In the absence of a 5′ cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3′ untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3′ cap-independent translational enhancers (3′ CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3′ CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5′ end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3′ CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

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During cap-dependent translation of eukaryotic mRNAs, initiation factors interact with the 5′ cap to attract ribosomes. When animal viruses translate in a cap-independent fashion, ribosomes assemble upstream of initiation codons at internal ribosome entry sites (IRES). In contrast, many plant viral genomes do not contain 5′ ends with substantial IRES activity but instead have 3′ translational enhancers that function by an unknown mechanism. A 393-nucleotide (nt) region that includes the entire 3′ UTR of the Turnip crinkle virus (TCV) synergistically enhances translation of a reporter gene when associated with the TCV 5′ UTR. The major enhancer activity was mapped to an internal region of ∼140 nt that partially overlaps with a 100-nt structural domain previously predicted to adopt a form with some resemblance to a tRNA, according to a recent study by J.C. McCormack and colleagues. The T-shaped structure binds to 80S ribosomes and 60S ribosomal subunits, and binding is more efficient in the absence of surrounding sequences and in the presence of a pseudoknot that mimics the tRNA-acceptor stem. Untranslated TCV satellite RNA satC, which contains the TCV 3′ end and 6-nt differences in the region corresponding to the T-shaped element, does not detectably bind to 80S ribosomes and is not predicted to form a comparable structure. Binding of the TCV T-shaped element by 80S ribosomes was unaffected by salt-washing, reduced in the presence of AcPhe-tRNA, which binds to the P-site, and enhanced binding of Phe-tRNA to the ribosome A site. Mutations that reduced translation in vivo had similar effects on ribosome binding in vitro. This strong correlation suggests that ribosome entry in the 3′ UTR is a key function of the 3′ translational enhancer of TCV and that the T-shaped element contains some tRNA-like properties.

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3′ end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Ψ ₃ ) within the 3′-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Ψ ₃ and a second pseudoknot (Ψ ₂ ) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Ψ ₃ and a pseudoknot that connects H5 and the 3′ end (Ψ ₁ ). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3′ end, and subsets containing Ψ ₃ , H4a, and H4b or Ψ ₃ , H4a, H4b, H5, and Ψ ₂ , form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Ψ ₃ , and Ψ ₂ are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3′ untranslated region sequences such as translation and replication.

Plus-strand RNA viruses without 5′ caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3′ untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5′ UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5′ UTR sequences. Addition of 80S ribosomes to the 5′ UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5′ end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5′-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5′ and 3′ sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5′ and 3′ UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5′ UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5′ and 3′ UTRs of TCV.

Many plant RNA viruses contain elements in their 3′ untranslated regions (3′ UTRs) that enhance translation. The PTE ( Panicum mosaic virus -like translational enhancer) of Pea enation mosaic virus (PEMV) binds to eukaryotic initiation factor 4E (eIF4E), but how this affects translation from the 5′ end is unknown. We have discovered a three-way branched element just upstream of the PEMV PTE that engages in a long-distance kissing-loop interaction with a coding sequence hairpin that is critical for the translation of a reporter construct and the accumulation of the viral genome in vivo . Loss of the long-distance interaction was more detrimental than elimination of the adjacent PTE, indicating that the RNA-RNA interaction supports additional translation functions besides relocating the PTE to the 5′ end. The branched element is predicted by molecular modeling and molecular dynamics to form a T-shaped structure (TSS) similar to the ribosome-binding TSS of Turnip crinkle virus (TCV). The PEMV element binds to plant 80S ribosomes with a K _d (dissociation constant) of 0.52 μM and to 60S subunits with a K _d of 0.30 μM. Unlike the TCV TSS, the PEMV element also binds 40S subunits ( K _d , 0.36 μM). Mutations in the element that suppressed translation reduced either ribosome binding or the RNA-RNA interaction, suggesting that ribosome binding is important for function. This novel, multifunctional element is designated a kl-TSS (kissing-loop T-shaped structure) to distinguish it from the TCV TSS. The kl-TSS has sequence and structural features conserved with the upper portion of most PTE-type elements, which, with the exception of the PEMV PTE, can engage in similar long-distance RNA-RNA interactions.