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. 2022 Apr 13;96(7):e0173621.
doi: 10.1128/jvi.01736-21. Epub 2022 Mar 17.

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

Jingyuan Liu  1 , Anne E Simon  1
Affiliations

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

Jingyuan Liu et al. J Virol. .

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.

Keywords: 3′CITEs; RNA structure; eIF4G-binding structure; noncanonical translation; translation enhancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Structures in CYVaV domain 3. (A) Genome organization of umbravirus PEMV2 and three class 2 ulaRNAs. ORF1 and the −1PRF extension of OR1 (ORF2) encode replication-required proteins including the p81 RdRp. OULV and FULV2 contain an additional ORF (ORF5) that contains motifs found in some movement proteins (30). CYVaV has two deletions in the analogous ORF5 region that, along with additional changes, eliminate translation of the ORF. (B) Structure of full-length CYVaV. The three domains (D1, D2, and D3) are indicated. Green asterisk denotes location of the start codon for the p21 ORF1 and the two red asterisks denote locations of stop codons for ORF1 and ORF2. The bridge stem at the base of D2 that juxtaposes D1 and D3 is indicated. Numbers are from reference and refer to specific secondary structure elements. (C) Structure and sequence of CYVaV D3. Residues are colored according to their SHAPE reactivity (30). Pseudoknot 1 (ψ1) is shown. Names of other structures are from reference . Line drawings at right are the putative structures for the same region in FULV2 and OULV determined by comparative modeling based on the CYVaV D3 structure. An extra segment in FULV2 D3, which is not found in other class 2 ulaRNAs, is bracketed. Inset at right is the 3′ end of PEMV2. The three hairpins and two pseudoknots that comprise the 3′TSS 3′CITE are shown.
FIG 2
FIG 2
Effect on translation of CYVaV 3′ UTR deletions. (A) Names of deletion mutants and the deleted regions are shown. The positions of RNA structures in CYVaV D3 are indicted on top. Portions included in the constructs are denoted by dark gray lines. (B) In vitro translation of full-length and deletion mutants of CYVaV in WGE. Positions of p21 and p81 are shown. Average values and standard deviations of p21 and p81 translation levels were obtained from three independent experiments and are normalized to WT p21 and p81 levels.
FIG 3
FIG 3
S14 is an eIF4G-binding 3′CITE. (A) Base conservation in class 2 ulaRNA S14. Conserved residues are indicated, with dark green and light green denoting conservation in eight or seven ulaRNAs, respectively. The ulaRNAs used for this alignment were CYVaV (JX101610), CYVaV-Delta (MT893741), CYVaV-RioBlanco (MT893740), OULV (MH579715), FULV1 (MW480892), FULV2 (MW480893), and Ethiopian maize-associated virus 1 and 2 (EMaV1/2, MF415880 and MN715238). Features referred to in the text are indicated. (B) Mutation analysis of CYVaV S14. Full-length CYVaV WT and mutant templates (mutations in red) were subjected to in vitro translation in WGE. Numbers in black denote levels of p21 and p81 obtained in WGE. Selected constructs were also assayed for accumulation in protoplasts (in green; A, accumulation). For both assays, values are presented as a percentage of WT with standard deviations obtained from three independent experiments. End points of the ISSLSΔB fragment used in panels C and D are indicated. (C) Trans-inhibition assay. Wild-type and mutant S14 fragments (ISSLS: positions 2452 to 2559, ISSLSΔB: positions 2484 to 2532) were added in a 10- or 25-fold molar excess along with full-length CYVaV gRNA template to WGE. Values are a percentage of the levels of p21 and p81 obtained in the reaction with no added fragments (lane -) from three independent experiments with standard deviations. (D) EMSAs using 2 nM radiolabeled RNA fragments. OPMV BTE (binds to eIF4G and eIF4F) and PEMV2 PTE (binds to eIF4E and eIF4F) were included as controls. Fragments were incubated with 200 nM BSA or 200/400 nM wheat eIF4F, eIF4G, or eIF4E at 30°C for 15 min and then exposed to UV light for 15 min. Eight percent SDS-PAGE was used for ISSLS and OPMV BTE, and 10% SDS-PAGE was used for ISSLSΔB and PEMV2 PTE.
FIG 4
FIG 4
ISSLS inhibition of translation in trans is not restored by addition of eI4G or eIF4F. CYVaV gRNA template and 10-fold excess OPMV BTE, CYVaV ISSLS, or ISSLSΔB fragments were added to WGE with and without 200/400 nM eIF4G or eIF4F. p21 levels are averages with standard deviations obtained from three independent experiments and are presented as a percentage of that obtained with no added RNA fragments or proteins (lane -).
FIG 5
FIG 5
CYVaV S1 promotes translation in the presence of the CYVaV 3′ UTR. (A) Secondary structure of CYVaV positions 1 to 60 and the PEMV2 5′ 89 nt. Start codons are in green. (B) Luciferase reporter construct ORFs are indicated by colored bars. Blue, CYVaV; green, PEMV2. UTRs are open rectangles the same color as the corresponding ORFs. Vector-derived sequence is denoted by a black bar. Relative luciferase activities were obtained from at least three experiments. (C) Mutations incorporated into S1 in construct C5′33+C3′U. (D) Reporter constructs containing the mutations shown in panel C were assayed for translation in protoplasts as described in panel B.
FIG 6
FIG 6
No LDI is discernible between the terminal loops of CYVaV S1 and ISSLS. (A) Base alterations in CYVaV S1 (left) and ISSLS (right) terminal loops. Mutated bases are in red and base numbers are indicated. (B) Luciferase reporter constructs used to assay translation of reporter constructs in protoplasts. See legend to Fig. 5 for details.
FIG 7
FIG 7
S1 of class 2 ulaRNAs is a translation enhancer. (A) OULV and FULV2 S1. Bases conserved with CYVaV S1 are in red. (B) Luciferase constructs containing S1 of CYVaV, OULV, or FULV2 upstream of F-luc reporter gene and the CYVaV 3′ UTR (C3′), PEMV2 3′ UTR (P3′), or vector-derived sequence (V). Data represent mean ± standard deviation from at least three independent experiments. (C) Northern blotting of WT CYVaV and CYVaV with S1 from FULV2 (CYVaVF-S1) at 18 h postinoculation. Each lane represents RNA extracted from different plants.
FIG 8
FIG 8
OULV and FULV SI can inhibit translation in WGE. (A) Trans-inhibition assay using CYVaV gRNA template and 10- or 25-fold molar excess of CYVaV, FULV2, or OULV S1. (B) Trans-inhibition assay using CYVaV gRNA template and 25-fold molar excess of CYVaV, FULV2, or OULV S1 with and without 200/400 nM eIF4G or eIF4F. p21 translation levels are averages with standard deviations of values obtained from at least three independent experiments and are presented as a percentage of that obtained with no added RNA fragments or proteins (lane -).
FIG 9
FIG 9
Structural alignments between some class 2 ulaRNAs ISSLS (left) and previously reported ISS (boxed, right). EMaV, Ethiopian maize-associated virus; MNSV, maize necrotic streak virus; MWLMV, maize white line mosaic virus; MNSV-264, melon necrotic spot virus. Bases conserved with CYVaV are in red. Circled residues denote ISS-conserved sequences. Bases in ISS that engage in long-distance pairing with 5′ sequences are shaded in blue.
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