Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome

Abstract

The genus Cardiovirus (family Picornaviridae) currently comprises the species Encephalomyocarditis virus (EMCV) and Theilovirus. Cardioviruses have a positive-sense, single-stranded RNA genome that encodes a large polyprotein (L-1ABCD-2ABC-3ABCD) that is cleaved to produce approximately 12 mature proteins. We report on a conserved ORF that overlaps the 2B-encoding sequence of EMCV in the +2 reading frame. The ORF is translated as a 128-129 amino acid transframe fusion (2B*) with the N-terminal 11-12 amino acids of 2B, via ribosomal frameshifting at a conserved GGUUUUY motif. Mutations that knock out expression of 2B* result in a small-plaque phenotype. Curiously, although theilovirus sequences lack a long ORF in the +2 frame at this genomic location, they maintain a conserved GGUUUUU motif just downstream of the 2A-2B junction, and a highly localized peak in conservation at polyprotein-frame synonymous sites suggests that theiloviruses also utilize frameshifting here, albeit into a very short +2-frame ORF. Unlike previous cases of programmed -1 frameshifting, here frameshifting is modulated by virus infection, thus suggesting a novel regulatory role for frameshifting in these viruses.

Fig. 1.

Conservation at polyprotein-frame synonymous sites in three cardiovirus clades. (A) Twenty-one EMCV sequences, including mengovirus. (B) Twelve TMEV and RTV sequences. (C) Twenty-two SAFV sequences. Panel 1 shows a map of the EMCV coding region, along with the overlapping gene 2B*, the site of frameshifting, and the site of StopGo cotranslational separation at the 2A-2B junction. Panels 2–3, 6–7, and 10–11 show the positions of stop codons (blue triangles) in the +1 and +2 reading frames relative to the polyprotein reading frame, and alignment gaps (green rectangles), in all the aligned sequences. Note the conserved absence of stop codons in the +2 frame in EMCV in the 2B* region (panel 3) and in the +1 frame in TMEV/RTV in the L* region (panel 6). Panels 4–5, 8–9, and 12–13 depict the conservation at polyprotein-frame synonymous sites in a 15-codon sliding window. Panels 4, 8, and 12 depict the probability that the degree of conservation within a given window could be obtained under a null model of neutral evolution at synonymous sites, whereas panels 5, 9, and 13 depict the absolute amount of conservation as represented by the ratio of the observed number of substitutions within a given window to the number expected under the null model. The range of features detected by this type of analysis depends partly on the sliding window size: 15 codons is optimal for features with a width of 15 codons and was used to illustrate the very localized conservation peak in the TMEV/RTV and SAFV alignments. In EMCV, the conservation in the 2B* region is even more pronounced if one uses a larger window size—indeed, the total conservation summed over the 2B* ORF has a corresponding p-value of approximately 10^-32. Conversely, features that cover significantly fewer than 15 codons may not be apparent in this analysis. Note that, due to codon usage, the lengths of “random” (i.e., noncoding) ORFs tend to be longer in the +1 frame than in the +2 frame (34).

Fig. 2.

Representative nucleotide and peptide sequences. (A) Nucleotide sequences flanking the proposed site of frameshifting (confirmed in EMCV) in representative cardiovirus sequences (GenBank accession numbers shown at left). The codons that encode NPG|P of the StopGo cassette are highlighted in pale blue. The slippery heptanucleotide where -1 frameshifting occurs is highlighted in pale orange. The stop codon that terminates the very short +2-frame ORF in TMEV and SAFV is indicated in red (in EMCV, the +2-frame ORF is much longer; see Fig. 1A). (B) Amino acid sequence of the transframe protein 2B* in mengovirus DQ294633. The two antigens used for Abs anti-N and anti-C are highlighted in yellow. Peptide sequences detected by mass spectrometric analysis of 2B* purified from infected cells are indicated with underscores (anti-N IP) and overscores (anti-C IP). The amino acids, VF, corresponding to the site of frameshifting are indicated in bold. (C) Amino acid sequences of the much shorter predicted transframe peptide in representative TMEV and SAFV sequences.

Fig. 3.

RNA structures predicted from alignment folding. The G GUU UUY frameshift site in EMCV and corresponding predicted frameshift site in TMEV are boxed. A potential lower extension to the stem in EMCV is indicated by underscores, but this extension is not completely conserved throughout the EMCV alignment. The EMCV CRE is also shown, with the 5′-most loop nucleotides, AAACAC, that are conserved throughout the cardioviruses highlighted in bold. Similar nucleotides in the shift-site 3′-proximal stem-loops are also highlighted in bold. Numbers represent genomic coordinates of selected nucleotides within the sequences indicated by GenBank accession numbers.

Fig. 4.

Immunodetection of 2B* and 2B. (A) 2B/2B* anti-N Western blot (WB) of protein lysates from BHK-21 cells either mock-infected (mock) or infected (MOI 10) with WT mengovirus (vMC0) for 5 h. As controls for the apparent molecular masses of 2B and 2B*, in vitro translated (IVT) 2B and 2B*—as well as a negative control (Co) with no input RNA added—were also immunoblotted. The right-hand panel shows radiolabeled in vitro translated 2B and 2B*. (B) Western blots of anti-N and anti-C immunoprecipitates prepared from mock- or vMC0-infected (MOI 1.0) BHK-21 cells as indicated by − or +. Cell lysates were prepared 8 h p.i. and incubated with IgG beads ± either anti-N or anti-C as indicated. Control cell lysates removed before the addition of beads or Abs are shown in the far right lanes marked “lysates.” The 2B/2B* N-terminal and 2B* C-terminal antigens are indicated in Fig. 2B.

Fig. 5.

Extent of the 3′ sequence that contributes to frameshift stimulation. Dual luciferase assay showing frameshifting efficiencies following transfection of WT and slip-site mutated (SS) pDluc constructs with 45, 50, or 125 nt 3′ of the slip site. Relevant IFCs were also transfected in parallel. Eighteen hours later, transfectants were either mock-infected or infected with vMC0 (MOI 2.0) for 7 h. Cells were then lysed and luciferase activities measured. Error bars represent the standard deviation of four independent experiments, in each of which each construct was transfected in quadruplicate.

Fig. 6.

Analysis of 2B* knockout mutants. (A) Schematic representation of the mengovirus region expressed by in vitro translation to test whether the SS, PTC1 or PTC2 mutations affect StopGo separation. (B) SDS-PAGE of radiolabeled in vitro translation reactions. LV has a mutation in the StopGo region (NPG|P to NPLV) that completely inhibits StopGo separation of 2B from 1D-2A. SS, PTC1, and PTC2 have the same mutations as the mutant mengoviruses of the same names (see main text). IFC is an in-frame control in which the WT slip site sequence has been mutated from G GTT TTT to A GTG TTT T so that, instead of 2B, a protein identical to transframe 2B* is translated. (C) Mean plaque diameter determined by measuring 100 plaques formed by standard plaque assay after infection with each WT or mutated mengovirus as indicated. Error bars indicate standard deviations. Inset: anti-N and anti-C Western blots of anti-N immunocomplexes on lysates prepared from BHK-21 cells infected with either WT mengovirus (MOI 1.0) for 8 h or each indicated mutated mengovirus (MOI 1.0) for 22 h. (D) Pulse labeling of vMC0, vMC0-SS, vMC0-PTC1, vMC0-PTC2 (MOI 10), and mock-infected BHK-21 cells at 8 and 22 h p.i.. The positions of the major mengovirus proteins are shown to the left. Arrowheads indicate positions of host proteins that are absent or decreased in cells infected with vMC0 when compared to mock-infected cells. There were no labeled proteins in the WT lane at 22 h p.i. because cells infected with vMC0 for 22 h had already undergone CPE.

Fig. 7.

Partial reversion of the mutated slip site restores 2B* expression and plaque size. (A) Anti-N Western blot of lysates prepared from BHK-21 cells infected for 8 h (MOI approximately 10) with either WT or the mutant mengoviruses (SS, PTC1, and PTC2) as indicated. P1 indicates the first passage from a virus stock, whereas P10 and P20, respectively, indicate 10 and 20 serial passages of a virus stock. A higher exposure is shown for a region below 10 kDa, showing a PTC2-specific product that may represent the approximately 7.1-kDa truncated 2B*. (B) Mean plaque diameter determined by measuring 100 plaques formed by standard plaque assay after infection with WT mengovirus, or slip-site mutated mengovirus that had been serially passaged once (P1) or eight times (P8) as indicated. Error bars indicate standard deviations.

Fig. P1.

Map of the EMCV genome, showing the polyprotein L-1ABCD-2ABC-3ABCD, the overlapping gene 2B*, and the site of programmed ribosomal frameshifting.