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Plus-strand RNA viruses frequently employ -1 programmed ribosomal frameshifting (-1 PRF) to maximize their coding capacity. Ribosomes can frameshift at a slippery sequence if progression is impeded by a frameshift stimulating element (FSE), which is generally a stable, complex, dynamic structure with multiple conformations that contribute to the efficiency of -1 PRF. As FSE are usually analyzed separate from the viral genome, little is known about cis-acting long-distance interactions. Using full-length genomic RNA of umbravirus-like (ula)RNA citrus yellow vein associated virus (CY1) and translation in wheat germ extracts, six tertiary interactions were found associated with the CY1 FSE that span nearly three-quarters of the 2.7 kb genomic RNA. All six tertiary interactions are conserved in other Class 2 ulaRNAs and two are conserved in all ulaRNAs. Two sets of interactions comprise local and distal pseudoknots that involve overlapping FSE nucleotides and thus are structurally incompatible, suggesting that Class 2 FSEs assume multiple conformations. Importantly, two long-distance interactions connect with sequences on opposite sides of the critical FSE central stem, which would unzip the stem and destabilize the FSE. These latter interactions could allow a frameshifting ribosome to translate through a structurally disrupted upstream FSE that no longer blocks ribosome progression.

 

Two-dimensional drawing of nucleic acid structures, particularly RNA structures, is fundamental to the communication of nucleic acids research. However, manually drawing structures is laborious and infeasible for structures thousands of nucleotides long. RNAcanvas automatically arranges residues into strictly shaped stems and loops while providing robust interactive editing features, including click-and-drag layout adjustment. Drawn elements are highly customizable in a point-and-click manner, including colours, fonts, size and shading, flexible numbering, and outlining of bases. Tertiary interactions can be drawn as draggable, curved lines. Leontis-Westhof notation for depicting non-canonical base-pairs is fully supported, as well as text labels for structural features (e.g., hairpins). RNAcanvas also has many unique features and performance optimizations for large structures that cannot be correctly predicted and require manual refinement based on the researcher’s own analyses and expertise. To this end, RNAcanvas has point-and-click structure editing with real-time highlighting of complementary sequences and motif search functionality, novel features that greatly aid in the identification of putative long-range tertiary interactions, de novo analysis of local structures, and phylogenetic comparisons. For ease in producing publication quality figures, drawings can be exported in both SVG and PowerPoint formats. URL: https://rnacanvas.app 

Two-dimensional drawing of nucleic acid structures, particularly RNA structures, is fundamental to the communication of nucleic acids research. However, manually drawing structures is laborious and infeasible for structures thousands of nucleotides long. RNAcanvas automatically arranges residues into strictly shaped stems and loops while providing robust interactive editing features, including click-and-drag layout adjustment. Drawn elements are highly customizable in a point-and-click manner, including colours, fonts, size and shading, flexible numbering, and outlining of bases. Tertiary interactions can be drawn as draggable, curved lines. Leontis-Westhof notation for depicting non-canonical base-pairs is fully supported, as well as text labels for structural features (e.g. hairpins). RNAcanvas also has many unique features and performance optimizations for large structures that cannot be correctly predicted and require manual refinement based on the researcher's own analyses and expertise. To this end, RNAcanvas has point-and-click structure editing with real-time highlighting of complementary sequences and motif search functionality, novel features that greatly aid in the identification of putative long-range tertiary interactions, de novo analysis of local structures, and phylogenetic comparisons. For ease in producing publication quality figures, drawings can be exported in both SVG and PowerPoint formats. URL: https://rnacanvas.app.

 

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3′ structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3′ cap-independent translation enhancers (3′CITEs), as umbraviruses can have different 3′CITEs in the central region of their lengthy 3′UTRs, and most also have a particular 3′CITE (the T-shaped structure or 3′TSS) near their 3′ ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3′CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3′CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3′CITEs that differentially affect the structure and translation of different umbraviruses. 

The 3′ untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3′UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3′ cap-independent translation enhancers (3′CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3′TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3′ terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed “Trio”, that are exclusively found in a subset of umbraviruses and are located just upstream of the 3′TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3′ terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3′CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement. 

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. 

ABSTRACT Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae . OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA D ). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3′ untranslated region of OPMV contains two 3′ cap-independent translation enhancers (3′ CITEs), a T-shaped structure (TSS) near its 3′ end, and a Barley yellow dwarf virus -like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5′ sequences in vivo , which differs from how umbravirus 3′ CITEs were used in a previous study. Similarly to most 3′ CITEs, the OPMV BTE links to the 5′ end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae . We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5′ end just upstream from a previously predicted xrRNA D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo . We also characterized the OPMV BTE structure, a 3′ cap-independent translation enhancer (3′ CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3′ CITEs. 

It is with great sadness and sympathy for his family and the plant virology community that we convey the passing of Michael Goodin unexpectedly in December 2020 [...]