![Detection of CP-RT in vitro and in vivo
(A) Schematic representation of sgRNA4 and its UGG and 2st mutant clones under a T7 promoter. (B) SDS-PAGE of proteins translated in wheatgerm extracts from in vitro transcripts of sgRNA4 and its two mutants. Mock–no RNA added. After drying the gel, proteins radioactively labelled with [³⁵S]Met were detected using a phosphorimager. (C) Schematic representation of the AV2 RNA3 with a tag (grey rectangle) appended to the 3′ end of the CP-RT gene. (D) Detection of CP and CP-RT-myc by western immunoblotting in plants infected with AV2 or AV2-myc. Samples were collected from the inoculated leaf and the 2nd upper non-inoculated leaf at 4 and 7 dpi, and the 3rd upper non-inoculated leaf (see Fig 3B) at 14 dpi. (E) Detection of CP and CP-RT-myc by western blotting in extracts of plants infected with AV2, AV2-myc or AV2-2st-myc. Samples were collected from the 2nd upper non-inoculated leaf at 4 dpi (see Fig 3B). Positions of CP and CP-RT-myc are indicated on the left. Sizes of molecular weight markers are indicated on the right in panels B, D and E. Ponceau red staining of the large Rubisco subunit (RbcL) was used as a loading control in panels D and E.](https://www.researchgate.net/publication/381007662/figure/fig4/AS:11431281251400965@1718224019140/Detection-of-CP-RT-in-vitro-and-in-vivo-A-Schematic-representation-of-sgRNA4-and-its_Q320.jpg)
May 2024
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65 Reads
Ilarviruses are a relatively understudied but important group of plant RNA viruses that includes a number of crop pathogens. Their genomes comprise three RNA segments encoding two replicase subunits, movement protein, coat protein (CP), and (in some ilarvirus subgroups) a protein that suppresses RNA silencing. Here we report that, in many ilarviruses, RNA3 encodes an additional protein (termed CP-RT) as a result of ribosomal readthrough of the CP stop codon into a short downstream readthrough (RT) ORF. Using asparagus virus 2 as a model, we find that CP-RT is expressed in planta where it functions as a weak suppressor of RNA silencing. CP-RT expression is essential for persistent systemic infection in leaves and shoot apical meristem. CP-RT function is dependent on a putative zinc-finger motif within RT. Replacing the asparagus virus 2 RT with the RT of an ilarvirus from a different subgroup restored the ability to establish persistent infection. These findings open up a new avenue for research on ilarvirus silencing suppression, persistent meristem invasion and vertical transmission.
![Mutational analysis of the cucumber mosaic virus (CMV) 2b protein. (a) Previously determined or inferred functional residues or domains of the 2b protein are indicated on a map of the 110 amino acid Fny-CMV 2b orthologue. Sequences longer than one amino acid are depicted as gray boxes: the N- and C-terminal domains; the RNA-binding domain; the KSPSE phosphorylation sequence; the GSEL and RHV sequences; and the nuclear export sequence (NES). However, the N-terminal MEL sequence and nuclear localization sequence (NLS) 1 and 2 are shaded in darker gray to indicate that they overlap with the N-terminal and RNA-binding domains, respectively. Single amino acid residues with known biological effects are indicated by arrowheads: S28, which is an additional phosphorylation site [22]; L55, which is required for 2b self-interaction [23]; and E104, which causes cytotoxicity in E. coli due to DNA binding [24], suggesting a biological function. The biological roles of sequences or residues are indicated on the left. The region spanning residues 38 to 94 of the 2b protein, inferred to contain amino acid residue(s) required for interaction with Argonaute 1 (AGO1), is indicated by a dashed line and was proposed by Duan et al. [21]. The map is updated from a previous iteration [11] to include more recent information [22]. (b) Deletion mutations are indicated by gray lines below the amino acid sequences for the Fny-CMV and LS-CMV 2b proteins, while insertions or substitution mutations are indicated above. Numbers refer to the residues of the 110 amino acids in the Fny-CMV 2b protein. Mutant 2b proteins were truncated from the N-terminus of the first 17 (2bΔ1–17) or 55 (2bΔ1–55) amino acids. Truncations were also made from the C-terminus, deleting 16 (2bΔ95–110), 26 (2bΔ85–110), 28 (2bΔ83–110), 37 (2bΔ74–110), 42 (2bΔ69–110), 46 (2bΔ65–110), 50 (2bΔ61–110), and 55 (2bΔ56–110) amino acids. In-frame internal deletions were also made between residues 83 to 93 (2bΔ83–93), 56 to 65 (2bΔ56–65), 56 to 60 (2bΔ56–60), and 39 to 48 (2bΔ39–48). Alanine substitution mutations were also made to replace the Fny-CMV 2b sequence from residues 56 to 65 with ten alanine residues (2b56aaa65) and residues 56 to 60 with five alanine residues (2b56aaa60). Two chimeric 2b proteins were generated. In 2bFny/LS(56–60), the mutations Y58H and Q59G were introduced into the Fny-CMV 2b sequence to recapitulate the sequence of the LS-CMV 2b orthologue between residues 56 and 60. The chimeric protein 2bLS/Fny(83–93) was created by introducing residues 83–93 from the Fny-CMV 2b protein into the LS-CMV 2b protein background. Note that the wild-type LS-CMV 2b protein does not contain a corresponding sequence.](https://www.researchgate.net/publication/380145733/figure/fig1/AS:11431281247108937@1716611426966/Mutational-analysis-of-the-cucumber-mosaic-virus-CMV-2b-protein-a-Previously_Q320.jpg)
![Subcellular localization of mutant 2b proteins and the full-length 1a protein. Using agroinfiltration, C-terminal RFP or GFP fusion proteins derived from the full-length 2b protein (2b-RFP) or mutant 2b proteins lacking residues 1–17, 95–110, 85–110, 56–110, 83–93, 56–60, or substitutions LS/Fny (83–93), 56aaa60, or Fny/LS (56–60) were co-expressed with 1a-RFP or 1a-GFP fusion proteins in N. benthamiana leaves in the combinations shown. Fluorescent signals were imaged using confocal scanning laser microscopy. Fny 2b-RFP accumulated in the nucleus and cytoplasm, with a proportion co-localizing with the 1a-GFP (merged signal shown as yellow and typical examples indicated with white arrows in the top left image), consistent with previous results [19]. Deletion of the N-terminal 17 residues (1–17) in the 2b sequence or the 15 C-terminal residues (95–110) did not disrupt the co-localization of 2b and 1a proteins. Deletions from 85–110 and 56–110 resulted in an apparent loss of co-localization with the 1a protein. Smaller in-frame deletions of residues 83–93 and 56–60 also both resulted in an apparent loss of co-localization between mutant 2b proteins and the 1a protein.](https://www.researchgate.net/publication/380145733/figure/fig2/AS:11431281247108938@1716611427730/Subcellular-localization-of-mutant-2b-proteins-and-the-full-length-1a-protein-Using_Q320.jpg)
![Pairwise sequence comparisons of PvEV1, 2 and 3 using sequences derived from the helicase region. Comparisons conducted in Sequence Demarcation Tool revealed clear demarcation between the three species. In this analysis, two comparator sequences (PvEV1 Nairobi MH567335 and PvEV1 Kirinyaga MH567348) previously identified as PvEV1 [31] and three PvEV2 sequences PvEV2 Nairobi 1 MH567336 and PvEV2 Nairobi 2 MH567339 [31] and PvEV2 Zambia MW534366 (downloaded from GenBank) were used to confirm the accuracy of the demarcation. Intraspecies sequence comparisons (Red) were PvEV1 95.7–100%, PvEV2 96.4–99.3%, and PvEV3 95.5–100%. Interspecies similarity (Blue) ranged between 54.4 and 64.7% when PvEV1 and PvEV2 were compared, 59.4–67.4% between PvEV1 and PvEV3 and 53.6–70.5% between PvEV2 and PvEV3](https://www.researchgate.net/publication/373046068/figure/fig2/AS:11431281188856410@1694796988198/Pairwise-sequence-comparisons-of-PvEV1-2-and-3-using-sequences-derived-from-the-helicase_Q320.jpg)
