Barley yellow dwarf virus (BYDV) utilizes a novel strategy for translation initiation.

Abstract

Most commonly, gene expression in uncapped RNA viruses is regulated by either an internal ribosomal entry site (IRES) or cap independent translation element (CITE), which are located mostly in the 5′ untranslated region (UTR) of viral mRNA. BYDV, which lacks both a cap and poly(A) tail, has a CITE (called BTE) present in its 3′UTR that is essential for efficient translation initiation at the 5′ proximal AUG. The mechanism of BYDV translation initiation is not understood.Using fluorescence anisotropy and gel mobility shift assays we demonstrated that the 40S ribosome has moderate binding affinity to the 3′ UTR (Kd~600nM) and has extremely low affinity for any other parts of mRNA. The binding affinity to 3′UTR increases nearly six fold (Kd~100nM) in the presence of the eIF4F‐4B‐4A‐ATP helicase complex. In order to identify the structural elements that interact with initiation factors or ribosomes, the secondary structure of both 3′ and 5′ UTR was probed using SHAPE technique. These results identified a very stable secondary structure for both 5′and 3′ UTRs. Primer extension inhibition (toe‐printing) was used to identify the initial 43S ribosome binding site. These results suggest a novel mechanism for 43S ribosome binding to the 3′BTE followed by subsequent transfer to the 5′end.