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Cap-independent translation of ORF1(p35)encoded by Tobacco bushy top virus

Deya Wang;Chengming Yu;Guolu Wang;Xuefeng Yuan

  Tobacco bushy top virus(TBTV),a member of Umbravirus,contains a positive single genomic RNA with 4 152 nts.The genome of TBTV lacked 5cap and3 poly(A) tail and encoded 4 proteins including components of replicase(p35 and p63),and movement proteins(p26 and p27).For the cap-independent translation of p35,previous report showed the existence of a BTE-like element in TBTV by using firefly luciferase(Fluc) reporter vector.We confirmed the BTE-like element in fulllength TBTV through in vitro translation in wheat germ extract(WGE).Furthermore) we used forward and reverse genetics methods to identify new elements regulating the expression of p35.The expression of p35 among different TBTV isolates showing severe or mild pathogenicity had 3 fold variations.Consequently,the regions altering the expression of p35 were mapped within 5terminal 500 nt and internal600 nt regions surrounding BTE-like elements by constructing chimeric TBTV.The 3proximal 120 nt regions located downstream of BTE-like element were also played role in the translation of p35 by deletion mutagenesis.The 3 terminal 120 nt regions contained 5 hairpins and three RNA pseudoknots through in-line probing and their roles on the translation of p35 will be further identified.In a word,it is suggested that at least three cis-elements including BTE-like element were associated with the cap-independent translation of p35.……