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Proc Natl Acad Sci U S A. Apr 15, 2008; 105(15): 5897–5902.
Published online Apr 11, 2008. doi:  10.1073/pnas.0800468105
PMCID: PMC2311343
Microbiology

An overlapping essential gene in the Potyviridae

Abstract

The family Potyviridae includes >30% of known plant virus species, many of which are of great agricultural significance. These viruses have a positive sense RNA genome that is ≈10 kb long and contains a single long ORF. The ORF is translated into a large polyprotein, which is cleaved into ≈10 mature proteins. We report the discovery of a short ORF embedded within the P3 cistron of the polyprotein but translated in the +2 reading-frame. The ORF, termed pipo, is conserved and has a strong bioinformatic coding signature throughout the large and diverse Potyviridae family. Mutations that knock out expression of the PIPO protein in Turnip mosaic potyvirus but leave the polyprotein amino acid sequence unaltered are lethal to the virus. Immunoblotting with antisera raised against two nonoverlapping 14-aa antigens, derived from the PIPO amino acid sequence, reveals the expression of an ≈25-kDa PIPO fusion product in planta. This is consistent with expression of PIPO as a P3-PIPO fusion product via ribosomal frameshifting or transcriptional slippage at a highly conserved G1-2A6-7 motif at the 5′ end of pipo. This discovery suggests that other short overlapping genes may remain hidden even in well studied virus genomes (as well as cellular organisms) and demonstrates the utility of the software package MLOGD as a tool for identifying such genes.

Keywords: P3, PIPO, Potyvirus, Turnip mosaic virus, frameshift

The genomes of many viruses are under strong selective pressure to compress maximal coding and regulatory information into minimal sequence space. Overlapping coding sequences (CDSs) are particularly common in such viruses, although they also occur in more complex genomes, including eukaryotes (1). Such CDSs can be difficult to detect using conventional gene-finding software (2), especially when short. The software package MLOGD, however, was designed specifically for locating short overlapping CDSs in sequence alignments and overcomes many of the difficulties with alternative methods (2, 3). MLOGD includes explicit models for sequence evolution in double-coding regions as well as models for single-coding and noncoding regions. It can be used to predict whether query ORFs are likely to be coding, via a likelihood ratio test, where the null model comprises any known CDSs, and the alternative model comprises the known CDSs plus the query ORF. We have been using MLOGD to search for new virus-encoded genes.

The family Potyviridae is the largest plant virus family and includes ≈30% of known plant viruses (4, 5). The genomic RNA is positive sense and ≈10 kb long. To date, all experimental and sequencing evidence has supported the existence of only one functional ORF in the six Potyviridae genera, with the exception of the genus Bymovirus, in which the ORF is divided between two genomic RNAs (5). The ORF encodes a large (340- to 395-kDa) polyprotein that is cleaved into ≈10 mature proteins (Fig. 1; reviewed in ref. 6). The genomic RNA has a covalently linked 5′-terminal protein (VPg) and a 3′ polyA tail. Cap-independent translation is mediated by the 5′UTR (7). Subgenomic transcripts are apparently not produced (8). Such a polyprotein expression strategy is common to many virus families, most notably the Picornaviridae. The potential for functional short overlapping ORFs in such viruses has been largely overlooked. However, when we applied MLOGD to the Potyviridae, we found evidence for an overlapping CDS within the P3 cistron. Here, we describe the bioinformatic evidence and experimental verification for the new CDS.

Fig. 1.
TuMV genome map (9,835 nt). In the plasmid p35STuMV-GFP, GFP is fused between P1 and HC-Pro. The position of the overlapping CDS, pipo, and the G2A6 motif are indicated.

Results

Bioinformatics.

The new CDS, pipo (Pretty Interesting Potyviridae ORF), was first identified in an alignment of Turnip mosaic virus (TuMV) (9) sequences, using the gene-finding software MLOGD (3). MLOGD has been tested extensively, using thousands of known virus CDSs as a test set, and it has been shown that, for overlapping CDSs, a total of just 20 independent base variations are sufficient to detect a new CDS with ≈90% confidence. The input alignment comprised the GenBank RefSeq NC_002509 and 20 of its genome neighbors (i.e., other TuMV genome sequences of RefSeq quality but not identical to the chosen RefSeq). In TuMV (9,835 nt; polyprotein coords 131..9625), pipo has coords 3079..3258 (60 codons; +2 frame relative to polyprotein; Fig. 1), which places it within the P3 cistron (coords 2591..3655). The total number of independent base variations within pipo is ≈92, thus providing MLOGD with a robust signal. The MLOGD output showed that pipo has a strong coding signature and the ORF is present across the alignment.

Further inspection revealed that the ORF (≥60 codons; +2 frame relative to polyprotein) is present in all 48 Potyvirus RefSeqs in GenBank. MLOGD, applied to the 48-RefSeq alignment, detected a very strong coding signature in the ORF, with ≈2,000 independent base variations now available to MLOGD. Scores outside of the ORF are negative, whereas, within the ORF, there are at least five nonoverlapping and hence completely independent high-positively scoring windows (Fig. 2).

Fig. 2.
(Upper) MLOGD sliding-window plot for an alignment of the 48 GenBank Potyvirus RefSeqs (polyprotein region only; CLUSTALW (10) amino acid alignment back-translated to nucleotide sequence). Reference sequence, ...

Starting from a highly conserved G1-2A6-7 motif (Fig. 3), there are no pipo-frame termination codons in any of the 48 RefSeqs for at least 60 codons. In 29 of 48 RefSeqs, there is an AUG codon 9 codons 3′ of the motif. Of the remaining 19 RefSeqs, 1 has an upstream AUG codon, 15 have downstream AUG codons (but only 3 are within 10 codons), and 4 have no AUG codon at all. Thus, AUG-initiation is unlikely. In 16 of 48 RefSeqs, the G1-2A6-7 motif begins with a pipo-frame stop codon, UGA, instead of GGA, thus precluding 5′ extension of the ORF. There is no well conserved termination codon position at the 3′ end—with ORF lengths (from the G1–2A6–7 motif) ranging from 60 to 115 codons. Of relevance to possible leaky scanning, pipo is preceded by many initiation codons, e.g., 57 AUG codons lie between the polyprotein AUG and pipo in the TuMV genome, of which 24 are in the polyprotein frame.

Fig. 3.
Pipo sequence. (A) Extract of 9 RefSeqs from an alignment of the 48 Potyvirus GenBank RefSeqs, showing the region around the 5′ end of pipo, which coincides with the annotated highly conserved G1-2A6-7 motif (see Fig. S8 for the full 48-RefSeq ...

Application of National Center for Biotechnology Information blastp (word size 2; virus RefSeq db) to PIPO revealed no similar amino acid sequences in GenBank, whereas tblastn identified only the pipo region in other Potyvirus sequences (as expected). Hydrophobicity plots are broadly similar for the N-terminal ≈60 aa: a hydrophobic peak followed by a hydrophilic peak and then a narrower hydrophobic peak. PIPO is rich in the basic residues Arg and Lys: Averaged over the 48 RefSeqs, their relative abundances are, respectively, 2.1 and 1.5 times higher in PIPO than in the polyprotein. Although predicted RNA secondary structures were found in individual RefSeqs, no widely conserved secondary structures were found in the vicinity of pipo [using alidot (11)].

There are four other Potyviridae genera with genomic RefSeqs in GenBank: Bymovirus (four RefSeqs × two RNA segments); Ipomovirus (one RefSeq), Rymovirus (three RefSeqs) and Tritimovirus (three RefSeqs). With the exception of Rymovirus and Potyvirus, the genera are highly divergent [typically 33–45% nucleotide identity between genera within the P3 cistron (12)]. In the genus Bymovirus, the longer genomic RNA (RNA1; ≈7,500 nt) contains a single polyprotein CDS that codes for the C-terminal eight proteins of the Potyvirus polyprotein—thus P3 becomes the N-terminal protein. In the P3 cistron, there is a G(G/C)A6 motif followed by a 74- to 88-codon +2 frame ORF, for which MLOGD detects a strong coding signature. In Tritimovirus, there is an A2G2A6-7 motif within the P3 cistron followed by a 134- to 137-codon +2 frame ORF, for which MLOGD again detects a strong coding signature. Similar results hold for an alignment of the Tritimovirus Wheat streak mosaic virus (WSMV) RefSeq with its four genome neighbors. In Rymovirus, there is a GA6GA motif within the P3 cistron followed by a 104- to 116-codon +2 frame ORF for which MLOGD detects a good coding signature. In Ipomovirus, there is a GA7 motif in the P3 cistron followed by a 99-codon +2 frame ORF. It is noteworthy that the frame of the G1-2A6-7 motif is not fully conserved (Fig. 3). In Potyvirus, the frame is generally (G) GAA AAA A(A) (spaces separate the polyprotein codons). In Bymovirus, the frame is GGA AAA AA. In Tritimovirus, two RefSeqs have G GAA AAA A, whereas the other has GGA AAA AAA. Further MLOGD results, GenBank accession numbers for all sequences used, and comparisons of the sizes and sequence divergences of P3 and PIPO are included in supporting information (SI) Figs. S1–S7 and Tables S1 and S2.

PIPO Knockouts.

To investigate whether pipo is an essential gene, we introduced mutations into an infectious clone of TuMV (p35STuMV-GFP) that expresses GFP for easy detection of virus-infected tissue. Two PIPO knockout mutants were generated. Each differs from WT GFP-expressing TuMV by just a single point mutation: GAC → GAU and UCG → UCU at NC_002509 coordinates 3103 and 3130, respectively (polyprotein-frame codons shown). These mutations are synonymous with respect to the polyprotein frame, but introduce premature termination codons (UGA) into pipo. The altered codon usage in the polyprotein ORF is not a factor because the new codons occur in the TuMV genome at frequencies similar (within 20%) to those they replace.

Nicotiana benthamiana plants were inoculated by bombardment with gold particles coated with p35STuMV-GFP DNA or the mutant derivatives or with water as a control. Each construct was bombarded into four plants. None of the eight plants bombarded with DNA containing mutations in PIPO showed any sign of infection up to 12 days postinoculation (dpi) (Fig. 4). There was no green fluorescence, and plants were as tall and healthy as uninoculated controls. In contrast, three of four WT-infected plants showed extensive infection (GFP fluorescence, stunted growth, and wilting) over the same time period. (The sole uninfected plant was bombarded with a smaller amount of DNA than the three infected ones and those bombarded with mutant TuMV DNAs.) RT-PCR with primers specific to p35STuMV-GFP, using lysate from infected plants at 12 dpi, confirmed that TuMV RNA was present only in WT-infected plants (Fig. 5).

Fig. 4.
N. benthamiana 12 days postinoculation (dpi). (Upper) Natural light. (Lower) UV light. From left: plants bombarded with (1) no DNA; (2) WT p35STuMVGFP virus (i.e., TuMV with GFP fused between P1 and HC-Pro); (3) and (4) p35STuMV-GFP PIPO knockout mutants ...
Fig. 5.
Detection of TuMV accumulation in plants by RT-PCR. Primers specific to p35STuMV-GFP were used to amplify cDNAs from RNA isolated from plants infected with WT TuMV (p35STuMV-GFP; lane 2), the two PIPO knockout mutants, p41 and p68 (lanes 2 and 3 respectively), ...

Immunoblotting.

Separate antisera were raised against two predicted 14-aa antigen sites within PIPO (AS 2–15: amino acids 2–15; AS 39–52: amino acids 39–52; Fig. 3C). Both antisera detected ≈25-kDa products in lysates from WT-infected plants (Fig. 6A). Neither antiserum detected an ≈6- to 7-kDa product, which is the predicted size of PIPO. Nonetheless, AS 39–52 clearly revealed an ≈7-kDa product generated by in vitro translation of an artificial transcript encoding only pipo (Fig. 6B) (pipo nucleotide sequence preceded by T7 promoter and followed by FLAG epitope; presumably initiates at the AUG codon 9 codons 3′ of the G2A6 motif, resulting in a 7-kDa product lacking the AS 2–15 antigen) demonstrating that AS 39–52, at least, would detect such a product if it were expressed in vivo.

Fig. 6.
Immunoblot detection of PIPO products. (A) An approximately 25-kDa PIPO fusion product. AS 2–15, antiserum against PIPO N-terminal peptide (amino acids 2–15). AS 39–52, antiserum against PIPO C-terminal peptide (amino acids 39–52). ...

Discussion

Possible Translational Mechanisms.

The detection with both antisera of an ≈25-kDa product and the absence of an ≈6- to 7-kDa product, appears to rule out independent ribosome entry into pipo (e.g., via an IRES or shunting). We propose that the G1-2A6-7 motif facilitates ribosomal frameshifting or transcriptional slippage (13), allowing expression of PIPO as a fusion product with the N-terminal region of P3 (hereafter P3N). In fact the predicted size of PIPO fused with P3N is 25.3 kDa, in good agreement with the observed ≈25-kDa band.

Many viruses harbor sequences that induce a portion of ribosomes to shift −1 nt and continue translating in the new (equivalent to +2) reading-frame (13). The −1 frameshift site consists of a slippery heptanucleotide typically fitting the consensus motif N NNW WWH. This is followed by a highly structured region, usually a pseudoknot, beginning 5–9 nt downstream (14). The G1-2A6-7 motif may serve as a slippery heptanucleotide (G GAA AAA, A AAA AAH or G AAA AAA, allowing for G:U repairings) in 47 of the 60 RefSeqs. However, although we could find potential downstream stimulatory RNA secondary structures in some RefSeqs, we could not find widely conserved structures. It remains to be seen whether some other process (e.g., long-range base pairing or the nascent P3 peptide sequence acting within the ribosomal exit tunnel) may, in this case, stimulate ribosomal frameshifting. We were unable to observe any frameshift product from in vitro translation of a construct comprising (in order) the T7 promoter, the TuMV 5′UTR, the TuMV P3 cistron with artificial in-frame initiation (AUG) and termination (UAA) codons, and the TuMV 3′UTR (Fig. S11 and data not shown) suggesting that, if ribosomal frameshifting occurs in TuMV, it may require distal sequence elements (cf. ref. 15).

Alternatively, the G1–2A6–7 motif may allow transcriptional slippage, whereby the viral polymerase “slips” on a region of repeated identical nucleotides, resulting in the insertion (or deletion) of extra nucleotides in a portion of transcripts. Transcriptional slippage has been documented in the Paramyxoviridae (e.g., Sendai virus) and the Filoviridae (e.g., Ebola virus) (16). In contrast to the Paramyxoviridae slippage site (generally A5–6G2–5 or A2GAG4–7), in the Potyviridae the Gs are 5′ of the As, which would appear to favor nucleotide deletions rather than insertions (because of the possibility of G:U repairings but not A:C repairings)—although insertional slippage on A7 is also possible [as in Ebola virus (17)]. To produce transcripts that allow PIPO expression, a 2-nt deletion or 1-nt insertion would be required. Mass spectrometry of cDNAs (nominally 62 nt, covering the G1-2A6-7 motif) derived from RNA extracted from an N. benthamiana plant infected with p35STuMV-GFP (Fig. 4) failed to detect transcripts with deletions or insertions (data not shown). Nonetheless, it is possible that some Potyviridae species or genera use transcriptional slippage, whereas others use ribosomal frameshifting [cf. DnaX (18)]. If so, then presumably either the sequence difference somehow prevents encapsidation of the modified RNAs or the slippage frequency is sufficiently low, so modified RNAs are not detected or have been disregarded as sequencing errors.

In any case, it is clear that the G1-2A6-7 motif is highly constrained. The polyprotein-frame codons—GAA and AAA (Fig. 3)—both have twofold degeneracy (GAA and GAG code for Glu; AAA and AAG code for Lys), yet AAG is never used at this location, and GAG is only used in two RefSeqs with motifs GAGA5 and GAGA6. Similarly, a WSMV mutant with a single point mutation G GAA AAA A → G GAG AAA A (synonymous in the polyprotein frame) is unable to infect plants systemically (19). Three other point mutations in the vicinity (at −6, +9, and +18 nt, respectively) did not affect infectivity (19).

Potential Protein Function.

Data published by Choi et al. (19) (see also ref. 20) provide interesting evidence for the function of PIPO. The authors constructed six WSMV mutants, each containing 10–16 synonymous (polyprotein frame) point mutations spaced along the 3′ half of the P3 cistron. All mutants had the same phenotype: (i) the ability to replicate was retained at 22–80% of WT; and (ii) the virus was able to establish infection foci limited to small clusters of cells that increased in size only slightly by 5 dpi, whereas infection foci produced by WT virus were much larger at 3 dpi and had coalesced by 5 dpi, as indicated in histochemical GUS assays. The authors postulated that the mutations disrupted an important RNA secondary structure involved in movement. However, we could find no evidence for a more widely conserved RNA secondary structure. In fact, all six mutants contain many nonsynonymous mutations in the pipo reading-frame. Importantly, a seventh mutant with synonymous mutations in the downstream CI cistron (3′ of the pipo termination codon) had WT phenotype. In contrast to the mutants described in ref. 19, no infection at all was detected for our TuMV PIPO knockout mutants (although local infection limited to just small clusters of cells may not have been apparent). Possible roles for PIPO include replication, movement, suppression of systemic silencing, or a combination of functions.

The P3 protein, whose cistron pipo overlaps (Fig. 1), is a ≈42-kDa nonstructural protein and is one of the least well characterized Potyvirus proteins. It may be involved in virus replication, pathogenicity, resistance breaking, and systemic infection (6, 9, 2123). It is interesting that previous investigations into the function and localization of P3 have produced conflicting results (6). Such discrepancies may in fact be a result of antibodies raised against P3 being sensitive also to P3N+PIPO in some studies but not in others. A survey of published material using antibodies against P3 produced no conclusive evidence either for or against PIPO (see Table S4). Furthermore, some phenotypes linked to mutations in the P3 cistron may result from alterations to P3N+PIPO or its translational mechanism rather than P3 itself. For example, a key virulence determinant of Zucchini yellow mosaic potyvirus has been mapped to the polyprotein-frame codon immediately 5′ of the GAA AAA A motif (24), mutations of which could affect the level of P3N+PIPO production.

Conclusions

We have demonstrated the existence of a new coding sequence, pipo, in the Potyviridae—a vast and extremely diverse family of plant viruses, many of which are of great agricultural significance (5). Evidence includes: (i) the ubiquitous presence of an ORF (≥60 codons) in all 60 Potyviridae GenBank RefSeqs; (ii) the ubiquitous presence of a strong MLOGD coding signature for this ORF; (iii) WSMV mutants in ref. 19 that disrupted PIPO but did not change the polyprotein amino acid sequence were unable to establish systemic infection, whereas mutants that did not disrupt PIPO had WT phenotype; (iv) our two TuMV PIPO knockout mutants, each differing from WT by a single point mutation synonymous in the polyprotein frame, were noninfectious; and (v) an ≈25-kDa product was detected in lysate from TuMV-infected plants by two separate antisera raised against different 14-aa domains in PIPO. The combination of our results (Potyvirus) with reinterpreted results from ref. 19 (Tritimovirus) gives broad phylogenetic support for these observations. Future work will address the mechanism by which pipo is translated and the function(s) of the PIPO fusion protein.

The Potyviridae have been grouped into the picorna-like virus superfamily, a loose grouping of single-stranded positive-sense RNA viruses that includes Picornaviridae, Caliciviridae, Comoviridae, Dicistroviridae, Potyviridae, and Sequiviridae (8). Overlapping and frameshift CDSs are almost unknown in the superfamily. Notable exceptions include Theilovirus (Picornaviridae) (25), Acyrthosiphon pisum virus (unclassified picorna-like virus) (26), and the Caliciviridae (27, 28) (see SI Text for further details). Identification of these CDSs was no doubt aided by their size (all are > 100 codons). The discovery of pipo, a short CDS embedded deep within the polyprotein cistron, raises the question of how many other such CDSs await discovery. We have demonstrated the utility of MLOGD as a tool to search for such “difficult” CDSs in all virus genomes.

Materials and Methods

Plasmids.

For in vivo translation, three plasmids were used: p35STuMV-GFP, p41, and p68. p35STuMV-GFP (29) contains the full CDS of TuMV with GFP fused between P1 and HC-Pro (Fig. 1). GFP is cleaved from the polyprotein, allowing in situ visualization of viral infection. Plasmids p41 and p68 are modified versions of p35STuMV-GFP, each containing a point mutation that is synonymous with respect to the polyprotein but introduces a stop codon in the pipo reading-frame (20 nt and 47 nt 3′ of the G2A6 motif for p41 and p68 respectively; Fig. 3). The mutations were achieved by PCR, using the GenTailor site directed mutagenesis system (Invitrogen) with primers 41F, 41R, 68F, and 68R (all primer sequences are given in Table S3) on the vector pTORFX, which is a PCR fragment containing pipo, generated from p35STuMV-GFP using primers F and R, cloned into pCR 2.1-TOPO (Invitrogen). The inserts were subcloned via KpnI and MfeI sites into pUC19X, resulting in pUC41 and pUC68. pUC19X is a pUC19-based vector containing 8 kb of the TuMV cDNA (from KpnI, which is in HC-Pro, to SalI, the end of the viral cDNA). Finally, the 8-kb fragments in pUC41 and pUC68, containing KpnI and SalI, were subcloned back into p35STuMV-GFP, resulting in p41 and p68.

The plasmid pTT7pipoFLAG was used for in vitro translation of pipo, to determine gel migration of isolated PIPO product and test the antiserum AS 39–52. The plasmid was derived from the TA cloning vector pGemT-easy (Promega) with an insert consisting of the TuMV pipo sequence with the T7 promoter fused to its 5′ end, the FLAG epitope fused to its 3′ end before the termination codon, and 99 nt of downstream sequence. The insert was released by digestion with EcoRI, resulting in T7pipo-FLAG, followed by gel purification for in vitro transcription and/or translation.

Inoculation of N. benthamiana via Gene Gun-Delivery of TuMV cDNA.

Before bombardment, 15 μg of 1-μm gold particles were washed and coated with 3 μg of plasmid DNA according to ref. 30 with variations as follows: The sample was continuously mixed on the vortexer while 50 μl of 50% glycerol, 25 μl of 2.5 M CaCl2, and 10 μl of 0.1 M spermidine were added. After 5–10 min of continuous mixing, the DNA-gold was washed with 70 μl of 70% isopropanol and once again with 100% isopropanol, followed by resuspension in 30 μl of 100% isopropanol. Ten microliters of the DNA-coated gold was pipetted onto each macrocarrier while the suspension was continuously shaken. Up to four plants were bombarded from each 1× tube of DNA-coated gold particles.

N. benthamiana plants used for bombardment were 5–7 weeks old and grown in a 22°C growth chamber with 14 h of daylight. Transformation was carried out with a PDS 1000/He biolistic gun (Bio-Rad), using the following parameters: 650 psi rupture disk pressure; a 6-cm target distance (from middle of launch assembly to target plate); a 6-mm gap; 1.2 cm from macrocarrier to stopping plate; and a 28-torr vacuum at rupture. All hardware and disposables for the biolistic gun were obtained from Bio-Rad.

Plant Tissue Preparation.

Fresh plant tissues were ground in liquid nitrogen followed by addition of TBS [10 mM Tris·HCl (pH 7.4), 300 mM NaCl, 5 mM EDTA, 1 mM PMSF, and 1 mM DTT) up to a tissue concentration of 1 mg/μl. The mixtures were then centrifuged at 10,000 × g for 10 min at 4°C and the supernatant was respun until clear. The clear supernatant was used for immunoblot analysis. In addition, quantitation of total protein in the lysate was done by using the Bradford assay (Bio-Rad).

Immunoblotting.

NuPAGE sample buffer (Invitrogen) was added to plant lysates, heated for 2 min at 100°C, and subjected to NuPAGE electrophoresis on 4–12% NuPAGE Bis-Tris gels with MES-SDS running buffer (Invitrogen) for 40 min. Samples were transferred to Hybond-ECL nitrocellulose membranes (Amersham), followed by blocking with 3% nonfat dry milk in 1× PBS-T for 30 min at room temperature. The blocked membranes were incubated overnight at 4°C with primary antiserum, washed, and incubated with horseradish peroxidase-conjugated goat secondary Ab (Bio-Rad) for 30 min. The bands were visualized by developing with enhanced chemiluminescence (Pierce). The following rabbit antisera (Genscript) were used: AS 2–15, raised against peptide sequence KKLSTNLGRSMERVC (TuMV PIPO amino acids 2–15 plus “C”; Fig. 3) and AS 39–52, raised against peptide sequence ANEKRSRFRRQIQRC (TuMV PIPO amino acids 39–52 plus “C”). In addition, 3.125 μl/ml wheat germ extract (Promega) was added during binding of AS 39–52 for higher specificity.

RT-PCR.

Plants were ground in liquid nitrogen and RNA was extracted by using Triazol (Invitrogen). Three μg of RNA were subjected to RT-PCR, using SuperScript II (Invitrogen) with primer R, which is specific to p35STuMV-GFP, 1,813 nt 3′ of the G2A6 motif. This was followed by PCR, using platinum Taq Hi-Fi (Invitrogen) with primers F and X99R. The PCR controls were p35STUMV-GFP for the positive controls and water for the negative control.

In Vitro Transcription and Translation.

In vitro translation of T7PIPO-FLAG was done first by transcribing the RNA, using the T7 Megascript kit (Ambion), followed by capping, using the ScriptCap m7G capping system (Epicentre). This was followed by in vitro translation where 0.2 pmol of RNA transcript were added to wheat germ extract (Promega) in a final reaction volume of 12.5 μl and translated for 2 h at 25°C. Total protein from the translation mix was separated on a precast 4–12% PAGE (Invitrogen), which was used for Western blot analysis.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Valérie Torney for assistance with bombardment, Jaime Holdridge for assistance with photography, and Krzysztof Treder for assistance with protein work. We thank Steve Whitham (Iowa State University) for providing the plasmid p35STuMV-GFP and valuable advice; we also thank the anonymous reviewers for their helpful comments. This work was supported by an award from Science Foundation Ireland and National Institutes of Health Grants R01 GM067104 (to W.A.M.) and R01 GM079523 (to J.F.A.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0800468105/DC1.

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