Eukaryotic initiation factor (eIF) 3 mediates Barley Yellow Dwarf Viral mRNA 3′–5′ UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation

摘要

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA virus that lacks the canonical 5′ 7-methylguanosine cap and a 3′ poly-A tail. Instead, BYDV utilizes a cruciform cap independent translation element (CITE) in its 3′UTR RNA (BYDV-like CITE or BTE) that binds eukaryotic translation initiation factor (eIF) 4F and recruits 40S ribosomal subunits in the presence of active helicase factors (eIF4A, eIF4B, eIF4F and ATP). A long-range, 5-nucleotide, base-pairing kissing loop interaction between the 3′BTE and a 5′UTR stem-loop is necessary for translation to initiate. The 40S–eIF complex does not bind to the BYDV 5′UTR, suggesting the involvement of additional factors. We identified eIF3 as a component of the 3′BTE recruited complex using affinity-tagged 3′BTE RNA pull-down assays. Fluorescence anisotropy binding and gel shift assays showed that the 3′BTE and 5′UTR RNAs can simultaneously and non-competitively bind eIF3 in the presence of active helicase factors forming a single, macromolecular complex. Further, quantitative studies showed eIF3 increased recruitment of the 40S subunit by more than 25-fold. We propose a new role for eIF3, where eIF3 bridges BYDV’s UTRs, stabilizes the long-range 5′-3′ interaction, and facilitates recruitment of the 40S–eIF complex to the 5′UTR, leading to translation initiation.