Study Of-1 Type Of Translation Frameshift For The Expression Of RNA-dependent RNA Polymerase(RdRp) Of Tobacco Bushy Top Virus

Tobacco bushy top virus（TBTV）,a member of the genus of Umbravirus in the family of Tombusviridae,and Tobacco vein distorting virus（TVDV）,a member of the family of Luteoviridae co-infected tobacco and caused tobacco bushy topdisease.TBTVhas a（+）ssRNA genome with 4,152 nucleotides.TBTV genome lacking 5’-cap and 3’-poly（A）encoded four putative open reading frames（ORFs）.ORF1 encoding 35KDa protein has 8 overlapped amino acids with ORF2 encoding 63 KDa proteins.RNA dependent RNA polymerase（RdRp）of TBTVwas expressed from ORF1 via-1 programmed ribosome frameshift（-1PRF）and its molecular weight is the sum of ORF1 and ORF2.Based on the previous identification on-1PRF expression of RdRp in TBTV,this study is focused on the identification of multiple levels of cis-elements and structural probing of these potential cis-elements invovled in-1PRF of RdRp.Based on the analysis of genome sequences,the potential hepta-nucleotide slippery sequence（the sequence motif is X XxY YYZ,where X can be any nucleotide,Y either A or U,and Z can be any nucleotide except G）was predicted to be located at ⁹⁴⁶GGAUUUU.Mutantion of G946A,U950C or U952Greduced the-1PRF level to the 20%⁴0%of wild type,which suggests the ⁹⁴⁶GGAUUUU is the hepta-nucleotide slippery sequence responsible for the-1 PRF of TBTV RdRp.To analysis the optimal distance between the hepta-nucleotide sequence and its downstream RNA cis-element,3 mutants were constructed as:deletion ofthree nucleotides(⁹⁵²UGC-De),insertion of 3 nucleotides(⁹⁵⁵CAU-In)and insertion of 6nucleotides(⁹⁵⁵CAUCAC-In).In vitro translation assay on these three mutants suggests that the optimal distance between hepta-nucleotide slippery sequences and its downstream RNA structure is 6 nt⁹ nt,which could ensure the normal efficiency of-1PRF for TBTV RdRp.Based on the Mfold prediction of the RNA structure in TBTV,the RNA structure loctaed at the downstream of hepta-nucleotide slippery sequences(⁹⁴⁶GGAUUUU)was analyzed using SHAPE structural probing.The SHAPE profile showed that there are three three stem-loops and one potential pseudoknot locating at the downstream of hepta-nucleotide slippery sequences.Mutagenesis analysis showed that all of these three stem-loops and this pseudoknot participate in the-1PRF of RdRp in TBTV.Through constructing 18 subclones covering the full-length of TBTV genome,potential long-distance RNA-RNA interaction between S5 and S18 was mapped using EMSA.S5（880-1120）includes the hepta-nucleotide slippery sequences and its downstream RNA domains,while S18 is located at the 3’terminal region of TBTV genome.Using In-line strcutural probing,the potential RNA-RNA interaction sites were primarily mapped through analyzing the structure change of S5 or S18 before and after RNA-RNA interaction.The RNA-RNA interaction sites within S5 were mapped at the first and third stem-loop downstream of hepta-nucleotide slippery sequences,while the RNA-RNA interaction site within S18 was located at the loop of Pr.Mutations in Pr loop suggests that ⁴¹³⁷ACU is the RNA-RNA interaction site,whose mutation decreased the-1 PRF to about 20%of wt.In summary,this study identified multi-elements associate with-1PRF of TBTV RdRp:the hepta-nucleotide slippery sequence⁹⁴⁶GGAUUUU,three stem-loops and one pseudoknot,6-9 nt optimal distance beween slippery sequence and downstream RNA structure,two long-distance RNA-RNA interaction between 3’Pr and local stem-loops downstream of slippery sequences.This study provides new model and date for-1PRF in（+）ssRNA virus and new potential targets for the prevention of virus disease caused by（+）ssRNA viruses expressing RdRp through-1PRF.