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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. 

We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.