Oscillating kissing stem–loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element

Abstract

The 3′-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5′-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem–loops between the BTE in the 3′-UTR and a BTE-complementary loop in the 5′-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5′-extensions of the 5′-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5′-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3′-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3′- and 5′-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.