152 in TBTV has similar pattern with the structure of3’terminal rgion of TCV.It is suggested that the second type of CITE in TBTV may be TSS.