The Complete Nucleotide-Sequences Of The Coat Protein Cistron And The 3' Noncoding Region Of A Newly-Identified Potyvirus Infecting Sweet-Potato, As Compared To Those Of Sweet-Potato Feathery Mottle Virus

Arch Virol (1994) _{139 327-336}

O Springer-Verlag 1994

Printed in Austria

The complete

nucleotide

sequences

of

the coat protein cistron

and the

3'

non-coding

region

of

a newly-identified

potyyirus

infecting

sweetpotato, as

compared

to

those

of

sweetpotato

feathery

mottle

virus

D. Colinet, J. Kummert, and P. Lepoivre

Faculté des Sciences Agronomiques, Laboratoire de Pathologie Végétale,

Gembloux, Belgium

Accepted August 10, 1994

Summary. Complementary

DNA

representing 728 nucleotides of the 3' end

of

the genomic

RNA

of sweetpotato virus G (SPV-G), a newly-identified potyvirus infecting sweetpotato, was cloned and sequenced. This sequence was combined

with that

previously determined

for the 5'

terminal

part of

the coat

protein cistron of the virus. The whole sequence contained a single open reading frame

(ORF)

of

1065 nucleotides,

with

the capacity

to

encode a coat

protein

of

355

amino acids, significantly larger than

that

ol

other potyviruses. The

ORF

was

lollowed

by

an untranslated region

of

222 nucleotides and a

poly

(A)

tail.

The coat protein ol SPV-G was only distantly related

to

that of known potyviruses,

with the

exception

of

sweetpotato feathery

mottle virus

(SPFMV).

Indeed, sequence

identity in

the C-terminal

three quarters

of

the coat protein

(more

than 80){)

and

in

the

3'

untranslated

region (more

Than 70"/.)

indicate

that

SPV-G should be considered as closely related to, though distinct from SPFMV.

This

subset relationship is

similar

to

that

previously reported

for

members

of

the

bean

yellow

mosaic

virus

subgroup

or

the

bean

common

mosaic virus

subgroup.

Introduction

Sweetpotato

may

be

affected

by

several potyviruses

_[12],

among

which sweetpotato

feathery

mottle

virus (SPFMV) and

sweetpotato

latent

virus (SPLV) were recently identified

in

sweetpotato clones originating

from

China, using a combined assay of reverse

transcription

and polymerase chain reaction

(RT-PCR)

with

degenerate

primers derived

from

conserved regions

in

the genome of the potyviruses

_[5,6].

RT-PCR

and sequence analysis of the variable

N-terminal

part of

the coat protein allowed a

third

distinct potyvirus

to

be

identified

in

sweetpotatoes

from China

_[6].

The

name sweetpotato

virus

G (SPV-G) has been tentatively assigned

to

this

virus.

Vîrology

. ::j:.:,,::, _i..

:,:,,j:: _{. ::.:,}

The

use

of

sequence

information

for

the

classification

of

potyviruses is

generally

more

reliable

than

schemes based

on

biological and

serological

properties,

which

reveal apparent relationships

that do not

always correlate

with

one another

_[15,17],

Comparisons of coat

protein

amino acid sequences

and

of

3'

non-coding nucleotide

sequences

have

been used

to

establish

taxonomic relationships among potyvirus members L7, 13, 15, 17, 1 8]. Sequence

comparisons show

a

bimodal

distribution of coat protein

sequence

identity

[15]. Known

distinct

potyviruses

vary

in

coat

protein

amino acid

sequence

similarity

between 38/,

to

7I/",

while known strains of viruses range from 90/"

to

997.

_[15].

Strains of the same virus share

83/"

to 99/"

nucleotide sequence

identity

in the 3' untranslated region, while distinct viruses share generally only

39/" to 53/"

identity

in

this

region

_[7].

In

order

to

further

examine

the

relationship

between

SPV-G and

other potyviruses we determined the nucleotide sequences

ol

the 3' terminal

part of

the coat protein cistron and

of

the

3'

untranslated

region

of

SPV-G. These

sequences were combined

with

that

previously determined

for

the 5' terminal

part

of the SPV-G coat protein cistron

_[6].

The complete SPV-G coat protein

amino acid

sequence

and

3'

non-coding

region

nucleotide

sequence were compared with those of other potyviruses, and more particularly with the Russet

Crack (RC) and the Common (C) strains ol

SPFMV

_[1],

and the Taiwan isolate

of SPLV (SPLV-T)

(D. Colinet, unpubl. results).

Materials

and methods

Plant material

Sweetpotato (I pomoea batatas L.) clone GN58 originated from China, Guangdong province (Dr. Fing ZuXia,Upland Crops Research Institute, Guangzhou) and was maintained in

Gembloux under greenhouse conditions.

RNA extraction and cDNA sYnthesis

Total RNA was extracted from symptomatic ieaves by the method of Chirgwin et al. _[4]. Single stranded cDNA was synthesised lrom 5 pg of totai RNA using the Amersham cDNA Synthesis Kit, with the following modifications:

-

oligo(dT) primer was replaced by 0.5pg of hybrid dT,r-adapter primer (s'-GACTCG-AGTCGACAGCGATTTTTTTTTTTTTTTTT- 3J _[9],

AGTCGACAGCGATTTTTTTTI

-

the reverse transcription reaction 30min. cDNA was diluted 10-fold

was incubated at 42'C for t h, and then at 52'C for

with sterile water.

Amplifcation of cDNA 3' end

Amplification of the cDNA was performed in a voiume

of

100pl of PCR buffer (10mM Tris-HCl

pH

9.0,2.5mM

MgClr,50mM

KCl,0.l7;

Triton x-100) containing 200pM each of dATP. dCTP, dGTP, dTTP, 0.1 nmol each of adapter primer (5'-GACTCGAGT-CGACAGCG-3')

_t9l

and specific primer, and 2.5 units

of

Triq DNA polymerase. Alter denaturation at94"C for 5 min, annealing at 50'C for 5min and elongationatT2oC for

at94"C for 30sec, annealing at 50"C for

lmin

and DNA synthesis

at72'C

for 3 min. A final 15 min elongation step at72'C was performed at the end of the 40 cycles. Amplification

products were analysed by electrophoresis of 10 pl of the reaction mixtures in a

l/,

agarose gel, in trisacetate-EDTA buffer

_[l4].

Bands were visualized by ethidium bromide staining.

Cloning and sequencing of the amplifed _fragment

After electrophoresis in 1/o agarose gel, the amplified fragment was excised and eluted with

the QIAEX Gel Extraction

Kit

from Qiagen. After digestion

with

BamHI and SalI, the DNA fragment was directionally cioned into the Bluescript plasmid.

Nucleotide sequence was obtained by subcloning the amplified fragment after cleavage

with

restriction enzymes. Double-stranded

DNA

sequencing

by

the

dideoxy chain termination method was performed using T7 DNA polymerase (Pharmacia) according to manufacturer's instructions.

Results

Amplifcation

of the

3'

terminal region of SPV-G genome

RACE

(Rapid

Amplification

of

cDNA

Ends) method

_[9]

was used

to

amplily

a

cDNA

fragment corresponding

to

the 3'

terminal

region of SPV-G genome'

The

RACE protocol

allows the 3' and 5' ends

of

a

cDNA to

be amplified

if

a

short

stretch

of

sequence located betrveen the ends

is known.

For

the 3' end,

RNA

is reverse transcribed using a

"hybrid"

primer consisting of oligo(dT) (17

residues)

linked

to a

unique

17-base

oligonucleotide

("adapter"

primer).

P1 HC-Pro P3 6K1 6K2 Nia

POLY(A)

POLY(A)

,/\

cPl .4s _{GATTATG_G_ôI_qCAATGATGAG}

Fig.

l.

Genetic map of the potyvirus genome showing the position and sequence of the

specific primer (CP 1.45) designed from the known sequence of the 5' terminal part of the coat protein cistron. The primer corresponds to codons 187-193 of the coat protein. The hatched box represents the part of the SPV-G genome previously amplified by RT-PCR

with degenerate primers _16). P1 First protein; HC-Pro helper component-protease; P3 third protein; 6K1 first 6K peptide;

CI

cytoplasmic inclusion protein; 6K2 second 6K peptide; Nic nuclear inclusion a protein; Nib nuclear inclusion b protein (RNA polymerase),

CP coat Protein

1.37

-

0.830.56

-Fig. 2. Agarose gel stained with ethidium bromide, showing the result ol amplification ol

the 3' terminal region of SPV-G genome.

I

DNA

size markers, lragment sizes (in kb) indicated on the ieft.2 Product amplified from SPV-G infected Ipontoea setoscr

2

Amplification

is subsequently performed using the adapter

pt'ir"ç.,

which binds

to

each

cDNA

at

its

3' end, and a

primer

specific

to

the gene

of

interest.

Total RNA

was isolated

from

leaves

of

Ipontoea setosa

grflted with

the sweetpotato clone

GN58

and shown

to

be inlected

with SPV-q

but

not

with

SPFMV

and SPLV, using PCR and

hybridization

assays

(unpu[I.

res.).

cDNA

was amplified using oligo(dT, _{,)-adapter and} amplification was caf ried out using

adapter

primer and

a

specific

primer (CP

1.45) designed

iro[n the

known

sequenceolthe5'terminal

partolthecoatproteincistronolSP\-Gt6l(Fig'

l).

The

three nucleotides 10

to

12

ol

the

specific

primer

CP

1.45 _J'vere modified

(GTG

to

TCC)

in

order

to

generate a

BamHI

site

(Fig.

1).

A 7ry

bp lragment was amplified (Fig.

2)

and cloned

into

the Bluescript

plasmid.

I I

Sequence analysis of the

SPV-G

PCR

product

I

Because

of

the

low fidelity ol the

Taq

DNA

polymerase, whicfr

may

lead to

misincorporations, the sequence ol the SPV-G amplified fragmer[t was deduced

from

three

independent clones.

This

sequence

was

combir[ed

with

that previously determined

for

the 5'-terminal

part of

the coat

profiein

cistron

ol

Spv-c

tot

I

The

complete nucleotide sequence

of

the coat protein cist{on and the

3'

untranslated region of SPV-G is presented

in

Fig.

3. There is ar| open reading

frame

(ORF)

extending

for

1065 nucleotides,

with

the capacitYlto encode 355

amino acids. The ORF is followed by an untranslated region o1122 nucleotides

and a

poly(A)

tail. The

DAG

box

involved

in

aphid

transmlssion

_[2]

was

located

at

position 7 of

the coat

protein amino

acid sequence

_loi

SPV-G,

lor

which aphid-transmissibility has been demonstrated (unpubl. ref.).The 3'

non-coding region

of

SPV-G

contains

the

consensus sequence{

AGTGAGG

(position

1238

to

1244) and

CCTC (position

1250

to

1253) se{arated

by

five nucleotides, which

could form a

step-loop structure possibly

hfvine

a

role

in

replication

_[3].

L_

TCT GCT GAA GAG ATA TAC GAT GCA GGA AAA ACA GGA AAC ACA GGA AGG GGA A6A GCA CGA GGT 63

S A E E I Y D A G K T G H T G R G R G R G 21

ACT GTG CCT CCG CCG CCG CCA CCC CCT GGG CCA CCA AGA ACA GGT GAC CTG CCT CCA GCA GTG 1?6

T VP PPP PP PGÀPR T GD L PPAU 12

CAG ACÂ GGA CCA TTA CCA CCA GGT GCA GÇC TCA AAÂ CCA CCT ATC ATT GAG GAA ATT CTG CAG 189

AT GPLP PGAASKPP I I EE I LO 63

CCA GA6 TCA CCG AGA ACG AAG GCA TTG CG6 6AA GCG AGA GGG MÂ GCT CCA GCA ACA ATT CCA 252

P E S P R T KA L R EAR GKAPAT I P 84

GAT AGT AGA GGG GTT GAT ACA TCA CAA ATA CCG AGT TTC ACA CCA GGT AGA 6AC CM ACA ATG 315

D S R G V D T S A I P S F T P G R D O T t't 105 ACA CCA ACC CCT CAA AGA ACA AGC ACT GAG GlG AGA GAT AGA GAT GTG MT GCT GGT ACT GTT 378

T P T P A R T S T E V R D R D V N A G T V 126 G6T ACT TTC ATA GTG CCA CGG CCC CAG ATA ACA CAT AGT ÀAG AAA ACA GCA CCA ATG GCA AAT I'11

G T F I V P R P O I T H S K K T A P I'{ A N 147 GGA AGA ATA GTA GTC AAT CTT GAC CAC TTG ACÂ ATC TAC GAC CCT GAA CM ACA AGT CTT TCA 501

G R I V V N L D H L T I Y D P E O T S L S 168

AAT ACT C6A GCA ACA CAG Gfu\ Cfuq TTT AAT GCT TG6 TAC GAG CGT GTC AGG GM GAT TAT GGA 567

N T R A T O E A F N A IJ Y E G V R E D Y G 189 GTG fudT GAT GAG CA,/\ ATG GGG ATA TTG CTC AAT GGG TTA ATG GTT TGG TGC ATC GAG fu\T GGA 630

V N D E O H G I L L H G L H V I'J C t E N G 210 ACA TCC CCA ,ÙqT ATT A,/\T GGA ATG TGG GTC ATG ATG GAT GGT GAT CAA CM GTT ACA TAT CCA 693

T S P N I N 6 It H V I.t I't D G D E O V T Y P ?31

ATA AAA CCT CTA TTG GAT CAT GCT GTC CCC ACA TTT AGG CAG ATA ATG ACA CAC TlT AGC GAC 756

I K P L L D H A V P T F R O I t"I T H F S O 25?

ATC GCT GAÂ CCG TAT ATT 6AA AAG CGC MT AGG ATC AAG GCA TAC ATG CCG AGG TAT GGT CTA 819

t A E A Y I E K R I,I R I K A Y t'I. P R Y G L 273 CÀA CGA AAC TTG ACG GAT ATG AGT ATT GCG CGA TAT GCG lTC GAT TTC TAT GAG CTG CAC TCG 882

O R II L T D H S I A R Y A F D F Y E L H S 294 AAC ACT CCT GTT C6G GCC AGA GAG GCA CAT ATG CAA AlG AAA GCA GCA 6CA CTT MG AÂC GCA 915

II T P V R A R E A H I.I O H K A A A L K H A 315 CÀÀ AAT CGG TTG TTT 6GT TTG GAC GGA AAC GTC TCC AC6 CAG GAA GAA GAT ACG GAG AGG CAC 1OO8

O }I R L F G L D G H V S T O E E D T E R H 336

ACA ACG ACT GAT GTT ACA AGG A,qT ATA CAT MC CTC TTG GGT ATG AGG GGT GTG CAG TAAACAA 1072

TTTDVTRH I HIILLGHRGVA 355

TATATTGGCTCGTACCTTTTAATTTCAGTTTCGCCTTCAGTGTAATTTAAATTCGTATCTTTCAGTCCCGAAGAACCTGGTTT 1155

GGT GCAGAGCT TAAT GAGGT GT TACCT CTAT CT T T GCATT GGAGAAGGGATCT T T CTAT TACGTAT CAlAAGG6ACT CT TAÀA 1 238

GTGAGGTTTTACCTCGTAAGAÂAÂGCCTTTTTGGTTCGTGATCGAGCC POIY (A) 128é

Fig.3. Sequence of the SPV-G coat protein and 3' untranslated region' The predicted

u-ino

acià sequence

of

the single ORF is presented above the nucleotide sequence' Underlined nucleotides indicate ihe region from which the specific primer was derived.

comparison of the coat protein sequence of

sPV-G with

sPFMV

(-RC

and

-C),

SPLV-T

and other potyuiruses

The SPV-G coat

protein

shared

66/.

and

65/"

identity

with

SPFMV-RC

and

-C,

respectively, a1

the amino acid

level

(Fig.

a).

The

sequence

identity

with

SPLV-T and other

potyviruses

was

less

than 60/". The

C-terminal

three-quarters (residues 116

to

355)

ofthe

SPV-G coat protein sequence shared 83/"

ind

S2/"

identity with

SPFMV-RC and -C,

respectively,

and

less

than 70/"

with

SPLV-T

and other potyviruses

at

the amino acid level'

SPV. G SP FI'IV - RC sPFtlV- C SPLV-T PPV PVY SCMV TEV SPV. G SPFI"IV-RC SP FMV- C SPLV- T PPV PVY SCMV TEV SPV. G SP FMV. RC SP FMV. C SPLV - T PPV PVY scl"tv TEV SPV-G SPFI{V-RC sPFilV-C SPtV. T PPV PVY scHV TEV SPV. G SP FMV. RC SP Ft'IV. C SPLV. T PPV PVY scMv TEV

lslnlile----rY

l.lrlrl*

r E-

-

F K ttu

lslr

c o P E

- -

F K L_l ADET...-IL ADEREDEEEV AID---Tl AG---ÏV sG---TV KTGNTGRGRGRGTVPPPPPPPGAPRT GD AD -.. AN-. KV-.-KP--_ ----svvT GS- - -AG--- GGGGNAGT AD.-OPPATGAAAOGGAO... PPATGAAA..' ilA DT DA DA OA DA

r- [p-F nl v o T G p L [p-Fl c e n s rlF p] r _{[-le [Ël} r L o

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r

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L-ni r

FT

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ol'

K p K

N'lo

ol-

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-lf'l'lrlrlrlv

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nlrln nlnlrl -le e elc K p K G n

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_lloll

Rlol!

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R K K R R K K R nlp n

r iFlofilnlclvlolSo I

P s F T P G R D o T r'l T P T P a n r[s]r e

olro,

_'

tltltltlol*lot

tl*E----

- KEs

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vcnsplxc

olrylvTflebl'Ld*lo r

slx c

-

-

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- E Ks MR

svs

Pa R

PPOTNTPTO-. - -..P6PSGKGKEVI SAKGGE I I RTS

::ll

::::::::::

::l

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O P P T T O G S O.L P - - . O G G A T G G G G A O T G A G G T G S V T G G .-.A6KKKDOKDDKVAEOA

rrM

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IN VD VN VO v t'l T TL SV YC TT TS TF TF TH KI TF TA RO RO RL SL Rl! RL OY ALN II{H VLN VLH VVN A T A A A A I 0 T T T s F D u o D N E D N POITHS VKMNAN VKLHTG LRAIPT TKATITS IKAIÏS LKAI'{SK INATIAT IKCGA VKGKA SKGAT AKGOD 11 RGEY R R R K R H I L H H L L K T K R H H H t H D E E K s K K A s s A A A A A N 0 R N o D D o ï T R A A D A E A TSLS FEVA HNIG LD I T VDLS IDIS ODIS IDLS TRSO P0sc TOSA TREE THEQ YEGV YEGV YEAV YEAI HOAV AK TR ÏR TR AR RE KG KG KN KR R l,l KK MT xlôle o ololt o

tt

olEA G DHOQ TDDE GETE DDTO NEEO I t K H R K R K E D D E G slLl.tN sll.tll pTvltN TVVMS KILLII

LHVIJCIEHGTSPIIING L I.IVIJ C I E N G T S P II I }I G LMVIJCIEII6TSPNI}IG v rJ c

t

E |l GlTls P Nll xlG v !r c

I

E lt GlTls P Nll NIG v rJ c

I

E N CITIS P Nlv Nlc v u c

r

E N Glcls P Nll slG v rJ c

t

E H GITIS P NIL Nlc SPV. G SP F HV. RC sP Fl'rv - c SPLV- T PPV PVY scr,rv TEV SPV- G SP FItV. RC SPFMV. C SPLV. T PPV PVY SCMV ÏEV SPV. G SPFMV. RC SP Ft',lv - C SPLV. T PPV PVY SCMV TEV SPV. G SP FI,IV. RC SP FI.IV. C SPLV. T PPV PVY SCMV TEV VSYPI VEHPI VÊYPL TVFPL VSYPL LIOF LLDH IVEN VIEN MVEN LV Lll LH LH Flt lll I}l VN IS LH DT ET NE Uts ED T T T L T ,[ùlu vlulr ulul' olulv

,lr

lu ululu

rlult

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 K K c o F F L F L o R o o o K tl H M K tl Y H I T T E E K T E Y K S R KH YIA CVE YIE YIE YIE OP KA EP ER RP LO LI LO LQ LT LT LR LT IT lts YS MG YS MS r lnl LIAI

'[!

LA LA LA LA LS XVS }IVG GIS NVG NVG VAR I.IAH I',I H H MT H TEEN OKOD AEEN OEEN AEED DRN I NRNI.{ SPSH SRNI.{ NRHI.I RGI H VT HT VT I't N LT N T T R ï R R K A A I A R K K K R R K R o K H H o o N G H N P T T AT DG ÏE AG AH tt TT TL cl TL VRGV VKNI'I VOOHH VRO

Fig. 4. Alignment of the deduced amino acid sequence of the coat protein ol SPV-G with

those of SPFMV-RC, -C, SPLV-T, and other potyviruses. Dashes indicate the gaps for

optimum alignment. Only identical residues of SPV-G and of both strains of SPFMV and those which are identical in the five other potyviruses are boxed

Comparison of the SPV-G and

SPFMV (-RC

and

-C)

3' non-coding sequences

The 3'

non-coding

portion of

the SPV-G

genome had

the

same

length

(222

nucleotides) and sharedT3%identity

with

those of

SPFMV-RC

and -C

(Fig.5)'

The sequence

identity

between SPV-G and other potyvirus

in

this region was

less

than

55l".

I.IMD GD E OVT Y P I KP L LD HAVP T

I.{MD GD E O VT Y P I K P L L D H AVP T

I.ID GD E OV T Y P I K P L L D H AV P T

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tlolv

s Y P llK PIL

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o K A Y t.{ P R Y G L O R N L T D I'I S KAYI'IPRYGLORNLTDHS KAYMPRYGTARNLTD}'IS Y l,{ P R v clt- oln

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Y I.I P R Y GII OIR NIL TID

Y tl P R Y GIL I IR NIL RID

Y M P R Y GIL olR NIL TID

Y r.t P R Y GIL olR Hll TID

RYAFDFYETHS RYAFDFYELHS RYATDFYELHS R Y A F D F Y EIV TIS R Y A F D F Y EIH TIS R Y A F D F Y EIV TIS R Y A F D F Y EII'I NIS R Y A F D F Y EIL TIS EAHIlOMKAAA

r

n

nlo

H K A A A EAHMOI{KAAA E AIY rla M K A A A LKNA LKNA LKNA LTNT LRNV LKSA VRGS VRNS E AIH I IO M K A A A e

alr

rlo l.r K A A A E AJH IqJO M K A A A E AIH MIO M K A A A NRLFGLDG NRLFGLDG NRLFGLDG RLFGLDG RLFGLDG RLFGLDG RLFGLDG RLFGLDG

NVSTOEEDT ERHT TTDVTRN I HNL LGI'IRGVO ItVS T O E ED T E R H T T TD VT RN I H}I L L GI{R GVO

N V S T O E E D T E R H T T T D V T R II I +I N L L G I'I R G VO T E R H TIA TID V

T E R H TID GID V

rlo e e

xlr

e n n

rlr

elo vls p _{s HIHIT} LIL G

rlo

r r

xlr

r

n H TIA clD vls R N r,{lHlS LIL G

SPV.G TAÂÂCAATATATTGGClCGTACCTT-...T]AATTTCAGTTTC...'6CCTTCAGTGTAATTTAÂATÏCGTATClTTCAGT

SPFI.IV.RC GGAC CCTC CAGT GA ATAC G ATC A6TAT ' G

SPFI{V.C GGAC CCTC CAGT G ATAC T6 ATC AGTAT ' G

SPV. G CCCGAAGAÀCCT 6GT T T GGT GCA6AGCT TAAT GA6GT GT TACCT C TAT C T T T GCAT T GGAGAA6GGAT CÏ T Ï CTAT TACGT

SPFI.IV.RC .GA GA TAAGG GATA T GGTA T TCGC

SPFI.,IV.C .GA GAA lAAGG GATA T GGTA T TCGC

SPV.G ATCATAAGGGACTCTTAA-AAGTGAGG..TTTTACCTCGTAAGAAAAGCCTTTTTTG6TTCGTGATCGAGCC POtY (A)

sPFr.rv-Rc - --AG PotY (A)

sPFMv-c - - -AG _PolY (A)

Fig. 5. Alignment of the nucleotide sequence ol the 3' untranslated region ol the SPV-G

genome with those oISPFMV-RC and -C. Dashes indicate the gaps lor optimum alignment

Discussion

The deduced coat protein amino acid sequence ol the newly identified potyvirus inlecting sweetpotato

lrom

China

(SPV-G) rvas significantly longer

than

that

of otheidescribed potyviruses (Fig. a). Moreover, coat protein sequence identity

between

SPV-G and other

potyviruses was

in

the

range

oi

distinct

viruses.

However, sequence

identity

above

80/"

in

the

conserved

coat

protein

core

region revealèd relatively close relationships

_[1,8]

and allowed the establish-ment of subgroupings

within

the aphid-transmitted potyviruses

ll3, l'lf

. indeed,

il

the

N-terminal

divergent sequences are omitted, the overall

identity

between

the C-terminal three quarters

ol

the coat

protein

ol

SPV-G i5 more

than

80/"

with both

strains

of

SPFMV,

but

less

than

70\

with

SPLV-T and

other potyviruses,

thus

suggesting

that

SPV-G is

more closely related

to

SPFMV

than

to

other

potyviruses.

Thus SPV-G and

SPFMV form

a

closely related subgroup similar to those previously reported

lor

the bean yellow mosaic virus

subgroup

_[16,

18] and the bean common mosaic virus subgroup

_[10,

ll,

16].

For comparison, the two strains

oISPFMV

shared 84l" identity ior the complete

coat protein and

90.5%

identity

in

the C-terminal

three quarters

ol

the

coat

protein at

the

amino

acid level

_[1].

The relationship between SPV-G and both strains of

SPFMV

was confirmed

by the

73ii

sequence

identity

observed

in

the

3' non-coding region,

which

is

Ultow the

â37;

to

99\

identity found

for

accepted strains

of

viruses

_[7],

but

above the

maximum identity

ol

50% generally given

for

distinct

potyviruses.

Such intermediate

level

ol

sequence

identity

is

sometimes observed

within

subgroups such as the bean yellow mosaic virus subgroup [16, 18].

In

contrast, the 3' non-coding regions of

SPFMV

strains RC and C were

98/"

homologous

tll

In

conclusion, coat protein and 3' non-coding region Sequence data indicate

that

SPV-G is closely related

to

SPFMV,

though

it

should be considered as a

distinct potyvirus.

Indeed,

the

length

ol

the SPV-G coat protein

(355 amino

acids) is longer

than

that of SPFMV

(316

amino

acids). Moreover, the levels

both

strains

of SPFMV

are much higher

than

between

SPFMV

and SPV-G.

It

remains

to

be determined whether the sequence identities described above between SPV-G and

SPFMV

are

linked

to

similarities

in

terms

of

biological

or

serological properties.

Acknowledgements

We thank Dr. Semal for helplul discussions and critical reading ol this manuscript and Dr. Burny in whose laboratory part of this rvork was carried out' This work rvas financially supported by the EEC (Project STD3 n'TS3-CT91-0013).

l.

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Authors' address:

D.

Colinet, Faculté des Sciences Agronomiques' Laboratoire de

Pathologie Végétale, 13, Avenue Marechal Juin, 8-5030 Gembioux, Belgium. Received MaY 19' 1994