Context-influenced cap-independent translation of Tombusvirus mRNAs in vitro

Tomato bushy stunt virus (TBSV) possesses a positive-strand RNA genome that is not 5′-capped or 3′-polyadenylated. Previous analysis revealed that the TBSV genome contains a 3′-cap-independent translational enhancer (3′CITE) in its 3′-untranslated region (3′UTR) that facilitates translation of viral mRNAs in vivo. A long-range 5′–3′ RNA–RNA interaction between the 3′CITE and the 5′UTR of viral mRNAs is necessary for function, and this RNA bridge has been proposed to mediate delivery of translation-related factors bound to the 3′CITE to the 5′-end of the message. Although fully functional when assayed in plant protoplasts, the TBSV 3′CITE was previously found to be unable to activate translation in vitro in wheat germ extract (wge). In the current report we have determined that (i) another Tombusvirus, Carnation Italian ringspot virus (CIRV), contains a TBSV-like 3′CITE that is active in wge; (ii) the CIRV 3′CITE functions in vitro in a manner analogous to the TBSV 3′CITE in vivo; (iii) the TBSV 3′CITE is able to competitively inhibit CIRV 3′CITE-dependent translation in wge and (iv) the TBSV 3′CITE can enhance translation in wge when present in short viral messages. These results reveal the contrasting activities of different TBSV-like 3′CITEs in vitro and shed light on the nature of the defect in TBSV.