An overlapping essential gene in the Potyviridae

Betty Y.-W. Chung*†, W. Allen Miller†, John F. Atkins*‡, and Andrew E. Firth*§

*BioSciences Institute, University College Cork, Cork, Ireland; †Department of Plant Pathology, 351 Bessey Hall, Iowa State University, Ames, IA 50011; and ‡Department of Human Genetics, University of Utah, Salt Lake City, UT 84112

Communicated by Paul Ahlquist, University of Wisconsin, Madison, WI, February 4, 2008 (received for review October 31, 2007)

The family Potyviridae includes >30% of known plant virus spe- +2 frame 0 frame cies, many of which are of great agricultural significance. These GGAAAAAA PIPO viruses have a positive sense RNA genome that is Ϸ10 kb long and ’ NIa− NIa− ’ 5 P1 HC−Pro P3 CI VPg Pro NIb coat 3 contains a single long ORF. The ORF is translated into a large 6K16 K2 Ϸ GFP polyprotein, which is cleaved into 10 mature proteins. We report 131 3079 3258 9625 the discovery of a short ORF embedded within the P3 cistron of the polyprotein but translated in the ؉2 reading-frame. The ORF, Fig. 1. TuMV genome map (9,835 nt). In the plasmid p35STuMV-GFP, GFP is termed pipo, is conserved and has a strong bioinformatic coding fused between P1 and HC-Pro. The position of the overlapping CDS, pipo, and signature throughout the large and diverse Potyviridae family. the G2A6 motif are indicated. Mutations that knock out expression of the PIPO protein in Turnip mosaic potyvirus but leave the polyprotein amino acid sequence evidence for an overlapping CDS within the P3 cistron. Here, we unaltered are lethal to the virus. Immunoblotting with antisera describe the bioinformatic evidence and experimental verifica- raised against two nonoverlapping 14-aa antigens, derived from tion for the new CDS. the PIPO amino acid sequence, reveals the expression of an Ϸ25- kDa PIPO fusion product in planta. This is consistent with expres- Results sion of PIPO as a P3-PIPO fusion product via ribosomal frameshift- Bioinformatics. The new CDS, pipo (Pretty Interesting Potyviridae ing or transcriptional slippage at a highly conserved G A motif 1-2 6-7 ORF), was first identified in an alignment of Turnip mosaic virus at the 5؅ end of pipo. This discovery suggests that other short (TuMV) (9) sequences, using the gene-finding software overlapping genes may remain hidden even in well studied virus MLOGD (3). MLOGD has been tested extensively, using thou- genomes (as well as cellular organisms) and demonstrates the sands of known virus CDSs as a test set, and it has been shown utility of the software package MLOGD as a tool for identifying such genes. that, for overlapping CDSs, a total of just 20 independent base variations are sufficient to detect a new CDS with Ϸ90% confidence. The input alignment comprised the GenBank P3 ͉ PIPO ͉ Potyvirus ͉ Turnip mosaic virus ͉ frameshift RefSeq NC࿝002509 and 20 of its genome neighbors (i.e., other TuMV genome sequences of RefSeq quality but not identical to he genomes of many viruses are under strong selective the chosen RefSeq). In TuMV (9,835 nt; polyprotein coords Tpressure to compress maximal coding and regulatory infor- 131..9625), pipo has coords 3079..3258 (60 codons; ϩ2 frame mation into minimal sequence space. Overlapping coding se- relative to polyprotein; Fig. 1), which places it within the P3 quences (CDSs) are particularly common in such viruses, al- cistron (coords 2591..3655). The total number of independent though they also occur in more complex genomes, including base variations within pipo is Ϸ92, thus providing MLOGD with eukaryotes (1). Such CDSs can be difficult to detect using a robust signal. The MLOGD output showed that pipo has a conventional gene-finding software (2), especially when short. strong coding signature and the ORF is present across the The software package MLOGD, however, was designed specif- alignment. ically for locating short overlapping CDSs in sequence align- Further inspection revealed that the ORF (Ն60 codons; ϩ2 ments and overcomes many of the difficulties with alternative frame relative to polyprotein) is present in all 48 Potyvirus methods (2, 3). MLOGD includes explicit models for sequence RefSeqs in GenBank. MLOGD, applied to the 48-RefSeq evolution in double-coding regions as well as models for single- alignment, detected a very strong coding signature in the ORF, coding and noncoding regions. It can be used to predict whether with Ϸ2,000 independent base variations now available to query ORFs are likely to be coding, via a likelihood ratio test, MLOGD. Scores outside of the ORF are negative, whereas, where the null model comprises any known CDSs, and the within the ORF, there are at least five nonoverlapping and alternative model comprises the known CDSs plus the query hence completely independent high-positively scoring windows ORF. We have been using MLOGD to search for new virus- (Fig. 2). encoded genes. Starting from a highly conserved G A motif (Fig. 3), there The family Potyviridae is the largest plant virus family and 1-2 6-7 are no pipo-frame termination codons in any of the 48 RefSeqs includes Ϸ30% of known plant viruses (4, 5). The genomic RNA Ϸ for at least 60 codons. In 29 of 48 RefSeqs, there is an AUG is positive sense and 10 kb long. To date, all experimental and Ј sequencing evidence has supported the existence of only one codon 9 codons 3 of the motif. Of the remaining 19 RefSeqs, 1 functional ORF in the six Potyviridae genera, with the exception has an upstream AUG codon, 15 have downstream AUG codons of the genus Bymovirus, in which the ORF is divided between two (but only 3 are within 10 codons), and 4 have no AUG codon at genomic RNAs (5). The ORF encodes a large (340- to 395-kDa) polyprotein that is cleaved into Ϸ10 mature proteins (Fig. 1; Author contributions: B.Y.-W.C. and A.E.F. contributed equally to this work; B.Y.-W.C., reviewed in ref. 6). The genomic RNA has a covalently linked W.A.M., J.F.A., and A.E.F. designed research; B.Y.-W.C. and A.E.F. performed research; 5Ј-terminal protein (VPg) and a 3Ј polyA tail. Cap-independent B.Y.-W.C., W.A.M., J.F.A., and A.E.F. analyzed data; and B.Y.-W.C., W.A.M., J.F.A., and A.E.F. translation is mediated by the 5ЈUTR (7). Subgenomic tran- wrote the paper. scripts are apparently not produced (8). Such a polyprotein The authors declare no conflict of interest. expression strategy is common to many virus families, most §To whom correspondence should be addressed. E-mail: a.fi[email protected]. notably the Picornaviridae. The potential for functional short This article contains supporting information online at www.pnas.org/cgi/content/full/

overlapping ORFs in such viruses has been largely overlooked. 0800468105/DCSupplemental. MICROBIOLOGY However, when we applied MLOGD to the Potyviridae, we found © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800468105 PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5897–5902 Downloaded by guest on September 29, 2021 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, NC࿝002509 (TuMV); window size, 40 codons; step size, 20 codons. Each window is represented by a small circle (showing the likelihood ratio score for that window), and gray bars show the width (ends) of the window. The null model, in each window, is that only the polyprotein frame is coding, whereas the alternative model is that both the polyprotein frame and the window frame are coding. Positive scores (i.e., above the horizontal dashed line) favor the alternative model. The positions of stop codons in each frame are shown below the likelihood scores and the positions of alignment gaps (green) in all 48 RefSeqs are shown at the top. Only the ϩ1 (red) and ϩ2 (purple) frames are shown because the ϩ0 frame is the polyprotein frame that is included in the null model. Scores are generally negative with occasional random scatter into low positive scores, except for the pipo region, which has consecutive high-positively scoring windows and a corresponding ORF present across the alignment. Note that other apparent ‘‘gaps’’ in the stop codon annotation mainly correspond to alignment gaps. See ref. 3 for details of MLOGD software. (Lower) Zoom-in showing pipo and the flanking Ϯ400 nt. Window size, 10 codons; step size, 5 codons. Note that pipo contains at least five nonoverlapping and hence completely independent high-positively scoring windows. Formally, and within the MLOGD model, P Ͻ 10–100.

all. Thus, AUG-initiation is unlikely. In 16 of 48 RefSeqs, the Ipomovirus (one RefSeq), Rymovirus (three RefSeqs) and Tri- G1-2A6-7 motif begins with a pipo-frame stop codon, UGA, timovirus (three RefSeqs). With the exception of Rymovirus and instead of GGA, thus precluding 5Ј extension of the ORF. There Potyvirus, the genera are highly divergent [typically 33–45% is no well conserved termination codon position at the 3Ј nucleotide identity between genera within the P3 cistron (12)]. end—with ORF lengths (from the G1–2A6–7 motif) ranging from In the genus Bymovirus, the longer genomic RNA (RNA1; 60 to 115 codons. Of relevance to possible leaky scanning, pipo Ϸ7,500 nt) contains a single polyprotein CDS that codes for the is preceded by many initiation codons, e.g., 57 AUG codons lie C-terminal eight proteins of the Potyvirus polyprotein—thus P3 between the polyprotein AUG and pipo in the TuMV genome, becomes the N-terminal protein. In the P3 cistron, there is a of which 24 are in the polyprotein frame. G(G/C)A6 motif followed by a 74- to 88-codon ϩ2 frame ORF, Application of National Center for Biotechnology Informa- for which MLOGD detects a strong coding signature. In Triti- tion blastp (word size 2; virus RefSeq db) to PIPO revealed no movirus, there is an A2G2A6-7 motif within the P3 cistron similar amino acid sequences in GenBank, whereas tblastn followed by a 134- to 137-codon ϩ2 frame ORF, for which identified only the pipo region in other Potyvirus sequences (as MLOGD again detects a strong coding signature. Similar results expected). Hydrophobicity plots are broadly similar for the hold for an alignment of the Tritimovirus Wheat streak mosaic N-terminal Ϸ60 aa: a hydrophobic peak followed by a hydro- virus (WSMV) RefSeq with its four genome neighbors. In philic peak and then a narrower hydrophobic peak. PIPO is rich Rymovirus, there is a GA6GA motif within the P3 cistron in the basic residues Arg and Lys: Averaged over the 48 RefSeqs, followed by a 104- to 116-codon ϩ2 frame ORF for which their relative abundances are, respectively, 2.1 and 1.5 times MLOGD detects a good coding signature. In Ipomovirus, there higher in PIPO than in the polyprotein. Although predicted isaGA7 motif in the P3 cistron followed by a 99-codon ϩ2 frame RNA secondary structures were found in individual RefSeqs, no ORF. It is noteworthy that the frame of the G1-2A6-7 motif is not widely conserved secondary structures were found in the vicinity fully conserved (Fig. 3). In Potyvirus, the frame is generally (G) of pipo [using alidot (11)]. GAA AAA A(A) (spaces separate the polyprotein codons). In There are four other Potyviridae genera with genomic RefSeqs Bymovirus, the frame is GGA AAA AA. In Tritimovirus,two in GenBank: Bymovirus (four RefSeqs ϫ two RNA segments); RefSeqs have G GAA AAA A, whereas the other has GGA

5898 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800468105 Chung et al. Downloaded by guest on September 29, 2021 Fig. 3. Pipo sequence. (A) Extract of 9 RefSeqs from an alignment of the 48 Potyvirus GenBank RefSeqs, showing the region around the 5Ј end of pipo, which coincides with the annotated highly conserved G1-2A6-7 motif (see Fig. S8 for the full 48-RefSeq alignment). Viruses: NC࿝001445, plum pox; NC࿝001555, tobacco etch; NC࿝001616, potato Y; NC࿝001671, pea seed-borne mosaic; NC࿝002509, turnip mosaic; NC࿝002634, soybean mosaic; NC࿝003397, bean common mosaic; NC ࿝004039, potato A; NC࿝003398, sugarcane mosaic. (B) Similar [GA]-rich tracts from the 5Ј end of pipo in representative RefSeqs from other Potyviridae genera (see Fig. S8 for other RefSeqs). Viruses: NC࿝001814, ryegrass mosaic; NC࿝002350, wheat yellow mosaic; NC࿝001886, wheat streak mosaic; NC࿝003797, sweet potato mild mottle. (C) The TuMV/p35STuMV-GFP PIPO peptide and nucleotide sequences, with the two antigen sites underlined in the peptide sequence and the two knockout point mutation sites underlined in the nucleotide sequence.

AAA AAA. Further MLOGD results, GenBank accession Nicotiana benthamiana plants were inoculated by bombard- numbers for all sequences used, and comparisons of the sizes and ment with gold particles coated with p35STuMV-GFP DNA or sequence divergences of P3 and PIPO are included in supporting the mutant derivatives or with water as a control. Each construct information (SI) Figs. S1–S7 and Tables S1 and S2. was bombarded into four plants. None of the eight plants bombarded with DNA containing mutations in PIPO showed PIPO Knockouts. To investigate whether pipo is an essential gene, any sign of infection up to 12 days postinoculation (dpi) (Fig. 4). we introduced mutations into an infectious clone of TuMV There was no green fluorescence, and plants were as tall and (p35STuMV-GFP) that expresses GFP for easy detection of healthy as uninoculated controls. In contrast, three of four WT-infected plants showed extensive infection (GFP fluores- virus-infected tissue. Two PIPO knockout mutants were gener- cence, stunted growth, and wilting) over the same time period. ated. Each differs from WT GFP-expressing TuMV by just a (The sole uninfected plant was bombarded with a smaller single point mutation: GAC 3 GAU and UCG 3 UCU at ࿝ amount of DNA than the three infected ones and those bom- NC 002509 coordinates 3103 and 3130, respectively (polypro- barded with mutant TuMV DNAs.) RT-PCR with primers tein-frame codons shown). These mutations are synonymous specific to p35STuMV-GFP, using lysate from infected plants at with respect to the polyprotein frame, but introduce premature 12 dpi, confirmed that TuMV RNA was present only in WT- termination codons (UGA) into pipo. The altered codon usage infected plants (Fig. 5). in the polyprotein ORF is not a factor because the new codons occur in the TuMV genome at frequencies similar (within 20%) Immunoblotting. Separate antisera were raised against two pre- to those they replace. dicted 14-aa antigen sites within PIPO (AS 2–15: amino acids

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 p41 and p68; and (5) nonbombarded plants. In each of (1–5), only one of four replicates is depicted. Only WT-infected plants showed any sign of infection (three of four replicates) with extensive GFP fluorescence, stunted growth, and wilting by 12 dpi. Note that the small yellowish-green patches visible on some leaves under UV—e.g., Lower (1) and (4),

are necrosis caused by bombardment (cf. natural light photos). Some patches on the soil also appear green under UV. However, GFP fluorescence is clearly MICROBIOLOGY distinguishable from both of these. High resolution photos are in Fig. S9 and Fig. S10.

Chung et al. PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5899 Downloaded by guest on September 29, 2021 consists of a slippery heptanucleotide typically fitting the con- sensus motif N NNW WWH. This is followed by a highly structured region, usually a pseudoknot, beginning 5–9 nt down- stream (14). The G1-2A6-7 motif may serve as a slippery hep- tanucleotide (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 Fig. 5. Detection of TuMV accumulation in plants by RT-PCR. Primers specific sequence acting within the ribosomal exit tunnel) may, in this to p35STuMV-GFP were used to amplify cDNAs from RNA isolated from plants case, stimulate ribosomal frameshifting. We were unable to infected with WT TuMV (p35STuMV-GFP; lane 2), the two PIPO knockout observe any frameshift product from in vitro translation of a mutants, p41 and p68 (lanes 2 and 3 respectively), and both negative (water; construct comprising (in order) the T7 promoter, the TuMV lane 5) and nonbombarded (lane 6) controls, all at 12 dpi. The PCR controls 5ЈUTR, the TuMV P3 cistron with artificial in-frame initiation were plasmid p35STuMV-GFP for the positive control (lane 7) and water for Ј the negative control (lane 8). Marker DNAs are in lane 1. TuMV RNA was (AUG) and termination (UAA) codons, and the TuMV 3 UTR present only in WT-infected plants. (Fig. S11 and data not shown) suggesting that, if ribosomal frameshifting occurs in TuMV, it may require distal sequence elements (cf. ref. 15). 2–15; AS 39–52: amino acids 39–52; Fig. 3C). Both antisera Alternatively, the G1–2A6–7 motif may allow transcriptional detected Ϸ25-kDa products in lysates from WT-infected plants slippage, whereby the viral polymerase ‘‘slips’’ on a region of (Fig. 6A). Neither antiserum detected an Ϸ6- to 7-kDa product, repeated identical nucleotides, resulting in the insertion (or which is the predicted size of PIPO. Nonetheless, AS 39–52 deletion) of extra nucleotides in a portion of transcripts. Tran- clearly revealed an Ϸ7-kDa product generated by in vitro trans- scriptional slippage has been documented in the Paramyxoviridae lation of an artificial transcript encoding only pipo (Fig. 6B)(pipo (e.g., Sendai virus) and the Filoviridae (e.g., Ebola virus) (16). In nucleotide sequence preceded by T7 promoter and followed by contrast to the Paramyxoviridae slippage site (generally A5–6G2–5 Ј FLAG epitope; presumably initiates at the AUG codon 9 codons or A2GAG4–7), in the Potyviridae the Gs are 5 of the As, which 3Ј of the G2A6 motif, resulting in a 7-kDa product lacking the AS would appear to favor nucleotide deletions rather than insertions 2–15 antigen) demonstrating that AS 39–52, at least, would (because of the possibility of G:U repairings but not A:C detect such a product if it were expressed in vivo. repairings)—although insertional slippage on A7 is also possible [as in Ebola virus (17)]. To produce transcripts that allow PIPO Discussion expression, a 2-nt deletion or 1-nt insertion would be required. Possible Translational Mechanisms. The detection with both anti- Mass spectrometry of cDNAs (nominally 62 nt, covering the sera of an Ϸ25-kDa product and the absence of an Ϸ6- to 7-kDa G1-2A6-7 motif) derived from RNA extracted from an N. product, appears to rule out independent ribosome entry into benthamiana plant infected with p35STuMV-GFP (Fig. 4) failed pipo (e.g., via an IRES or shunting). We propose that the to detect transcripts with deletions or insertions (data not shown). Nonetheless, it is possible that some Potyviridae species G1-2A6-7 motif facilitates ribosomal frameshifting or transcrip- tional slippage (13), allowing expression of PIPO as a fusion or genera use transcriptional slippage, whereas others use ribo- product with the N-terminal region of P3 (hereafter P3N). In fact somal frameshifting [cf. DnaX (18)]. If so, then presumably the predicted size of PIPO fused with P3N is 25.3 kDa, in good either the sequence difference somehow prevents encapsidation agreement with the observed Ϸ25-kDa band. of the modified RNAs or the slippage frequency is sufficiently Many viruses harbor sequences that induce a portion of low, so modified RNAs are not detected or have been disre- garded as sequencing errors. ribosomes to shift Ϫ1 nt and continue translating in the new In any case, it is clear that the G A motif is highly (equivalent to ϩ2) reading-frame (13). The Ϫ1 frameshift site 1-2 6-7 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 3 G GAG AAA A (synony- mous in the polyprotein frame) is unable to infect plants systemically (19). Three other point mutations in the vicinity (at Ϫ6, ϩ9, and ϩ18 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 con- taining 10–16 synonymous (polyprotein frame) point mutations Fig. 6. Immunoblot detection of PIPO products. (A) An approximately spaced along the 3Ј half of the P3 cistron. All mutants had the 25-kDa PIPO fusion product. AS 2–15, antiserum against PIPO N-terminal same phenotype: (i) the ability to replicate was retained at peptide (amino acids 2–15). AS 39–52, antiserum against PIPO C-terminal 22–80% of WT; and (ii) the virus was able to establish infection peptide (amino acids 39–52). Ϫ, lysate from noninfected plants. ϩ, lysate from foci limited to small clusters of cells that increased in size only plants infected with WT TuMV (p35STuMV-GFP). Lanes were loaded with slightly by 5 dpi, whereas infection foci produced by WT virus equal amounts of tissue; similar results were obtained when lanes were loaded with equal total protein (data not shown). (B) ϩ, detection of an were much larger at 3 dpi and had coalesced by 5 dpi, as indicated Ϸ7-kDa product with AS 39–52 from in vitro translation of pipo nucleotide in histochemical GUS assays. The authors postulated that the sequence preceded by T7 promoter and followed by FLAG epitope, demon- mutations disrupted an important RNA secondary structure strating that the antiserum would detect such a product if it were expressed involved in movement. However, we could find no evidence for in vivo. Ϫ, negative control (no RNA). a more widely conserved RNA secondary structure. In fact, all

5900 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800468105 Chung et al. Downloaded by guest on September 29, 2021 six mutants contain many nonsynonymous mutations in the pipo modified versions of p35STuMV-GFP, each containing a point mutation that is reading-frame. Importantly, a seventh mutant with synonymous synonymous with respect to the polyprotein but introduces a stop codon in the Ј mutations in the downstream CI cistron (3Ј of the pipo termi- pipo reading-frame (20 nt and 47 nt 3 of the G2A6 motif for p41 and p68 nation codon) had WT phenotype. In contrast to the mutants respectively; Fig. 3). The mutations were achieved by PCR, using the GenTailor described in ref. 19, no infection at all was detected for our 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 TuMV PIPO knockout mutants (although local infection limited is a PCR fragment containing pipo, generated from p35STuMV-GFP using to just small clusters of cells may not have been apparent). primers F and R, cloned into pCR 2.1-TOPO (Invitrogen). The inserts were Possible roles for PIPO include replication, movement, suppres- subcloned via KpnI and MfeI sites into pUC19X, resulting in pUC41 and pUC68. sion of systemic silencing, or a combination of functions. pUC19X is a pUC19-based vector containing 8 kb of the TuMV cDNA (from The P3 protein, whose cistron pipo overlaps (Fig. 1), is a KpnI, which is in HC-Pro, to SalI, the end of the viral cDNA). Finally, the 8-kb Ϸ42-kDa nonstructural protein and is one of the least well fragments in pUC41 and pUC68, containing KpnI and SalI, were subcloned characterized Potyvirus proteins. It may be involved in virus back into p35STuMV-GFP, resulting in p41 and p68. replication, pathogenicity, resistance breaking, and systemic The plasmid pTT7pipoFLAG was used for in vitro translation of pipo,to infection (6, 9, 21–23). It is interesting that previous investiga- 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 tions into the function and localization of P3 have produced (Promega) with an insert consisting of the TuMV pipo sequence with the T7 conflicting results (6). Such discrepancies may in fact be a result Ј Ј ϩ promoter fused to its 5 end, the FLAG epitope fused to its 3 end before the of antibodies raised against P3 being sensitive also to P3N PIPO termination codon, and 99 nt of downstream sequence. The insert was re- in some studies but not in others. A survey of published material leased by digestion with EcoRI, resulting in T7pipo-FLAG, followed by gel using antibodies against P3 produced no conclusive evidence purification for in vitro transcription and/or translation. either for or against PIPO (see Table S4). Furthermore, some phenotypes linked to mutations in the P3 cistron may result from Inoculation of N. benthamiana via Gene Gun-Delivery of TuMV cDNA. Before alterations to P3NϩPIPO or its translational mechanism rather bombardment, 15 ␮gof1-␮m gold particles were washed and coated with 3 than P3 itself. For example, a key virulence determinant of ␮g of plasmid DNA according to ref. 30 with variations as follows: The sample Zucchini yellow mosaic potyvirus has been mapped to the was continuously mixed on the vortexer while 50 ␮l of 50% glycerol, 25 ␮lof ␮ polyprotein-frame codon immediately 5Ј of the GAA AAA A 2.5 M CaCl2, and 10 l of 0.1 M spermidine were added. After 5–10 min of continuous mixing, the DNA-gold was washed with 70 ␮l of 70% isopropanol motif (24), mutations of which could affect the level of ␮ ϩ and once again with 100% isopropanol, followed by resuspension in 30 lof P3N PIPO production. 100% isopropanol. Ten microliters of the DNA-coated gold was pipetted onto each macrocarrier while the suspension was continuously shaken. Up to four Conclusions plants were bombarded from each 1ϫ tube of DNA-coated gold particles. We have demonstrated the existence of a new coding sequence, N. benthamiana plants used for bombardment were 5–7 weeks old and pipo,inthePotyviridae—a vast and extremely diverse family of grown in a 22°C growth chamber with 14 h of daylight. Transformation was plant viruses, many of which are of great agricultural significance carried out with a PDS 1000/He biolistic gun (Bio-Rad), using the following (5). Evidence includes: (i) the ubiquitous presence of an ORF parameters: 650 psi rupture disk pressure; a 6-cm target distance (from middle (Ն60 codons) in all 60 Potyviridae GenBank RefSeqs; (ii) the of launch assembly to target plate); a 6-mm gap; 1.2 cm from macrocarrier to ubiquitous presence of a strong MLOGD coding signature for stopping plate; and a 28-torr vacuum at rupture. All hardware and disposables for the biolistic gun were obtained from Bio-Rad. this ORF; (iii) WSMV mutants in ref. 19 that disrupted PIPO but did not change the polyprotein amino acid sequence were unable Plant Tissue Preparation. Fresh plant tissues were ground in liquid nitrogen to establish systemic infection, whereas mutants that did not followed by addition of TBS [10 mM Tris⅐HCl (pH 7.4), 300 mM NaCl, 5 mM disrupt PIPO had WT phenotype; (iv) our two TuMV PIPO EDTA, 1 mM PMSF, and 1 mM DTT) up to a tissue concentration of 1 mg/␮l. The knockout mutants, each differing from WT by a single point mixtures were then centrifuged at 10,000 ϫ g for 10 min at 4°C and the mutation synonymous in the polyprotein frame, were noninfec- supernatant was respun until clear. The clear supernatant was used for tious; and (v)anϷ25-kDa product was detected in lysate from immunoblot analysis. In addition, quantitation of total protein in the lysate TuMV-infected plants by two separate antisera raised against was done by using the Bradford assay (Bio-Rad). different 14-aa domains in PIPO. The combination of our results (Potyvirus) with reinterpreted results from ref. 19 (Tritimovirus) Immunoblotting. NuPAGE sample buffer (Invitrogen) was added to plant lysates, gives broad phylogenetic support for these observations. Future heated for 2 min at 100°C, and subjected to NuPAGE electrophoresis on 4–12% work will address the mechanism by which pipo is translated and NuPAGE Bis-Tris gels with MES-SDS running buffer (Invitrogen) for 40 min. Samples were transferred to Hybond-ECL nitrocellulose membranes (Amersham), the function(s) of the PIPO fusion protein. followed by blocking with 3% nonfat dry milk in 1ϫ PBS-T for 30 min at room The Potyviridae have been grouped into the picorna-like virus temperature. The blocked membranes were incubated overnight at 4°C with superfamily, a loose grouping of single-stranded positive-sense primary antiserum, washed, and incubated with horseradish peroxidase- RNA viruses that includes Picornaviridae, Caliciviridae, Como- conjugated goat secondary Ab (Bio-Rad) for 30 min. The bands were visualized by viridae, Dicistroviridae, Potyviridae, and Sequiviridae (8). Over- developing with enhanced chemiluminescence (Pierce). The following rabbit lapping and frameshift CDSs are almost unknown in the super- antisera (Genscript) were used: AS 2–15, raised against peptide sequence KKLST- family. Notable exceptions include Theilovirus (Picornaviridae) NLGRSMERVC (TuMV PIPO amino acids 2–15 plus ‘‘C’’; Fig. 3) and AS 39–52, raised (25), Acyrthosiphon pisum virus (unclassified picorna-like virus) against peptide sequence ANEKRSRFRRQIQRC (TuMV PIPO amino acids 39–52 ␮ (26), and the Caliciviridae (27, 28) (see SI Text for further plus ‘‘C’’). In addition, 3.125 l/ml wheat germ extract (Promega) was added details). Identification of these CDSs was no doubt aided by their during binding of AS 39–52 for higher specificity. size (all are Ͼ 100 codons). The discovery of pipo, a short CDS RT-PCR. Plants were ground in liquid nitrogen and RNA was extracted by using embedded deep within the polyprotein cistron, raises the ques- Triazol (Invitrogen). Three ␮g of RNA were subjected to RT-PCR, using Super- tion of how many other such CDSs await discovery. We have Script II (Invitrogen) with primer R, which is specific to p35STuMV-GFP, 1,813 demonstrated the utility of MLOGD as a tool to search for such nt 3Ј of the G2A6 motif. This was followed by PCR, using platinum Taq Hi-Fi ‘‘difficult’’ CDSs in all virus genomes. (Invitrogen) with primers F and X99R. The PCR controls were p35STUMV-GFP for the positive controls and water for the negative control. Materials and Methods Plasmids. For in vivo translation, three plasmids were used: p35STuMV-GFP, In Vitro Transcription and Translation. In vitro translation of T7PIPO-FLAG was p41, and p68. p35STuMV-GFP (29) contains the full CDS of TuMV with GFP done first by transcribing the RNA, using the T7 Megascript kit (Ambion), fol-

fused between P1 and HC-Pro (Fig. 1). GFP is cleaved from the polyprotein, lowed by capping, using the ScriptCap m7G capping system (Epicentre). This was MICROBIOLOGY allowing in situ visualization of viral infection. Plasmids p41 and p68 are followed by in vitro translation where 0.2 pmol of RNA transcript were added to

Chung et al. PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5901 Downloaded by guest on September 29, 2021 wheat germ extract (Promega) in a final reaction volume of 12.5 ␮l and translated Treder for assistance with protein work. We thank Steve Whitham (Iowa for2hat25°C. Total protein from the translation mix was separated on a precast State University) for providing the plasmid p35STuMV-GFP and valuable 4–12% PAGE (Invitrogen), which was used for Western blot analysis. advice; we also thank the anonymous reviewers for their helpful com- ments. This work was supported by an award from Science Foundation ACKNOWLEDGMENTS. We thank Vale´rie Torney for assistance with bom- Ireland and National Institutes of Health Grants R01 GM067104 (to W.A.M.) bardment, Jaime Holdridge for assistance with photography, and Krzysztof and R01 GM079523 (to J.F.A.).

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