A primary determinant of cap-independent translation is located in the 3 '-proximal region of the tomato bushy stunt virus genome

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the p rototype member of the genus Tombusvirus. The genomes of members of this ge nus are not polyadenylated, and prevailing evidence supports the absence of a 5' cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3' terminus of the TBSV genome was implicated as a determ inant of translational efficiency (S.K. Oster, Il, Wu and K. A. White, J. V irol, 72:5845-5851, 1998), In the present report, we provide evidence that a 3'-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation, Our results indicate that (i) a 5' cap structure can substitute functionally for the absence of region 3 .5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3. 5 from viral and chimeric mRNAs has no appreciable effect on message stabil ity; (iii) region 3.5 represents part of a larger 3' proximal element, desi gnated as the 3' cap-independent translational enhancer (3'CITE), that is r equired for proficient cap-independent translation; (iv) the 3'CITE also fa cilitates cap-dependent translation; (v) none of the major viral proteins a re required for 3'CITE activity; and (vi) no significant 3'CITE-dependent s timulation of translation was observed when mRNAs were tested in vitro in w heat germ extract under various assay conditions. This latter property dist inguishes the 3'CITE from other characterized plant viral 3'-proximal cap-i ndependent translational enhancers, Additionally, because the 3'CITE overla ps with cis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.