Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs

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.

Short Internal Sequences Involved in Replication and Virion Accumulation in a Subviral RNA of Turnip Crinkle Virus

ABSTRACT cis-acting sequences and structural elements in untranslated regions of viral genomes allow viral RNA-dependent RNA polymerases to correctly initiate and transcribe asymmetric levels of plus and minus strands during replication of plus-sense RNA viruses. Such elements include promoters, enhancers, and transcriptional repressors that may require interactions with distal RNA sequences for function. We previously determined that a non-sequence-specific hairpin (M1H) in the interior of a subviral RNA (satC) associated with Turnip crinkle virus is required for fitness and that its function might be to bridge flanking sequences (X. Sun and A. E. Simon, J. Virol. 77:7880-7889, 2003). To establish the importance of the flanking sequences in replication and satC-specific virion repression, segments on both sides of M1H were randomized and subjected to in vivo functional selection (in vivo SELEX). Analyses of winning (functional) sequences revealed three different conserved elements within the segments that could be specifically assigned roles in replication, virion repression, or both. One of these elements was also implicated in the molecular switch that releases the 3′ end from its interaction with the repressor hairpin H5, which is possibly involved in controlling the level of minus-strand synthesis.