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Cis-elements of-1 programmed ribosome frameshift responsible for the expression of RdRp in Tobacco bushy top virus

Chengming Yu;Deya Wang;Guolu Wang;Xuefeng Yuan

  Tobacco bushy top virus(TBTV) has a(+) ssRNA genome with 4 152 nucleotides.The viral genome without 5-cap and 3-poly(A) encoded four putative open reading frames(ORFs),in which ORF1(p35) and ORF2(p63) are components of replicase based on common charactericstic of umbravirus.Through in vitro translation of TBTV in wheat germ extract(WGE),it showed that ORF2(p63) was expressed through-1 type frameshift mechanism and existed as ORFl-fused pattern resulting in the production of p98,which may function as RNA-dependent RNA polymerase(RdRp).Through comparation of sequences,mutagenesis and in vitro translation,we identified the essential heptanucleotide slippery sequences at946 GGATTTT,whose site-mutations decreased the frameshift ratio to about 30%of wild type.We also tested the optimal distance between heptanucleotide slippery sequences and potential RNA structure located 6 nt downstream,showing that 9 nt is the upper limit.Furthermore,long distance RNA-RNA interaction was found though electrophoretic mobility shift assay(EMSA).The 3terminal 200 nt could form RNA-RNA interaction with potential RNA structure located downstream of heptanucleotide slippery sequences.In-line probing showed that one of the RNA-RNA interaction sites is located at the 3proximal hairpin,which is also responsible for replication in similar genus.The local RNA structure and detailed long distance RNA-RNA interaction,which is essential for-1type frameshift,will be further identified.……