Concerted action of two 3' cap-independent translation enhancers increases the competitive strength of translated viral genomes.

Several families of plant viruses evolved cap-independent translation enhancers (3'CITE) in the 3' untranslated regions of their genomic (g)RNAs to compete with ongoing cap-dependent translation of cellular mRNAs. Umbravirus Pea enation mosaic virus (PEMV)2 is the only example where three 3'CITEs enhance translation: the eIF4E-binding Panicum mosaic virus-like translational enhancer (PTE) and ribosome-binding 3' T-shaped structure (TSS) have been found in viruses of different genera, while the ribosome-binding kl-TSS that provides a long-distance interaction with the 5' end is unique. We report that the PTE is the key translation promoting element, but inhibits translation in cis and in trans in the absence of the kl-TSS by sequestering initiation factor eIF4G. PEMV2 strongly outcompeted a cellular mRNA mimic for translation, indicating that the combination of kl-TSS and PTE is highly efficient. Transferring the 3'-5' interaction from the kl-TSS to the PTE (to fulfill its functionality as found in other viruses) supported translationin vitro, but gRNA did not accumulate to detectable levels in protoplasts in the absence of the kl-TSS. It was shown that the PTE in conjunction with the kl-TSS did not markedly affect the translation initiation rate but rather increased the number of gRNAs available for translation. A model is proposed to explain how 3'CITE-based regulation of ribosome recruitment enhances virus fitness.