Proc. Natl. Acad. Sci. USAVol. 88, pp. 7887-7891, September 1991Microbiology

Amino acid substitutions in the coat protein result in loss of insecttransmissibility of a plant virus

(potyvirus/aphid/site-directed mutagenesis)

PRABHA L. ATREYA, CHINTAMANI D. ATREYA, AND THOMAS P. PIRONE*Department of Plant Pathology, University of Kentucky, Lexington, KY 405460091

Communicated by Robert J. Shepherd, June 10, 1991 (received for review April 23, 1991)

ABSTRACT Amino acids near the N terminus of the coatprotein of tobacco vein mottling virus were deleted or alteredby site-directed mutagenesis to determine the effect on aphidtransmissibility of the virus. Deletion of a three amino acidsequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. Themutation Ala -- Thr in this triplet drastically reduced trans-mission, whereas the mutation Asp -- Asn had no effect, andthe mutation Asp -' Lys consistently reverted to the wild-typeresidue. The mutation Lys-- Glu, in the residue adjacent to theglycine ofthe triplet, drastically reduced transmission, whereasthe mutation Gin -+ Pro, seven residues downstream from theglycine had no effect. Comparison of the sequences of otherpotyviruses suggests that the presence ofa glycine residue at thethird position of the Asp-Ala-Gly triplet is critical for aphidtransmissibility and that certain changes in the residues adja-cent to this position abolish or greatly reduce aphid transmis-sibility.

The potyviruses comprise a large group of plant viruses thatcause serious diseases in major crops worldwide (1). Naturalspread of potyviruses is accomplished by specific vectors,the homopterous insects, aphids. Current evidence suggeststhat potyvirus aphid transmission is in part determined bytwo viral-encoded proteins, the helper component and thepotyvirus capsid protein (2). Experimentally, potyvirusesmay also be transmitted to plants by "mechanical" (manual)inoculation, and repeated mechanical inoculation often leadsto loss of aphid transmissibility (3, 4). It was proposed sometime ago (5) that loss of aphid transmissibility could be due toaltered properties ofthe potyvirus coat protein, but it was notuntil the amino acid sequences of the coat proteins of anumber of potyviruses became available that specific se-quences that might be involved in aphid transmissibility couldbe proposed.The N terminus of the coat protein of potyviruses has been

shown to be exposed on the surface of the particle (6, 7), andenzymatic removal of the N-terminal domain results in lossof aphid transmissibility (8, 9). Harrison and Robinson (10)pointed out that an amino acid triplet, Asp-Ala-Gly, ispresent near the N terminus of the coat protein of tobaccoetch virus and several other aphid-transmissible potyviruses,but that in a non-aphid-transmissible isolate of tobacco etchvirus, the sequence Asp-Ala-Ser is present. Since then,comparisons of the coat protein sequences of aphid-transmissible and non-aphid-transmissible isolates of severalother potyviruses have revealed differences in the second orthird amino acids of this triplet (11-15). Although in mostcases there are other amino acid differences in the coatproteins, changes in the Asp-Ala-Gly triplet seem to be aconsistent feature of nontransmissible potyvirus isolates.

Direct evidence that an amino acid substitution in the thirdposition of the Asp-Ala-Gly triplet can affect aphid transmis-sibility was recently provided from our laboratory (16) byusing a non-aphid-transmissible isolate of tobacco vein mot-tling virus (TVMV-NAT). TVMV-NAT differs, in the coatprotein region, from aphid-transmissible TVMT (TVMV-AT)by a single nucleotide; this alters a single amino acid residue,Gly -- Glu in the Asp-Ala-Gly triplet. A cDNA fragmentrepresenting the coat protein gene of TVMV-NAT wassubstituted into a full-length cDNA clone ofTVMV-AT, andtranscribed RNA was inoculated to tobacco plants. Aphidswere unable to transmit the resultant hybrid virus, whichcontained the sequence Asp-Ala-Glu in the coat protein.To test the hypothesis that other specific amino acid

changes in the Asp-Ala-Gly triplet result in the loss of aphidtransmissibility, we used site-directed mutagenesis to alteramino acid residues in the Asp-Ala-Gly triplet of the poty-virus TVMV-AT. The effect of changing other residues in theN-terminal region of the coat protein was also tested. In thisreport, we present the results of these experiments anddiscuss them in view of the above hypothesis.

MATERIALS AND METHODSThe virus isolate TVMV-AT has been described (16). Thegenome-length cDNA clone, pXBS7, derived fromTVMV-AT RNA (17), is referred to as pDAGK in this reportto indicate the amino acid residues in the coat protein that arethe focus of this report.

Construction of Genome-Length cDNA Plasmids ContainingCoat Protein Mutations. The subclone pTVMV-CP was con-structed by digestingpDAGK at the Sal I and Spe I restrictionsites that flank the coat protein gene at nucleotide positions7883 and 9251, respectively (16); the released DNA fragmentwas gel purified and cloned into Sal I- and Spe I-digestedplasmid BlueScript II KS+ (Stratagene), using standardprocedures (18). Site-directed mutagenesis was done by themethod of Kunkel et al. (19) using the oligonucleotide prim-ers listed in Table 1, the dut- ung- Escherichia coli strainBD2399 helper phage M13K07, and dut' ung+ strain JM101.The clones containing the mutant coat protein genes wereidentified as described (20, 21). The mutant coat proteingenes were inserted into genomic-length plasmid pDAGK byreplacing the wild-type Sal I-Spe I restriction fragment withgel-purified Sal I-Spe I fragments from the above mutant coatprotein subclones (Fig. 1). The mutations in the full-lengthclones were confirmed by dideoxynucleotide chain-termination sequencing using Sequenase version 2.0 (UnitedStates Biochemical).In Vito Transcription and Inoculation of Plants with RNA.

pDAGK and the mutant plasmids were linearized with Sst I,

Abbreviations: TVMV, tobacco vein mottling virus; TVMV-AT,aphid-transmissible TVMV; TVMV-NAT, non-aphid-transmissibleTVMV.*To whom reprint requests should be addressed.

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Table 1. Oligonucleotide primers used for site-directed mutagenesis in the TVMV coat protein geneNucleotide position inTVMV genome*

Mutated At which Nucleotide Oligonucleotide primer complementaryplasmid Mutated primer anneals change to the cloned TVMV genometpADAGK (8438-8446) 8428-8456 Deletion 5'-CCTTGTCCTTTACTGTATCA-3'pNAGK 8438 8429-8448 G A 5'-TCCCAGCATtTACTGTATC-3'pKAGK 8438 8429-8447 G - A 5'-TTCCCAGCcTtTACTGTATC-3'

8440 T-. GpDTGK 8441 8433-8449 G - A 5'-CTTCCCAGtATCTACTG-3'pDAGE 8447 8440-8454 A -- G 5'-TTGTCCTcCCCAGCA-3'pDAGK-P 8466 8457-8474 A-- C 5'-CTAGTTTCgGATCCCTfG-3'*The nucleotide sequence of the wild-type coat protein gene is as follows: (8426) 5'-AGUGAUACAGUAGAUGCUGG-GAAGGACAAGGCAAGGGAUCAGAAACUAG-3' (8474).tLowercase letter indicates the mismatched nucleotide.

which does not cleave within the TVMV genome (Fig. 1), andthe protruding 3' ends were removed with T4 DNA polymer-ase before in vitro transcription reactions. Capped transcriptsfrom the linearized plasmid templates containing the T7promoter were synthesized with T7 RNA polymerase in a100-lId reaction according to the supplier's instructions (Strat-agene). Reactions were incubated at 370C for 1 hr andimmediately used for mechanical inoculation of Nicotianatabacum L. cv. Kentucky 14 (Ky 14) seedlings as describedby Domier et al. (17). Plants that developed symptoms wereassayed by immunodiffusion and by immunoblot analysis ofSDS/PAGE-separated proteins to confirm the presence ofTVMV.Aphid Transmission Assays. The general procedures for

rearing and handling of aphids and assay plants have beendescribed (16). Aphids were allowed a 5- to 10-min acquisi-tion access period on plants that had been transcript-inoculated 10-14 days previously. Aphids were placed on testplants in groups of 10 and were allowed an overnight inoc-ulation access period. The aphids were then killed by spray-

0 2 4

ing with an insecticide, and the plants were placed in a growthroom for symptom development.

Isolation and Sequencing of RNA from Infected Plants.Plants that became infected as a result of mechanical inoc-ulation with the transcripts, as well as those that subse-quently became infected by aphid inoculation, were tested toverify the presence ofthe mutation in the viral RNA. Sampleswere taken from the transcript-inoculated plants at the sametime that aphid transmission tests were made. Samples weretaken from aphid-inoculated plants as soon as symptomsappeared (=7 days after inoculation). Total RNA was iso-lated from infected leaf tissue, and cDNA to TVMV coatprotein-specific RNA was amplified by PCR and then se-quenced. Briefly, first-strand cDNA was synthesized in a20-,ul volume containing 2 ,ug of total leaf RNA, avianmyeloblastosis virus reverse transcriptase and 2.5 pmol of a27-residue-long reverse primer, (5'-GTCGAGCTCTTACAC-CCCCTTAACACC-3'), specific for the 3' terminus of theviral coat protein gene. This reaction mixture was then madeup to 100 ,ul in a buffer containing Taq polymerase for PCR

6 8 9.5kbSa(l7 SpeI(7883) (9251) SstlIV}1171

(9220)\22

pDAGK SDTVDAGKDKARDQKLI/,/ J. VKGV

pDAEK SDTVDAEKDKARDQKL tVKGV

pADAG SDTV--- KDKARDQKLi / KGKGVpNAGK SDTVNAGKDKARDQKLi I JVKGVpKAGK SDTVKAGKDKARDQKL4/t VKGVpDTGK SDTVDTGKDKARDQKLi VKVKGVpDAGE SDTVDAGEDKARDQKLJ /t VKGV

pDAGK-P SDTVDAGKDKARDPKL4/gVKGV

(2741) (3005)

FIG. 1. Representation of transcription plasmids containing coat protein mutations in genome-length (9.5 kilobases) TVMV-cDNAdownstream of a 17 promoter. Arrow on T7 promoter indicates direction of transcription. Numbers in parentheses at top represent nucleotidepositions of restriction enzyme sites and of coat protein gene. Sst I site shown is the site used for linearizing the plasmid before run-offtranscription. Below, wild type and mutant sequences of coat protein are shown in detail. pDAGK is a genome-length plasmid containing cDNAofwild-type TVMV-AT coat protein sequence (same as pXBS7) (17). pDAEK is a plasmid containing coat protein sequence of naturally occurringTVMV-NAT (16). pA&DAG through pDAGK-P are various TVMV full-length cDNA plasmids containing site-directed mutations in the coatprotein gene. Amino acid sequences in N- and C-terminal regions ofcoat protein are shown in single-letter code. Underlined amino acids indicatethe mutation. Numbers below amino acid sequence indicate positions of N- and C-terminal residues of coat protein in TVMV polyprotein.

I -r-

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(22). A 19-residue-long forward primer (5'-ATCTGCAG-GAGATACTCG-3') and the reverse primer were then addedto the reaction, and the viral cDNA sequence was amplifiedin a thermal-cycler. The double-stranded DNA obtained fromthe amplification reaction was then subjected to an asym-metric amplification reaction using the reverse primer (23);this resulted in single-stranded (ss) DNA that was thensequenced by using Sequenase version 2.0 with the forwardprimer.

Quantitative ELISA and Infectivity Assays. The possibleeffect of the mutations on the amount of virus in infectedplants was tested by immunological and infectivity tests.Systemically infected leaves from the transcript-inoculatedplants used as sources for virus acquisition by aphids wereused for both types of assay. Each mutant was directlycompared with the wild-type virus in both types of test, andat least two separate determinations were made for eachmutant.For ELISA tests, 300-mg tissue samples were ground in a

mortar with 1.2 ml of sample buffer (24). The samples werecentrifuged in a microcentrifuge for 30 sec, and a series of5-fold dilutions, made in sample buffer, was then processedfor direct ELISA as described (24) using antiserum to TVMVcoat protein.For infectivity tests, 600-mg tissue samples were ground in

a mortar with 600 A.l of 100 mM potassium phosphate buffer,pH 7.0. A series of 5-fold dilutions, from 1:25 through 1:3025was then used to inoculate KY 14 tobacco plants, 10 plantsfor each dilution. Relative infectivity was assessed by com-paring the number of plants infected at each dilution for eachmutant.

RESULTSDesign of the TVMV Coat Protein Mutants. The sequences

at the N terminus ofthe wild-type and the mutantTVMV coatproteins are listed in Table 2. Among the mutants listed,TVMV-NAT is the naturally occurring variant describedearlier (16). In the coat protein of this isolate, the glycineresidue in the Asp-Ala-Gly triplet was changed to a glutamateresidue due to a spontaneous point mutation at nucleotide8445 of the TVMV genome. The six other mutants in Table2 were created by site-directed mutagenesis as described.TVMV-ADAG is a deletion mutant designed to determine

the effect of complete removal of the Asp-Ala-Gly (DAG)sequence on aphid transmissibility. The aspartate residue ofthe Asp-Ala-Gly triplet is highly conserved among potyvi-ruses, and the only reported change is to asparigine (25-27;Table 3). Therefore, in addition to the Asp -- Asn change(TVMV-NAG), an Asp -* Lys change (TVMV-KAG) was

made to test whether other amino acids at this location aretolerated without adverse effects on aphid transmission.TVMV-DTG contains the Asp-Thr-Gly sequence that natu-

rally occurs in two non-aphid-transmissible potyviruses (14,33; Table 3). The Lys -+ Glu mutation (TVMV-DAGE) was

created to assess the effect of a mutation adjacent to theAsp-Ala-Gly region, and the mutation Gln -+ Pro (TVMV-

DAG-P) at amino acid position 2754 of the TVMV polypro-tein (14th amino acid from the N terminus of TVMV coatprotein) was created to determine the effect of an amino acidchange in the N-terminal region but not within or adjacent tothe Asp-Ala-Gly triplet.Aphid Transmission ofTVMV Coat Protein Mutants. Table

2 shows that aphid transmission was completely abolished forthe TVMV-ADAG mutant, in which the Asp-Ala-Gly tripletwas deleted from the coat protein. Sequence analysis ofTVMV RNA from the transcript-inoculated plants showedthat the deletion mutation was retained in the coat proteingene.The TVMV-NAG mutant remained highly aphid transmis-

sible, and sequence analysis of the coat protein gene showedthe presence of the mutation both in the transcript-inoculatedplants and in the plants infected as a result of aphid trans-mission. At first, the TVMV-KAG mutant also seemed highlyaphid transmissible. However, analysis of the coat proteingene in transcript-inoculated plants, as well as in the plantsinoculated by aphids, immediately after the symptoms ap-peared, revealed only wild-type sequence, although the mu-tation was present in the run-off transcript RNA used forinoculation. Thus, reversion to the Asp-Ala-Gly sequenceevidently occurred after inoculation. This reversion wasobserved in each of three separate transcription and inocu-lation experiments.The TVMV-DTG mutant had drastically reduced aphid

transmissibility, as did the TVMV-DAGE mutant (Table 2).Finally, the Gln -- Pro mutation (TVMV-DAG-P), which is

seven amino acid residues away from the Asp-Ala-Gly triplet,had no effect on aphid transmissibility. The mutations inTVMV-DTG, TVMV-DAGE, and TVMV-DAG-P all wereshown to be retained in both the transcript-inoculated and theaphid-inoculated plants.

Effects of the Mutations on Virus Concentration and Symp-tom Expression. Because decreased aphid transmission ofpotyviruses can result from low levels of virus in infectedleaves (35), ELISA and quantitative infectivity assays weredone to determine the relative virus content ofplants infectedwith the coat protein mutants. There were no differences inthe amount of viral antigen as measured by ELISA (data notshown). The relative concentrations of virus, as measured byinfectivity dilution curves, were similar to those reported byAtreya et al. (16) and were not different for the mutant andwild-type viruses (data not shown). The mutations had nodiscernible effect on symptoms produced on KY 14 tobaccoinoculated either mechanically or by aphids.

Table 2. Effect of amino acid deletion or substitutions in the N terminus of coat protein onaphid transmissibility of TVMV from infected plantsTVMV N-terminal coat

designation protein sequence* Transmissiont % StatustAT SDTVDAGK 178/200 89 Wild-type sequenceNAT SDTVDAEK 0/200 0 Natural mutationADAG SDTV---K 0/170 0 Deletion retainedNAG SDTVNAGK 44/50 88 Mutation retainedKAG SDTVKAGK 74/80 92 Mutation revertedDTG SDTVDIGK 5/170 3 Mutation retainedDAGE SDTVDAGF 1/200 0.5 Mutation retainedDAG-P SDTVDAGK ... E 26/30 87 Mutation retained*Single-letter symbol is used for amino acids; underlined amino acid indicates mutation or deletion.tNumber of infected plants per number of test plants; 10 aphids were used per test plant.tPresence of mutation was checked by nucleotide sequence analysis.

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Table 3. Amino acid sequence of N terminus of the coat proteinof representative potyviruses and aphid transmissibility of virus

AphidVirus/ N-terminal amino acid transmiss-isolate sequence Reference ibility

TVMV-AT SDTVD&GKDK 28 YesTVMV-NAT SDTVDAEKDK 16 NoTEV-AT SGTVD&GADA 6 YesTEV-NAT GGTVDASADV 6 NoPVY-D ANDTIDAGESS 29 YesPVY-18 ANDTIDAGDNK 30 NoPVY-RR ANDTIDDGGSS 31 Yes*PeMV ANDTIDTGGNS 32 NRTuMV#1 AGETLDAGLTD 15 YesTuMV #31 AGETLDANLTD 15 NoBYMV SDQEQLN&GEEK 25,26 NRCYVV SDKEKLNVGEQQ 27 NRPRSV-P SKNEAVDAGLNE 14 YesPRSV-W SKNEAVDTGLNE 14 NotZYMV-AT SGTQPTVADAGATK Yes*ZYMV-NAT SGTQPTVADTGATK 33 NoZYMV-CT SGTQPTVSDAGATK 34 NotSMV-G2 SGKEKEGDMDAGKDP 13 YesSMV-G7 SGKEKEGEHDAGKDP 13 YesSMV-N SGKEKEGDMD&DKDP 12,13 NoPPV-AT ADEREDEEEVDAGKPL 11 YesPPV-NAT ADEREDEEEVD&LQPP 11 No

TVMV, tobacco vein mottling virus; TEV, tobacco etch virus;PVY, potato virus Y; PeMV, pepper mottle virus; TuMV, turnipmosaic virus; BYMV, bean yellow mosaic virus; CYVV, cloveryellow vein virus; PRSV, papaya ringspot virus; ZYMV, zucchiniyellow mosaic virus; SMV, soybean mosaic virus; PPV, plum poxvirus; NR, not reported. Singe-letter symbol is used for amino acids.*B. Raccah (The Volcani Center, Bet Dagan, Israel), personalcommunication.

tE. Hiebert (Univ. of Florida, Gainesville), personal communication.tR. Grumet (Michigan State University, East Lansing), personalcommunication.

DISCUSSIONThe results described here confirm the hypothesis that aminoacid residues in the coat protein Asp-Ala-Gly triplet play arole in TVMV aphid transmissibility and demonstrate thatamino acid residues C-terminal to this triplet may also affectaphid transmissibility.The substitution Asp -. Asn in the first amino acid of this

generally conserved sequence did not lead to a loss of aphidtransmissibility of TVMV. The sequences Asn-Ala-Gly andAsn-Val-Gly have been reported to occur near the N termi-nus of two other potyviruses (25-27; Table 3), but the aphidtransmissibility of these isolates was not reported.There was a surprising but consistent reversion of the

Asp -- Lys mutation back to the wild-type residue afterinoculation and presumably infection oftobacco. Thus, thereappears to be strong selection pressure against either theparticular nucleotides or the particular amino acid changemade at this location. Two base changes were required forreversion to wild-type, whereas reversion did not occur in thesingle-base mutants, including TVMV-NAG, which differedfrom TVMV-KAG by only one base (Table 1). At the aminoacid level, the Asp -k Asn mutation, which is not selectedagainst, represents a change from an acidic to a neutralresidue, whereas the Asp -- Lys mutation is a change froman acidic to a basic residue. Because the mutation is presentin the RNA transcript used for inoculation, the reversionobviously occurs in the plant, after the inoculation stage ofthe infection process. It will be quite interesting to determineat which point the mutation is selected against.

The substitution Ala -- Thr in the second residue of theAsp-Ala-Gly triplet has been reported for non-aphid-transmissible isolates of zucchini yellow mosaic virus andpapaya ringspot virus (33, 14; Table 3), as well as in an isolateof pepper mottle virus, the aphid transmissibility of whichwas not reported (32; Table 3). In our experiments, thissubstitution in TVMV resulted in a drastic reduction, but notin a total loss, ofaphid transmissibility, and we speculate thatextensive testing of the papaya ringspot virus and zucchiniyellow mosaic virus isolates might reveal a very low level ofaphid transmission. Evidently not all substitutions in thisposition result in loss of aphid transmissibility, as the se-quence Asp-Asp-Gly occurs in an aphid-transmissible isolateof potato virus y (31; Table 3). The sequence Asn-Val-Glyoccurs in a clover yellow vein virus isolate, the aphidtransmissibility of which was not reported (27; Table 3).The most commonly reported difference is in the third

position of the Asp-Ala-Gly triplet, and substitutions ofglutamate, serine, asparagine, aspartate, or leucine residuesfor the glycine residue, are all associated with loss of aphidtransmissibility (Table 3). Thus it seems reasonable to con-clude that a glycine residue at this position may be critical foraphid transmissibility.The amino acid residue that follows the Asp-Ala-Gly is not

highly conserved; alanine, glutamate, glycine, leucine, andlysine residues are present at this location in various poty-virus isolates that are aphid transmissible (Table 3). For plumpox virus there is a difference between this residue in aphid-transmissible and non-aphid-transmissible isolates, but this isassociated with the deletion of a 15-amino acid sequencefollowing the leucine, in plum pox virus-non-aphid-transmissible (11). Therefore the effect of the Lys -- Glusubstitution in the coat protein on aphid transmissibility ofTVMV (Table 2) was quite surprising. The sequence Asp-Ala-Gly-Glu occurs in potato virus Y-D, which is reportedlyaphid transmissible (Table 3), although no quantitative dataon aphid transmission were given (29). This apparent dis-crepancy could be resolved if potato virus Y-D is transmis-sible at a very low level; alternatively, it could be that thecontext within which the glutamate residue occurs may alterthe effect on aphid transmissibility. There are non-aphid-transmissible potyvirus isolates in which the Asp-Ala-Glytriplet is conserved (30, 34). For potato virus Y-18, theresidue after the glycine differs from that in transmissibleisolates (30, Table 3), whereas zucchini yellow mosaic vi-rus-CT differs from an aphid-transmissible isolate in theresidue preceding the aspartate of the triplet (34, Table 3).Whether loss of aphid transmissibility is due to these or othermutations that affect amino acid residues in the N terminusof the coat protein or to mutations in the helper componentgene (36), which codes for a protein essential for aphidtransmission of potyviruses, remains to be determined.The experiments described here demonstrate that certain

amino acid substitutions at specific positions in the N termi-nus of the TVMV coat protein can reduce or abolish aphidtransmissibility of the virus. Comparisons of the effect ofthese substitutions with those in the Asp-Ala-Gly region ofother potyvirus coat proteins suggests that some generalconclusions can be drawn, but it also demonstrates thatfurther mutational analyses in the Asp-Ala-Gly and otherN-terminal residues of the coat protein are needed. Atpresent, it seems likely that any change in the third residueof the Asp-Ala-Gly triplet will result in loss of aphid trans-missibility. This result may be of practical significance in thecreation of virus resistance via the development oftransgenicplants that contain the potyvirus coat protein gene (37).

We thank Shari Dutton for technical assistance. This research wassupported by United States Department of Agriculture CompetitiveGrant 89-37263-4593, USDA-ARS Contract 58-43YK-90023, and a

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