Identification of Novel 5′ and 3′ Translation Enhancers in Umbravirus-Like Coat Protein-Deficient RNA Replicons

Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). ABSTRACT Translation of plant plus-strand RNA viral genomes that lack a 5′ cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 3′ untranslated region (UTR). 3′CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 3′ end that is nearly always transferred to the 5′ end via a long-distance kissing-loop interaction between sequences in the 3′CITE and 5′ hairpins. We report here the identification of a novel 3′CITE in coat protein-deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 3′ UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 3′CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5′ end. We also report that the ∼30-nucleotide (nt) 5′ terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3′CITE or the 3′CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 3′CITE. These findings introduce a new type of 3′CITE and provide the first information on translation of ulaRNAs. IMPORTANCE Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 3′ cap-independent translation enhancer (3′CITE), which does not connect with the 5′ end by a long-distance RNA:RNA interaction that is typical of 3′CITEs. ulaRNA 5′ terminal hairpins can also enhance translation in association with cognate 3′CITEs or those of related ulaRNAs and, to a lesser extent, with 3′CITEs of umbraviruses, or even independent of a 3′CITE. These findings introduce a new type of 3′CITE and provide the first information on translation of ulaRNAs.