Fig 1.
Organization of PEMV1 genome and subgenomic mRNA.
(A) Linear representation of PEMV1 genome showing encoded ORFs (grey boxes) for p0, p1, p2, coat protein (CP) and coat protein-readthrough domain (CP-RTD). Proteins translated from the genome are shown beneath it as tan and green bars. P1/2 RdRp protein is expressed via programmed -1 frameshifting within the p1 ORF. Black arrow beneath the genome indicates the transcription initiation site for the subgenomic (sg) mRNA. The black square at the 5′-end of the genome represents the VPg. (B) PEMV1 sg mRNA encoding CP and CP-RTD. Corresponding translation products are indicated below as blue bars. CP-RTD is expressed via programmed readthrough of the CP UGA stop codon. Relative positions of the proposed readthrough-regulating proximal readthrough element (PRTE) and distal readthrough element (DRTE) are shown as red circles. (C) RNA secondary structure model of full-length PEMV1 sg mRNA, as predicted by RNAStructure using default settings [34] and rendered using RNA2Drawer [57]. Labelled are the 5′ and 3′ ends, PRTE, DRTE, CP stop codon, SL1, SL3, SL4 and the pink-orange intervening (POI) domain. The red circles on the folded structure correspond to the regions circled on the linear sg mRNA in panel B. The proposed long-distance RNA-mediated interaction between PRTE and DRTE is indicated by a red double-headed arrow, and spans approximately 700 nt.
Fig 2.
SHAPE-guided RNA secondary structures of PRTE and DRTE.
(A) SHAPE-guided and alternative folds for the PRTE in PEMV1. Relative SHAPE reactivities of individual nucleotides are colour-coded (see key) in the SHAPE-guided fold (left). The CP stop codon, SL1, and SL3 are labeled. An alternative fold in which SL2 forms is shown on the right, with a double-headed arrow depicting the proposed interconversion between the two conformations. The stem of SL3 is highlighted in blue, while orange and red highlights denote sequences that have complementary partner segments in the DRTE, which is shown in panel B. (B) SHAPE-guided fold of DRTE in PEMV1. Nucleotide reactivities are colour-coded and segments complementary to red and orange RNA segments in the PRTE are indicated. (C) SL2 and SL3 PRTE equivalents predicted in citrus vein enation virus (CVEV, NC_021564), with corresponding orange, red, and blue segments highlighted.
Fig 3.
Assessing the red long-distance RNA-RNA interaction.
(A) Secondary structures for the PRTE alternative fold and DRTE in PEMV1. The intervening 668 nucleotides between SL3 and SL4 are depicted by a connecting dashed line. (B) Wheat germ extract (wge) in vitro translation assay testing wt and mutant PEMV1 sg mRNAs. In mutants sg1 and sg2, the CP start codon and the CP stop codon, respectively, were inactivated (AUG → CAG and UGA → GGA). The sg mRNAs tested are indicted above each lane and the identities of the translated viral proteins are indicated on the left. The X-designated doublet band likely represents translation initiation at internal start codons in the CP ORF, and their probable readthrough products are indicated by the arrowhead. (C) and (E) Compensatory mutations introduced in the sg mRNA to test the red interaction. Nucleotide substitutions are shown in white. (D) and (F) In vitro translation analyses of the sg mRNAs shown in panels C and E, respectively. Average relative readthrough (Rel. RT) levels (±SE) calculated from three independent trials are shown below each lane. (G) Northern blot analysis of total nucleic acids isolated from pea protoplasts transfected with wt and HA-tagged mutant PEMV1 genomes. gHA, gHA7, gHA8, gHA9 and gHAns each contain a triple HA tag inserted 6 amino acids from the CP N-terminus. Tagged genomic mutants gHA7, gHA8 and gHA9 contain the same compensatory mutations as shown in panel E, and genomic mutant gHAns has the same CP stop codon knockout substitution as mutant sg2 in panel B. Substitutions in the DRTE in gHA8 and gHA9 lead to an arginine to serine amino acid change in CP-RTD. Positions of the genome (g) and sg mRNA (sg) are shown on the left side of the blot. Average sg levels (±SE) were calculated from three independent trials and are displayed below each lane. An ethidium bromide-stained rRNA loading control is shown below the Northern blot. (G) Western blot analysis of total proteins extracted from the same pea protoplast infections as in panel G. Identities of the detected viral proteins are indicated on the left and averaged Rel. RT levels (±SE) from three independent trials are shown under each lane. Ponceau S-stained loading control of the blot is shown below.
Fig 4.
Assessing the orange long-distance RNA-RNA interaction.
(A) RNA secondary structures of PRTE and DRTE in PEMV1. (B) and (D) Compensatory mutations introduced in the sg mRNA to test the orange interaction. Nucleotide substitutions are shown in red. (C) and (E) In vitro translation analyses of the wt and mutant sg mRNAs shown in panels B and D, respectively. Averaged Rel. RT levels (±SE) collected from three independent trials are shown below each lane.
Fig 5.
Assessing the local SL3 (blue) in the PRTE.
(A) RNA secondary structures of PRTE and DRTE in PEMV1. A nucleotide mono-variation (U to C) in the SL3 of four enamoviruses that maintains base pairing is shown (boxed). Alfalfa enamovirus-2 (AEV-2, KY985463.1), bean enamovirus-1 (BEnV-1, MZ361924), birdsfoot trefoil virus-2 (BFTV-2, NC_048296) and red clover enamovirus-1 (RCEV-1, MN412742). (B) SL3 of citrus vein enation virus (CVEV, NC_021564). Covariations in the SL3 stem that maintain pairing are boxed. (C) Compensatory mutations introduced into SL3, with substitutions depicted in red. (D) Results of in vitro translation reactions for the sg mRNAs shown in panel C. Averaged Rel. RT levels (±SE) calculated from three independent trials are shown below each lane. (E) Proposed RNA secondary structure when the red and orange long-distance interactions between the PRTE and DRTE occur. The linker sequence between red and blue helices is highlighted in pink, with corresponding substitutions in mutant sg mRNAs circled and indicated in red. (F) Results of in vitro translation reactions for mutant sg mRNAs shown in panel E. Averaged Rel. RT levels (±SE) calculated from three independent trials are shown below each lane.
Fig 6.
Assessing the pink long-distance RNA-RNA interaction.
(A) RNA secondary structures of PRTE and DRTE in PEMV1, including the POI domain (grey shading). The complementary sequences highlighted in pink represent a third long-distance RNA-RNA interaction between PRTE and DRTE. (B) and (D) Compensatory mutations introduced in the sg mRNA context to test the pink interaction. Nucleotide substitutions are shown in red. (C) and (E) In vitro translation analyses of the wt and mutant sg mRNAs shown in panels B and D, respectively. Averaged Rel. RT levels (±SE) collected from three independent trials are shown below each lane.
Fig 7.
Assessing SL4 and a potential fourth PRTE/DRTE interaction.
(A) RNA secondary structure model for the readthrough structure when red, orange, and pink complementary sequences are paired. (B) Conservation of SL4 in DRTEs among the members of the genus Enamovirus: PEMV1, AEV-2, BFTV-2, RCEV-1, BEnV-1, and CVEV. In the top row, mono- and co-variations that maintain base pairing of SL4 are shown in green. (C) RNA secondary structures of PRTE and DRTE in PEMV1, with substitutions targeting the green PRTE sequence and the stem of SL4 boxed and shown in red. Segments in the putative fourth PRTE/DRTE long-distance interaction are highlighted in green. (D) In vitro translation analyses of the wt and mutant sg mRNAs. The boxed area represents results from the SL4 stem compensatory mutants shown to the right in panel C. Averaged Rel. RT levels (±SE) collected from three independent trials are shown below each lane. (E) Conservation of the green pairing between the PRTE and DRTE among enamoviruses. The green PRTE sequence is 100% conserved (except for CVEV), while complementary green DRTE sequences have variations that maintain (green) or potentially destabilize (red) the base-pairing of the green partner sequences. (F) Compensatory mutations introduced in PEMV1 sg mRNA that target the long-distance PRTE/DRTE green base-pairing interaction. (G) In vitro translation analyses of the wt and mutant sg mRNAs shown in panel F. Averaged Rel. RT levels (±SE) collected from three independent trials are shown below each lane.
Fig 8.
Assessing the PRTE/DRTE interaction in PLRV.
(A) RNA secondary structure model for a central region of the PLRV sg mRNA based on RNAStructure [34] and rendered using RNA2Drawer [57]. In the structure, small black circles represent nucleotides, with those corresponding to the PLRV CP UAG stop codon shown in red. Highlighted in orange are the PRTE and DRTE sequences previously proposed to base-pair and regulate CP readthrough in PLRV [13]. (B) and (C) Compensatory mutations introduced in PLRV sg mRNA that target the orange PRTE/DRTE base-pairing interaction, and mutant PLns, in which the CP stop codon was inactivated. (D), (E) and (F) In vitro translation analyses of the wt and mutant PLRV sg mRNAs shown in panels B and C. Averaged Rel. RT levels (±SE) collected from three independent trials are shown below each lane.
Fig 9.
Comparison of PLRV and PEMV1 readthrough structures.
(A) Predicted readthrough structure of PLRV showing the key orange PRTE/DRTE interaction. (B) Proposed readthrough structure for PEMV1, including local and long-distance interactions. (C) Predicted local RNA secondary structures for PRTE and DRTE [13] regions in PLRV. Nucleotides shown in red were targeted for compensatory mutational analysis in Fig 8.
Fig 10.
Model for assembly of the PEMV1 readthrough structure.
(A) Default structures for PRTE and DRTE. The helicase activity of a terminating ribosome extends over SL1 and unfolds it. (B) (i) The sequence refolds into an alternative conformation that includes SL2. (ii) SL2 pairs with SL4 via a red sequence kissing-loop interaction and nucleates the assembly process. (C) Other key interactions then form, such as pairing of the orange partner sequences. (D) Addition of the pink interaction generates an extended helical structure, stabilized by coaxial stacking at stem junctions, that promotes efficient readthrough production of CP-RTD. A potential fourth green PRTE/DRTE interaction may also occur (not depicted), which would extend the quasi-continuous helix to the stop codon. See text for details.