Identification and characterization of the functional elements within the tobacco etch virus 5 ' leader required for cap-independent translation

Translation in planes is highly cap dependent, and the only plant mRNAs kno wn to naturally lack a cap structure (m(7)GpppN) are viral in origin. The g enomic RNA of tobacco etch virus (TEV), a potycirus that belongs to the pic ornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even o n reporter mRNAs. We have carried out a deletion analysis of the TEV 5' lea der to identify the elements responsible for its regulatory function and ha ve identified two centrally located cap-independent regulatory elements (CI REs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is l ess 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the pr esence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can f unction in a 5'-distal position. Interestingly, the introduction of a stabl e stem-loop upstream of the TEV leader sequence or upstream of either CERE in dicistronic constructs markedly increased their regulatory function. The se data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in partic ular, is facilitated by proximity to the 5' end.