Download The Complete Nncleotide Sequence of Potato Virus X and Its

J. gen. Virol. (1988), 69, 1789-1798.
Printed in Great Britain
1789
Key words: PVX/nucleotide sequence/potexvirus
The Complete Nncleotide Sequence of Potato Virus X and Its Homologies
at the Amino Acid Level with Various Plus-stranded RNA Viruses
By M A R I A N N E
J. H U I S M A N , 1 H U U B J. M. L I N T H O R S T , 2 J O H N F. B O L 2
AND B E N J. C. C O R N E L I S S E N 1.
1 M O G E N International N. V., Einsteinweg 97, 2333 CB Leiden and 2 State University of Leiden,
Department of Biochemistry, Wassenaarseweg 64, 2333 A L Leiden, The Netherlands'
(Accepted 5 M a y 1988)
SUMMARY
Double-stranded c D N A of potato virus X (PVX) genomic RNA has been cloned and
sequenced. The sequence [6435 nucleotides excluding the poly(A) tract] revealed five
open reading frames (ORFs) which were numbered one to five starting at the 5'
terminus of the RNA. They encoded proteins of Mr 165588 (166K), 24622 (25K),
12 324 (12K), 7595 (SK) and 25 080 (coat protein), respectively. ORFs 1 and 2 were inphase coding regions. The ORF 1 product contained domains of homology with the
tobacco mosaic virus 126K and 183K products. The ORF 2 and 3 products showed
homologies with the barley stripe mosaic virus 58K and 14K proteins, the beet necrotic
yellow vein virus 42K and 13K products and the white clover mosaic virus 26K and
13K products, respectively. The significance of these homologies with respect to
putative functions of the PVX-encoded proteins are discussed.
INTRODUCTION
Potato virus X (PVX) is the type member of the potexvirus group. The flexuous rod-shaped
virions (Stols etal., 1970) contain a single-stranded RNA of 2.1 x 106 Mr (Bercks, 1970). At its 5'
terminus the RNA contains a mTGpppG cap structure (Sonenberg et al., 1978) and at its 3' end a
poly(A) tract (Morozov et al., 1983). In a rabbit reticulocyte cell-free system the genomic R N A is
translated into two predominant polypeptides with estimated Mr of 145K and 180K. It has been
suggested that the larger protein arises by readthrough of a 'leaky' termination codon (WodnarFilipowicz et at., 1980). PVX virions contain solely the genomic RNA. However, in infected
tissue two major subgenomic RNAs of 0.9 kb and 2.1 kb are present as well as four minor ones of
1.4, 1.8, 3.0 and 3.6 kb. All these subgenomic RNAs are T-coterminal (Dolja et al., 1987). It has
been suggested that in vivo the coat protein is translated from the 0.9 kb subgenomic RNA.
The nucleotide sequence of the genomic R N A of PVX has been partly determined; the 5'terminal 80 bases as well as 1300 nucleotides of the 3' end have been elucidated (Morozov et al.,
1983, 1987). Morozov et al. (1987) analysed the amino acid sequence of the proteins encoded by
the three open reading frames (ORFs) at the 3' end of the genome. They observed first of all that
the coat protein sequence of PVX showed homologies with coat protein sequences of other
potexviruses and even potyviruses, Secondly, they observed homologies between the PVX 12K
protein and the barley stripe mosaic virus (BSMV) 14K and the beet necrotic yellow vein virus
(BNYVV) 13K proteins. And thirdly, they observed homologies between the 8K protein and the
sequence of a similar protein encoded by potato aucuba mosaic virus (another potexvirus). In
this paper we report the complete nucleotide sequence and the organization of the PVX genome.
The deduced amino acid sequences of the PVX-encoded proteins are compared with those of
other viruses with capped plus-stranded R N A genomes. The possible functions of the PVXencoded proteins are discussed.
0000-8330 © 1988 SGM
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1790
M.J.
HUISMAN AND OTHERS
METHODS
Enzymes were purchased from Bethesda Research Laboratories and Amersham, and chemicals from Sigma.
Radiochemicals were from Amersham. Computer programs used were from the Genetics Computer Group of the
University of Wisconsin, Madison, Wis., U.S.A.
Virus purification and isolation of viral RNA. PVX (strain X3) was isolated from Nicotiana tabacum cv. Samsun
NN plants 2 weeks post-inoculation. Infected material was homogenized in 0-1 M-Tris-citric acid pH 9.0, 0.25 ~ 2mercaptoethanol. The supernatant was treated with 0.75 volumes of chloroform/n-butanol (1:1 v/v). The virus was
precipitated from the water phase by addition of 0.2 volumes of 3 0 ~ (w/v) polyethylene glycol. After
centrifugation the pellet was resuspended in 0.1 M-Tris~:itric acid pH 9.0. This suspension was ultracentrifuged
twice to purify the preparation. The virus was stored in aliquots at a concentration of 26 mg/ml in Tris-citric acid
pH 9.0 at - 8 0 °C. Purified virions were phenol-extracted twice in 2 ~ SDS, 1 x SSC (150 mM-NaH2PO4, 15 mMsodium citrate pH 7-0). The RNA was ethanol-precipitated twice, dissolved in H20 and stored at - 2 0 °C.
cDNA synthesis and cloning in 2gtlO. cDNA synthesis and cloning was performed precisely as described in the
instructions for use of the Amersham kits for cDNA synthesis and ,tgtl0 cloning, respectively. For first strand
cDNA synthesis an oligo(dT) primer was extended on PVX RNA by the use of reverse transcriptase. For the
second strand synthesis RNase H, Escherichia coli DNA polymerase I and T4 DNA polymerase were used (Gubler
& Hoffman, 1983). The double-stranded cDNA was treated with EcoRI methylase, provided with EcoRI linkers
and ligated to the EcoRI-digested 2gtl0 arms. The DNA was packaged in vitro to obtain phage particles and
recombinant phages were plated on E. coil NM514.
Isolation o f 2gtlO clones. The cDNA library was screened as described (Davis et al., 1986). Hybridization was
carried out at 55 °C with an oligonucleotide primer (corresponding to bases 6004 to 6024) and at 65 °C with nicktranslated recombinant plasmids. The hybridization solution used contained 5 x SSC, 5 x Denhardt's solution,
10~ dextran sulphate, 0.1 ~ SDS and 200 ~tg/ml denatured calf thymus DNA (Davis et al., 1986). Filters were
washed in 0.1 x SSC, 0.1 ~ SDS at the same temperature as was used for the hybridization. Phage DNA was
obtained essentially as described (Davis et al., 1980). For further analysis the phage inserts were subcloned into
pUC9. Subclones of PVX cDNA fragments in pUC9 were transformed to E. coli DH5ct according to the method
described by Dagert & Ehrlich (1979). Plasmid DNA was isolated as described by Holmes & Quigley (1981).
cDNA sequencing, cDNA sequencing was done by the dideoxy method (Sanger et aL, 1977) using [~t-35S]dATP
(Biggin et al., 1983). The cDNA was subcloned into M13 tg130/131 vectors (Kieny et al., 1983). Overlapping
restriction fragments were subcloned and sequenced to cover the sites that were used for subcloning. Most of the
DNA was sequenced in both orientations.
RNA sequencing. Direct sequencing of PVX R N A was used to sequence across the internal EcoRl sites in the
genome, across some of the sites that were used for subcloning into M 13 vectors, for parts of the genome devoid of
useful restriction sites and to obtain the sequence of the 5' terminus. The procedure used was as follows. PVX
RNA was annealed to a suitable primer (in a 100-fold molar excess) in a buffer containing 50 mM-Tris-HCl pH 8-3,
10 mM-MgCI2, 40 mM-KC1 by heating the mixture at 95 °C for 5 min and a subsequent incubation at 40 °C for 30
rain. Dithiothreitol to a concentration of 10 mM, 2.5 units of reverse transcriptase and 10 IxCi [ct-35S]dATP (sp. act.
600 mCi/mmol) were added. This mixture then was added to the termination mixtures. The final concentrations
for dCTP, dGTP and dTTP were 62.5 I~Mand for dATP 12.5 p.M. Each termination mixture contained one of the
dideoxy NTPs ddCTP, ddGTP or ddTTP at a final concentration of 18.75 ~tvl; in the case of ddATP the final
concentration was 6.25 ktM. The samples were incubated for 30 min at 40 °C and were chased with 0.3 volumes of
0-5 mM of all dNTPs.
RESULTS
Nucleotide sequence o f P V X genomic R N A
Double-stranded cDNA of PVX was synthesized and cloned into 2gtl0. Inserts of selected
clones w e r e s u b c l o n e d as E c o R I r e s t r i c t i o n f r a g m e n t s i n t o p U C 9 . T h e s e s u b c l o n e s w e r e u s e d for
f u r t h e r analysis. T h e P V X c D N A clones p X 5 1 , p X 2 5 a n d p X 6 3 3 a p p e a r e d to s p a n m o s t o f t h e
P V X g e n o m e (Fig. 1). T h e n u c l e o t i d e s e q u e n c e o f t h e s e t h r e e c l o n e s w a s d e t e r m i n e d b y use o f
t h e d i d e o x y s e q u e n c i n g m e t h o d o n r e s t r i c t i o n f r a g m e n t s s u b c l o n e d i n t o M 1 3 p h a g e s . T h e 29
b a s e s a t t h e e x t r e m e 5' e n d w e r e n o t i n c l u d e d in c l o n e p X 6 3 3 a n d w e r e d e t e r m i n e d b y d i r e c t
d i d e o x y s e q u e n c i n g o f t h e R N A b y e x t e n s i o n o f a s y n t h e t i c o l i g o n u c l e o t i d e c o m p l e m e n t a r y to
b a s e s 76 to 94. T h e c o m p l e t e n u c l e o t i d e s e q u e n c e o f P V X R N A is s h o w n in Fig. 2. C o m p a r i s o n
o f t h i s s e q u e n c e w i t h t h e p a r t i a l s e q u e n c e s r e p o r t e d before, i.e. t h e 5 ' - t e r m i n a l 80 n u c l e o t i d e s
a n d t h e 3 ' - t e r m i n a l 1300 n u c l e o t i d e s , r e v e a l s m i n o r d i f f e r e n c e s (Fig. 2). A t p o s i t i o n 67/68 we
f o u n d a G A s e q u e n c e w h e r e a s M o r o z o v et al. (1983) n o t e d a n A G s e q u e n c e a t t h a t p o s i t i o n .
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Nucleotide sequence of P V X
Lf
1791
166K
I
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I
723
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3349
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3349
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6437
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29
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pX633
1 kb
Fig. 1. Schematic representation of the organization of the PVX R N A genome and the localization of
the cDNA clones used to determine the nucleotide sequence. The 5' end of the PVX RNA contains a
mTGpppG cap structure (D,) and the 3' end a poly(A) tail (An). CP, Coat protein.
Since the first A U G codon is found at base 84 it is unlikely that this difference represents a
significant deviation. The differences in the 1300 nucleotides at the 3' terminus as compared
with the sequence reported by Morozov et al. (1987) include an insertion of a G G A triplet coding
for a glycine residue. The relevance of this amino acid insertion is discussed in the paragraph on
homologies.
Coding sequences on the P V X genome
The PVX genomic R N A contains five ORFs (Fig. 1). These are preceded by an 84 base
leader sequence. The first ORF (ORF 1) extends from the A U G at base 85 until the U A A stop
codon at base 4453 and encodes a protein of Mr 165 588 (166K). This cistron comprises about
two-thirds of the coding capacity of the virus. In phase with this first ORF a second one is found,
which starts at position 4486, 11 triplets downstream of the ORF 1 UAA stop, and ends at the
UAG codon at position 5164. ORF 2 thus encodes a protein with an Mr of 24622 (25K).
Seventeen bases upstream of the U A G stop codon of ORF 2 the translational start of a third
ORF is found, which thus partly overlaps ORF 2. The stop signal (UAG) for ORF 3 resides at
base 5492, and hence the ORF codes for a protein of Mr 12323 (12K). This 12K cistron shows
not only an overlap with ORF 2 at its 5' terminus but also an overlap with ORF 4 at its 3'
terminus (Fig. 1). This T-terminal overlap is 68 bases in length. The ORF 4 translational start
and stop signals are located at positions 5427 and 5637, respectively; this ORF encodes an Mr
7594 (8K) product. The last ORF on the PVX genome starts at an A U G codon 13 bases
downstream of the U G A stop codon of ORF 4. This last ORF encodes the PVX coat protein (Mr
25 080). The translational stop (UAA) at position 6361 is followed by an untranslated region of
76 nucleotides and the poly(A) tract.
Homologies found with other plus-stranded R N A viruses
Recently, the complete nucleotide sequence of another potexvirus, white clover mosaic virus
(WC1MV), has been elucidated (Forster et al., 1988). The organization of the WC1MV genome is
identical to that of PVX. Comparison of the amino acid sequence of the PVX-encoded products
with those of WCIMV shows the highest homology between the ORF 1-encoded proteins, i.e.
66 % by the computer program G A P (data not shown). Comparison of the primary structure of
the PVX ORF 1 product with the 126K and 183K proteins of tobacco mosaic virus (TMV) shows
that two out of three domains of homology that are found in the comparisons of these TMVencoded proteins with those of other viruses (Haseloff et al., 1984; Cornelissen & Bol, 1984;
Ahlquist et al., 1985) are also present in the 166K protein of PVX (Fig. 3). The C-terminal
domain of homology in the TMV 183K readthrough protein is characterized by the sequence
motif S / T G - - - T - - - N S / T [18 to 37 amino acids (aa)]GDD. This motif is conserved in virusencoded proteins putatively involved in R N A replication of most animal and plant plusstranded R N A viruses (Kamer & Argos, 1984; Cornelissen & Van Vloten-Doting, 1988). The Cterminal portion of the PVX 166K protein contains the sequence T G - - - T - - - N T ( 2 2 aa)GDD
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1792
M.J.
HUISMAN
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Fig. 2. The complete nucleotide sequence of IP:
the 88.99.165.207
genomic RNA of PVX; the amino acid sequence
On: Fri, 16 Jun 2017 08:42:16
Nucleotide sequence of P VX
1793
3361
K K O I G O V L I I N Y Q K A M G [ P K
~GAAGGACAUUGGGGACGUUCUI;UUUUUAAA(UACCAAAAAGCUAUGGGUUUGCCCAAA
l S E T I l 5 R H G V E F V K P C Q V T
4981 6AGUCUGAGACAAC~
,EUGUCCACGCAUGGUCUU
A
ifU
ifUUGUUAAGCCCUGCCAAGUGACU
3421
E R I P F S Q E V W ~ A I A H [ V Q S K
GAGCGUAUUCCUUUUUCCCAAGAGGUCUGGGAAGCUU(;U(;CCCACGAAGUACAAAGCAAG
G l [ L K V V T I V S A A P I E E I G O
5041 GGACUUGAGUUGI~AGUAGUCACUAUUGUGUCUGCCGCACCAAUAGAGGA-~AUUGGCCAG
3481
Y L S K S K C N L I N G I V R Q S P D F
UACCUCAGUAAGUCAAAGUGCAA(UUGAUCAAUI;{;{;A{UGUGAfiACAGAGCCCAGACUUC
S T A F Y N A I T R S K G t T Y V RAG
M S A 0 G
5101 UCCACAGCUUUCUACP&CGCUAUCACCAGGUCAAAGGGAUUGACAUAUGUCCGCGCAGGG
I
AC
3541
D E N K I M V F L K S Q W V I K V E K L
GAUGA~CAAGAUUAUGGUAUUCCUCAAGUCGCAGUGGGUCACAAAGGUGGAAAAACUA
3601
G L P K I K P G Q I I A A I Y Q Q I V M
GGUCUACCCAAGAUUAAGCCAGGUCAAACCAUAGCAGCCUUUUACCAGZAGACUGUGAUG
3661
L F G I M A R Y M R W F R Q A F Q P K E
CUUUUUGGAACUAUGGCUAGGUACAUGCGAUGGUU(A(;ACAGGCUUUCCAGCCAAAAGAA
3721
GUCUUCAUAAACUGUGAGACUACGCCAGAAGACAUGUCUGUAUGGGCCUUGAACAACUGG
VFIN6
I H * R L I (DA) P V N S E K V Y I V L G L S F
5161 ACAUAGACUGACCGCUCCGGUCAAUUCUGAAAAAGUGUACAUAGUAUUAGGUCUAUCAUU
A
A l V S I T F L L S R N S L P H V G D N
522] UGCUUUAGUUUCMUUACUUUCCUGCUUUCUAGAAAUAGUUUGCCCCACGUCGGUGAC~
C U
(L)
I H S L P H G G A Y R D G 7 K A I L Y N
5281 CAUUCACAGCUUGCCACACGGAGGAGCUUACAGAGACGGCACCAP&GC~UCUUGUAC~
TTP~DMSVWALNN~
U
A
UU
---
S P N l G S R V 5 l H N G K N A A F A A
5341 CUCCCC~(UU~UCUAGGGUCACGAGUGAGUCUACACAACGGAAAGAACGCAGCAUUUGCUGC
A
3781
NFSRP
L A N D Y I A F D Q S Q D G
~UUUCAGCAGACCUAGCUUAGCUAAUGACUACACAGCUUUCGACCAGUCUCAGGAUGGA
3841
AMLQF
V L K A K H H C I P E E I I
GCUAUGCUGCAAUUUGAGGUGCUCAAAGCCAAGCACCACUGCAUACCAGAGGAAAUCAUC
3901
QAYIO
K T N A Q I F L G T C S I M
C~GCAUACAUAGACAUUAAGACCAAUGCACAGAUUUUCCUAGGCACAUUAUCGAUUAUG
3961
RLTGE
P T F D A N T E C N I A Y T
CGCCUGACUGGUGAGGGUCCCACUUUUGAUGCAAACACUGAGUGCAACAUAGCUUACACC
V I K I T G E S I T V L A C K L D A E T
5521 GUGUCAUC~GAUUACUGGGGIU~UCAAUCACAGUGUUGGCUUGCA~&UUAGAUGCAG~
A
4021
H I K F D
P A G T A Q V Y A G D D S A
CACAC~GUUUGACAUCCCAGCCGGAACUGCUCAAGUUUAUGCAGGAGACGACUCCGCA
I
A 1 A D L K P L S V E R L S F H *
5581 CUAUAAAAGCCAUUGCCGAUCUCAAGCCACUCUCCGUUGAACGGUUAAGUUUCCAUUGAU
C CG
4081
LDCVP
V K H S F H R L E D K L L L
CUGGAUUGCGUUCCAGAAGUGAAGCAUAGUUUCCACAGGCUUGAAGACAAAUUACUCCUC
M S A P A S T T Q A T G S T T S T
564] ACUCG~GAUGUCAGCACCAGCUAGCACAACACAGGCCACAGGGUC~CUACCUC~CU
4]4]
K S K P V I T Q Q K K G S W P E F C G W
~GUC~GCCUGU~UCACGCAGCAAAAGAAAGGCAGUUG~CCUGAGUUUUGUGGUUGG
5701
4201
L I T P K G V M K D P I K L H V S L K L
CUGAUCACACCAAAAGGGGU~UGAAAGACCCAAUUAAGCUCCAUGUUAGCUUA~UUG
5761
4261
A E A K G E L K K C Q D S Y E I D L S Y
GCCG~GCU~GGGUG~CUC~G~AUGUCAAGAUUCCUAUGAAAUUGAUCUGAGUUAU
5821
A Y D H K D S L H D L F D E K Q C Q A H
4321
GCCUAUGACCAC~GGACUCUCUGCAUGACUUGUUCG~UGAGA~CAGOGUCAGGCACAU
4381
T L T C R T L I K S G R G T V S L P R L
ACACUCACUUGCAGGACACUAAUCAAGUCAGGGAGAGGCACUGUCUCACUUCCCCGCCUC
M E V N T Y L N A ] I L
V L L L T L L I Y G S K Y I S Q R N H T
540t CGUUUUGCUACUGACUUUGCUGAUCUAUGGAAGUAAAUACAUAUCUC~CGCAAUCAUAC
A
A
C
(1)(F)
V L V V T I I A V I S T S L V R T E P C
C A C G /¢ N 8 S 5 H *
5461 UUGUGCUUGUGGUAACAAUCAUAGCAGUCAUUAGUACUUECUUAGUGAGGACUGAACCUU
UC UC
(K)
Q A A W D L V R H C A O V G S S A Q T E
5881 CAGGC~CUUGGGACU~G~AGACACUGUGCUGAUGUGGGAUCAUCUGCUCAAACAGAA
AG
C
5941
4441
R N F L *
M D I L I
AG~CUUUCUUU~CCGUUAAUUUACCUUAUAGAUUUGAAUAAGAUGGAUAUUCUCAUC
4501
AG~GU~G~GUUUAGGUUAUUCUAGGACUUCUAAAUCUUUAGAUUCAGGACCUUUG
S S E K S L G Y S R T S K S L O S G P L
V V H A V A G A G K S I A L R K L I L R
GUAGUACAUGCAGUAGCCGGAGCAGGUAAGUCCACAGC~CUAAGGAAGUUGAUCCUCAGA
4621
H P T F T V H T L G V P D K V S I R T R
CACCC~CAUUCACCGUGCAUACACUCGGUGUCCCUGALAAGGUGAGUAUCAGAACUAGA
6121
6181
4681
G I Q K P G P I P F G N F A I L D E Y T
GGCAUACAG~GCCAGGACCUAUUCCUGAGG~(AAUUUEGCAAUCCUCGAUGAGUAUACU
474]
L D N I I R N S Y ~ A t l A D P Y Q A p
UUGGAC~CACCACAAGGAACUCAUA~CAGGCACUUUUU&CUGACCCUUAUCAGGCACCU
4801
4861
4921
(A)
R I D T G P Y S N G [ S R A R L A A A I
AUGAUA~U~AGGUCCUUAUUCC~CGGCAUCAGCAGAGCUAGACUGGCAGCAGCMUC
G
C CG
C
K E V C T L R Q F C M K Y A P V V W N W
6001 ~GAGGUGUGCACACUUAGGC~UUUUGCAUGAAGUAUGCUCCAGUGGUAUGG~CUGG
C
6061
4561
T T K T A G A T P A T A S G L F T I P D
ACCACGAAAACUGCAGGCGC~CUCCUGCCACAGCUUCAGGCCUGUUCACCAUCCCGGAU
A
A
(V)
(D)
G D F F S T A R A I V A S N A V A T N E
GGGGAUUUCUUU~UACAGCUCGUGCCAUAGUAGCCAGCAAUGCUGUCGC~CAJ~U~G
C
UG
G
C U
(E)
(N~
(T)
D L S K I E A I W K D M K V P T D T M A
GACCUCAGC~GAUUGAGGCUAUUUGGAAGGACAUG~GGUGCCCACAGACACUAUGGCA
G
C
A
A
M L T N N S P P A N W Q A Q G F K P E H
AUGUU~CU~C~CAGUCCACCUGCUAACUGGCAAGCACAAGGUUUC~GCCUGAGCAC
CG
G
K F A A F O F F N G V T N P A A I N P K
AAAUUCGCUGCAUUC~CUUCUUC~UGGAGUCACCAACCCAGCUGCCAUCAUGCCCAAA
[ G l
I 8 P P 8 E A E M N A A O T A A F
GAGGGGCUCAUCCGGCCACCGUCUGAAGCUGAAAUGAAUGCUGCCCAAACUGCUGCUUUU
U
C
V K I T K A R A O 5 8 D F A S L D A A V
6241 GUG~GAUUAC~GGCCAGGGCACAAUCCAACGACUUUGCCAGCCUAGAUGCAGCUGUC
6301
T R G R I l G T T 1 A [ A V V T l P P P
AI:UCGA~CI/CGUAI)CACUGG,q,4Cq/~C~C(~GCUGAGGC UGUUGUCACUCUACCAC CACCA
6361
UAACUACGUCUAC
AUAACCGACGCCUACCCCAGUUUCAUAGUAUUUUC
UGGUUUGAUUGU
6421
AUGAAUAAUAU~UAn
E F S L E P H F Y L [ I S F R V P R K V
GAGUUUAGCCUAGAGCCCCACUUCUACUUGGAAAEAUCAUUUCGAGUUCCGAGGA~GU6
N S ~ l L G tl
GCAGAUUUGAUAGCUGGCUGUGGCUU((JAUUU((;A(;AIUAALULA(A(;GAAGAAGGGCAU
A
0
L
I
A
6
£
~
I
U
I
I
1
6436
L E I T G 1 t K G I~ l I (; K V I A I I1 [
UUAGAGAUCACUGGCAUAUUCAAAGGG(
L(.( UA( UU{,(;AAA(;GUGAUAGCCAUUGAUGAG
derived therefrom is written above. The nucleotide differences between this sequence and the one
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by acid sequence are
published by Morozov et al. (1983,
1987) are
underneath,
and the effects on the amino
given in parentheses above the deduced amino
sequence.
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M. J. H U I S M A N A N D O T H E R S
and hence it fits the conserved motif. The middle part of the 166K protein contains a domain of
homology with the C-terminal part of the TMV 126K protein; the homologous domain starts
with the G -- G- GKS sequence which has been found to be conserved as G . . . . GKS/T in various
ATP- and GTP-binding proteins (Zimmern, 1987, and references therein). Interestingly, the
G - - G - G K S / T sequence motif is also found in the N-terminal part of the 25K protein (Fig. 4).
This protein bears considerable homology to the C-terminal half of the BNYVV 42K protein
(Bouzoubaa et al., 1986), to the C-terminal two-thirds of the 58K protein of BSMV (Gustafson &
Armour, 1986) and to the WC1MV 26K protein (Forster et al., 1988). The start of this extensive
region of homology is marked by the G - - G - G K S / T sequence motif (Fig. 4). The ORFs of
BNYVV, BSMV, WC1MV and PVX coding for the 13K, 14K, 13K and 12K proteins,
respectively, show appreciable homology at the aa level, which is centred around the sequence
motif GD(7 to 8 aa)GG-YBDGS/TB (B stands for K or R) (Gustafson & Armour, 1986;
Bouzoubaa et al., 1987). This homology has been observed before (Morozov et al., 1987);
however, because our sequence has an extra G G A triplet the extent of the homology has
considerably widened, since in our sequence of the PVX 12K protein, both the middle glycine
residues of the sequence motif are present (Fig. 5). The extra G G A codon in our PVX 12K
cistron may reflect a difference between the PVX isolates used by Morozov et al. (1987) and ours.
If so, the question arises as to the biological meaning of conserved aa sequences such as are
found in the sequence motif observed in the PVX 12K homologous proteins mentioned above.
Unfortunately, no evidence is presently available about whether or not these smaller ORFs are
expressed in vivo.
The PVX ORF 4 product (the 8K protein) shows homologies to similar proteins encoded by
other potexviruses, notably WC1MV and potato aucuba mosaic virus (Forster et al., 1988;
Morozov et al., 1987). The PVX coat protein has been demonstrated to have extensive aa
sequence homologies with some potexvirus and potyvirus coat proteins (Sawyer et al., 1987;
Morozov et al., 1983, 1987; Short et al., 1986; Forster et al., 1988).
DISCUSSION
Five ORFs are found in the sequence of the genomic RNA of PVX. However, upon addition
of PVX genomic RNA to an in vitro translation system the major products are just two large
polypeptides (Wodnar-Filipowicz et al., 1980). This has been suggested to be the result of
translational readthrough of a leaky stop codon. At the end of the 5'-proximal ORF which
encodes the 166K protein an ochre stop codon was revealed (Fig. 2). Readthrough of this stop
codon would lead to the production of an Mr 191480 (191 K) protein. Translational readthrough
of the ochre stop codon would lead to the production of both the 166K and the 191K product.
These two proteins could very well correspond to the 145K and 180K polypeptides found in
translation studies in vitro (Wodnar-Filipowicz et al., 1980). However, three considerations seem
to argue against translational readthrough in vivo. Firstly, to date, translational readthrough of
an ochre stop codon has never been observed. Secondly, the translational start for the 25K
protein is located 1971 bases upstream of the poly(A) tract, so the observation of a major
subgenomic RNA of 2.1 kb strongly suggests that this RNA is the subgenomic messenger for the
25K ORF 2 product (Dolja et al., 1987). The third argument against readthrough originates from
comparison of the PVX genome organization to that of WC1MV (Forster et al., 1988) where the
amber stop codon at the end of the 147K protein is followed, out of phase, by the second ORF
coding for a 26K protein.
Upon translation in vitro of the PVX RNA the predominantly detected products are two large
polypeptides. None of the other ORFs seems to be translated from the genomic RNA. Another
way to express cistrons is by producing subgenomic RNAs. Such RNAs have been detected for
other potexviruses e.g. daphne virus X (Guilford & Forster, 1986), narcissus mosaic virus (NMV;
Short & Davies, 1983), WCIMV (Forster et al., 1987) and clover yellow mosaic virus (Bendena et
al., 1987). For NMV and the M isolate of WC1MV the subgenomic RNA for the coat protein is
encapsidated, whereas for other potexviruses this has not been ascertained. As was pointed out
before, the ORF 2 product might well be translated from the 2.1 kb major subgenomic RNA,
whereas the coat protein might be translated from the other major subgenomic RNA of 0.9 kb
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Nueleotide sequence of P VX
1795
PVX
R\ '~
\ \
\
\ \ ",\\
//
//
/
/
\
\
\
~
/
\
\\ NX
\
\
X
166K
X
\
TMV
183K
.-/126K
~ $//
/ //
1I /
WC1MV
147K
Fig. 3. Schematic representation of regions of homology between the C-terminal domains of the T M V
126K and 183K proteins, and the two domains of the PVX 166K protein and the W C I M V 147K
protein. Black boxes represent homologies.
(a)
BSMV 58K
261
BNYVV42K
111 K ~ S D W T ' - A
w ~ v ~
,6
A L K P
T ~ ~
V
F L T
S G V P G S G K S T I V
R GV~ L I G ~ P I G ~ G
~l_~N~t!l~lV
v ~ ~]~1
T
•
KS T S
.
•
292
D
F
144
~ ~ ~ ~ ~ ~/Vl ~ a ~1. ~[L21~ ~ ~.[!1 49
(b)
BSMV58K
BNYVV 42K
,~I D E Y T~RLAE S A F ~ L ~ Q R RHR~S M~L L v F ~ v ~ . .
364
186 [ L VID ~ ~ E VH M
~| |~ V ~T A ~1 G HILIG V K NC I IV FI:2IPI:I:I:IL IN 222
330
WC1MV26K 79
II L D E ¥1G Q L P LLTJD L n SL~F E, I ~ f ~ D p y 0 , , l ~ E ] , ~ , l , ,
115
Fig. 4. Parts (a) and (b) show alignments o f two stretches of a m i n o acid sequences from the PVX 25K
(ORF 2) product with the 26K protein of W C I M V and the C-terminal parts of the B N Y V V 42K and the
BSMV 58K products. The homologous amino acid residues are boxed.
BSMV 14K
35 H ~ T
BNYVV 13K
35 H K T H S
PVX 12K
371N S L P H V G D . N I H S L P H [ G G A
MPVX 12K
37 N S L P H V G D
WCIMV 13K
33
Sequencemotif
E S ~ ~ D . N I H K F A N
DY
GVPTF
SN
N I H S L PIL
voo
G D . . . . . . . .
G G~I~Y R D G T R]S A D FIN S] 66
Y R DG
T KA
I L Y N S167
G A Y R D G T K A I L/~_~
65
G~Y
62
K D G T K
~
R
S R
G G . Y / D G / /
K
T K
Fig. 5. Alignment of the amino acid sequence of the PVX 12K (ORF 3) product with the published PVX
sequence (Morozov et al., 1987) ( M P V X 12K) and the BSMV 14K, the B N Y V V 13K and the WC1MV
12K products. A m i n o acid residues homologous to the P V X sequences are boxed.
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1796
M. J. H U I S M A N A N D O T H E R S
(Dolja et al., 1987). It is possible that the two small virus-encoded products of 12K and 8K are
translated from the 1.8 and 1.4 kb minor subgenomic RNAs, respectively.
Sequence comparisons have offered the opportunity to evaluate newly elucidated sequences,
and, in particular, to address the question of which functions the ORF products perform in the viral
replication cycle. The homologies between the PVX 166K and the TMV 126K/183K proteins
suggest a common function. The C-terminal domains homologous to those of the TMV 126K and
183K proteins are present in products encoded by other plus-stranded R N A viruses
(Cornelissen & Bol, 1984; Haseloff et aL, 1984; Ahlquist et al., 1985). Comparison of the aa
sequence of the PVX 166K protein with the TMV 126K and 183K proteins shows that the 166K
protein contains both these two blocks of homology. In this respect the PVX 166K protein
resembles the 237K protein of BNYVV (Bouzoubaa et al., 1987) which also contains both these
blocks of homology on one protein. The C-terminal domain of the TMV 183K protein is thought
to be of functional importance to the viral replicase (Kamer & Argos, 1984). Therefore, it is
conceivable that the PVX 166K protein is involved in viral replication. The comparison of
sequences gives us reason to believe that the PVX 25K, WCIMV 26K, BNYVV 42K and BSMV
58K proteins probably have some common function, although we do not know which function.
The same holds for the comparison of the PVX 12K, WC1MV 13K, BNYVV 13K and BSMV
14K proteins. The homologies observed between BNYVV, BSMV and the potexvirus-encoded
proteins suggest a closer evolutionary relationship between these viruses than hitherto assumed.
One of the PVX-encoded proteins performs a function in cell-to-cell spread of the virus. For
TMV this transportation function was elegantly shown to reside on the 30K protein (Meshi et
al., 1987). LS 1, a mutant of TMV is temperature-sensitive (ts) in cell-to-cell spread of the virus
(Nishigushi et al., 1978, 1980). The mutation responsible for the ts phenotype appeared to be a
single aa change in the 30K protein (Ohno et al., 1983; Meshi et al., 1987). Taliansky et al. (1982)
have shown that at the restrictive temperature systemic spread of LS1 could be observed
provided the leaves had been preinoculated with PVX. This suggests that the 30K protein
function can be provided in trans by a transportation function located on the genome of PVX.
Proteins encoded by the PVX genome that might fulfil a function in viral spread are the 25K,
12K and 8K proteins, because these proteins have not as yet been definitely assigned functions.
Initiation of encapsidation from the 5' end onwards would explain why the PVX subgenomic
RNAs are not encapsidated. The coat proteins of PVX, WCIMV and another potexvirus papaya
mosaic virus (PMV) show considerable aa sequence homologies (Short et al., 1986; Sawyer et al.,
1987; Harbison et al., 1988). For PMV it has been reported that the 5'-terminal sequences of the
genomic R N A can be folded into a secondary structure that could interact with the PMV coat
protein in such a way that in vitro assembly into virions could be observed (Lok & AbouHaidar,
1986). It can be envisaged that PVX and WC1MV RNAs are encapsidated in a similar fashion.
WC1MV R N A has been predicted to fold into a secondary structure resembling the one
described for PMV (Forster et al., 1988). Unlike the predictions for PMV and WCIMV the
computer program FOLD predicts an unpaired 5' end up to nucleotide 32 for the 5'-terminal 150
nucleotides of the PVX genomic RNA (data not shown). However, since the computergenerated secondary structures still lack experimental or phylogenetic support, it is premature to
speculate about the biological meaning of the differences found in the Y-terminal secondary
structures.
To make sure that the clones described represent a viable PVX molecule we are currently
placing the full length c D N A behind the T7 promoter by the use of a synthetic oligonucleotide
containing the T7 promoter sequence (Janda et al., 1987). This will enable us to produce R N A
transcripts in vitro which can be tested for infectivity.
Ms Anne-Marie Krebbers is gratefullyacknowledgedfor her endurance in preparing the manuscript. This work
was sponsored in part by STW with financial aid from The Netherlands Organization for Advancement of Pure
Research (ZWO).
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Nucleotide sequence o f P V X
1797
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