Journal of General Virology (1993), 74, 2225-2231. Printed in Great Britain
2225
Complete nucleotide sequence of pepper huasteco virus: analysis and
comparison with bipartite geminiviruses
I. Torres-Pacheco,~- J. A. Garz6n-Tiznado,~" L. Herrera-Estrella and R. F. Rivera-Bustamante*
Departamento de Ingenier[a Gendtica, Centro de Investigaci6n y de Estudios Avanzados del LP.N., Unidad Irapuato,
Apartado Postal no. 629, Irapuato, Gto. Mdxico
The complete nucleotide sequence of pepper huasteco
geminivirus (PHV) isolated in Northern Mexico was
determined. The PHV genome consists of two circular
ssDNA molecules of 2631 bases (PHV A) and 2589
bases (PHV B). PHV has a genome organization typical
of a bipartite geminivirus with four open reading frames
(ORFs) (AR1, ALl, AL2 and AL3) in component A and
two (BR1 and BL1) in component B. An unexpected
ORF was found in the complementary sense strand of
PHV A. This ORF, termed AL5, is found entirely inside,
but in the opposite orientation to AR1 (encoding the
coat protein). AL5 shows some homology to equivalent
but smaller ORFs predicted in other geminiviruses.
Phylogeny trees based on pairwise comparisons ofAR1,
AL2, AL3, BL1 and BR1 predicted proteins placed PHV
among the western hemisphere geminiviruses. A phylogeny tree based on ALl (replicase-encoding OR]=), on
the other hand, placed PHV with eastern hemisphere
geminiviruses, i.e. African cassava mosaic virus and the
Sardinia and Israel isolates of tomato yellow leaf curl
virus. Possible mechanisms for the 'hybrid or transition
nature' of PHV are discussed.
Introduction
Genetic analyses have suggested the possible function
for some ORF products. A R 1 0 R F is considered to be
the coat protein gene, although to our knowledge direct
evidence of this has been reported for only two cases [e.g.
sequencing of the African cassava mosaic virus (ACMV)
coat protein and immunological identification of the
tomato golden mosaic virus (TGMV) AR1 gene product
after expression in Escherichia coli] (Stanley & Gay,
1986; Murayama et al., 1991). The products of the ALl,
AL2 and A L 3 0 R F s have been implicated in several
steps of the replication process (Elmer et al., 1988;
Sunter et al., 1990). In addition, the AL2 protein has
been reported as a possible trans-activator needed for
expression of AR1 and BR1 genes (Sunter & Bisaro,
1991). The ORFs found in component B, BL1 and BR1,
on the other hand, have been suggested as being
responsible for the movement of the virus throughout the
plant (Lazarowitz, 1992; von Arnim & Stanley, 1992).
Recently we reported the cloning of both components
of pepper huasteco virus (PHV), a new whiteflytransmitted bipartite geminivirus affecting pepper crops
in northern Mexico (Garz6n-Tiznado et al., 1993). This
non-mechanically transmissible geminivirus has been
detected in several horticultural areas in the country,
usually in association with other non-characterized
geminiviruses. These complex viral diseases are currently
the most important phytopathological problem in
Mexican horticulture. Monomeric clones of PHV DNAs
(A + B) are infectious with a 60 to 70 % efficiency when
Geminiviruses are a group of plant viruses that have a
small 'twinned' isometric particle and a genome containing one or two ssDNA molecules. Most geminiviruses
can be classified into four subgroups according to their
host-range, insect vector and genome organization.
All bipartite geminiviruses described so far show
similar genomic organization with four open reading
frames (ORFs) in component A and two ORFs in
component B. Component A has one ORF (AR1) in
the positive or viral sense and three ORFs (ALl, AL2
and AL3) in the negative or complementary sense. In
component B, one ORF is found in each orientation
(BR1 and BL1). In both cases, the ORFs extend in
opposite directions from a 180 to 200 base common
region shared by both components. Some sequences in
this common region have been reported to interact with
the respective putative replicase (ALl protein) and are
unique for each geminivirus except for a conserved 30
base sequence element with a potential hairpin structure
(Lazarowitz, 1992).
Nucleotidesequencesof PHV are availablefromthe EMBLdatabase
with the followingaccession numbers: PHV A, X70418; PHV B,
X70419.
? Permanent address: Instituto Nacional de InvestigacionesForestales y Agropecuarias,ApartadoPostal no. 112,Celaya,Gto. Mrxico.
0001-1698 © 1993 SGM
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2226
L Torres-Pacheco and others
introduced into pepper plants by a biolistic procedure.
Neither A nor B D N A produces any symptoms when
inoculated alone (Garzdn-Tiznado et al., 1993).
Here, we report the complete nucleotide sequence of
PHV DNAs. We also compared PHV predicted proteins
with those of previously reported geminiviruses in an
attempt to address the question of the possible origin of
this important geminivirus problem in Mexico.
Methods
Sequencing of PHV. All standard procedures utilized during the
cloning and sequencingof viral DNA were according to Sambrook et
al. (1989). Viral inserts contained in plasmids pIGV21 and pIGV22
were sequenced using Sequenase 2.0 kits from United States
Biochemicals according to the manufacturer's instructions. In some
cases, restrictionfragments from pIGV21 and pIGV22 were subcloned
into Bluescript SK+ (Stratagene) for sequencing. Synthetic oligonucleotides were used as primers to sequence regions where no
convenient restriction sites could be found. Oligonucleotides were
produced in a DNA SynthesizerModel 381Afrom Applied Biosystems.
Computer analysis. Sequences were assembled and analysed using
GeneWorks2.2 and PCGene6.5 (Intelligenetics)and MacDNAsis 2.0
(Hitachi) softwarepackages. The followinggeminivirussequenceswere
obtained directly from the EMBL and GenBank databases and used
for comparisons: ACMV (X17095, X17096) (Stanley & Gay, 1986);
abutilon mosaic virus, AbMV (X15983, X15984) (Frischmuth et al.,
1990); beet curly top virus, BCTV(X04144)(Stanleyet al., 1986);bean
golden mosaic virus, Puerto Rico isolate, BGMV-PR (D00200,
D00201) (Howarth et al., 1985), Brazil isolate, BGMV-BZ (M88686,
M88687), and Guatemala isolate, BGMV-GA (M91604, M91605)
(Gilbertson et al., 1991); bean dwarf mosaic virus, BDMV (M88179,
M88180) (Hidayat et al., 1993); potato yellow mosaic virus, PYMV
(D00940, D00941) (Coutts et al., 1991); TGMV (K02029, M73794)
(Hamiltonet al., 1984;yon Arnim & Stanley, 1992);tomato yellowleaf
curl virus, Sardinia, TYLCV-S (X61153) and Israel isolates, TYLCVI (X15656) (Kbeyr-Pour et al., 1991; Navot et al., 199t); squash leaf
curl virus, SqLCV (M38182, M38183) (Lazarowitz& Lazdins, 1991).
Phylogeny trees were produced with the GeneWorks software
package which uses the UPGMA (unweightedpair group method with
arithmetic mean) procedure (Nei, 1987). The program algorithm does
pairwise comparisons and first clusters the two most similar sequences
and calculates the average similarityof those sequences. The program
then clusters the next two most similar objects.
Results and Discussion
P H V genornic organization
Sequence analysis revealed PHV as a typical bipartite
geminivirus whose genome is divided in two singlestranded molecules termed PHV A and PHV B.
Components A and B are 2631 and 2589 bases long,
respectively. Fig. 1 presents the nucleotide sequence o f
both components. Also shown is the 30 base stem-loop
region shared by all geminiviruses infecting dicotyledons
(GGCCAT/ACCGNT/AA/TTAATATTACCGGA/TTGGCC). Nucleotide 1 in both cases corresponds to the
first base of the 30 base conserved region. This region
contains the typical loop structure T A A T A T T A C , found
in hairpin structures of all monocotyledon- and dicotyledon-infecting geminiviruses, and is believed to be
involved in virus replication (Lazarowitz, 1987).
Similarly to all previously reported geminiviruses, the
base composition of PHV showed that thymidine is
present at the highest percentage, whereas cytosine is the
lowest in occurrence. Individually, the base composition
of each component was: PHV A, 26.25 % A, 19.45 % C,
23.82% G and 30.47% T; PHV B, 28.23% A, 18.35%
C, 21.71% G and 31.71% T.
The genomic organization of PHV is similar to that of
all bipartite geminiviruses. Fig. 2 illustrates the coding
capacity of PHV. Component A contains five putative
ORFs and component B contains two. The ORFs are
found in both orientations: viral or positive-sense and
complementary or negative-sense. The nomenclature
used for PHV ORFs is based on analogies with
previously reported geminiviruses.
Analysis o f P H V ORFs
Computer analysis suggests a subdivision for bipartite
geminiviruses into western (WH) and eastern (EH)
hemisphere geminivirus subgroups. W H geminiviruses
included BGMV, T G M V and AbMV whereas E H
geminiviruses were represented by A C M V and TYLCV
(Howarth & Vandemark, 1989). The analysis was based
on pairwise homologies of coat proteins (ARl-encoded)
and the product of the A L 1 0 R F (putative replicase). To
determine the situation of PHV among these subgroups,
the predicted protein from PHV AR1 was compared
pairwise with the equivalent proteins from several other
geminiviruses (Table i). PHV AR1 showed a high degree
of similarity (82 to 90%) with the W H geminivirus
subgroup, closest to the Guatemalan isolate of B G M V
(BGMV-GA) (Gilbertson et al., 1991). The degree of
similarity decreased (68 %) when the PHV AR1 protein
was compared with the equivalent proteins from members
of the E H subgroup: A C M V and the Sardinia and Israel
isolates of T Y L C V (TYLCV-S and TYLCV-I, respectively) (Kheyr-Pour et al., 1991; Navot et al., 1991). A
phylogeny tree based on pairwise homology data showed
an evident grouping of the viruses placing PHV, as
expected, with the W H cluster (Fig. 3 a). Alignments of
coat proteins from all viruses included in Fig. 3 revealed
several highly conserved domains (data not shown). In
contrast, no significant homology was detected when the
PHV AR1 protein was compared with coat proteins
from geminiviruses transmitted by leafhoppers, e.g.
BCTV (29 %), maize steak virus and wheat dwarf virus
(data not shown).
A similar analysis was applied to the putative replicase,
encoded by the A L l ORF, and several interesting
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Complete nucleotide sequence of PHV
2227
A
GGC•AT•CGTTAT••AATATTACCGGATGGCCGAC•G•TTA•CTTATCTATCCGTA•TGCTTTATTTGAATTAAAGATGTTACTTTTATGCTATCCAATGAA
100
GCGTAGCGTCTGGGAAGCTTAGTTATCAGTTCCAGACGTGGGGACCAAGTAGTGTATGA•CACTTTA.•-•-GACTGTCAGCTTTATAAATTGAAATTAAAAC 200
ATAAGTGGT~CATGTACCTTTAATTCA.6~TGCCTAAGCGTGATGCTCCTTGGCGATTAACGGCGGGGACCGCCAAGATTAGCCGAACTGGCAATAATTC
ACGGGCTCTTATCATGGGCCCGAGTACTAGCAGGGCCTCAGC-•TGGGTTAATCGCCCAATGTACAGGAAGCCCCGGATTTATCGTATGTACAGAACTCCG 400
GATGTGCCGAAAGGTTGTGAAGGTCCCTGTAAGG.•TCAATCG.•TFGAACAACGACATGACGTCTCTCATGTTGGTAAGGTTAT•TGTATATCCGACGTAA 500
CTCGTGGTAATGGTATTACCCATCGTGTTGGCA/~CGATTCTGCG~TAAGTCTGTCTATATTCTGGGCAAAATCTGGATGGATGAAAATATTAAGTTGAA 600
GAACCATACCAACAGTGTCATGTTTTGGTTG
GTTAGGGATAGGAGACCCTACGGTACGCCTATGGA'I"ITTGGCCAAGTCTTTAACATGTATGACAACGAG700
•••AGTA••GCTACTGTGAAGAACGATCTTCGGGATCGTTATCAAGTTATGCATAGATTcTATGCTAAGGTCACTGGTGGGCAATATGCAAGCAACGAGC 800
AAGCCTTGGTTAGGCGTTTCTGGAAGGTGAACAACCATGTTGTGTATAACCATCAAGAAGCTGGGAAATATGAGAACCACACGGAGAATGCGCTGTTATT
900
GTATATGGCATGTACTCATGCATCTAATCCCGTGTATGCAACACTCAAAATTCGGGTCTATTTTTATGACTCGATAATGAATTAATAAAGTTTGTATA•T I000
ATTTCATGATTCTCAAGTACAGCATTGACATAACG1-~TGTCTGTAGCAAACGAAACAGCCCTAA1TACATTGTTAACTGAAATAAGACCTAAGTTATCTA11~
GATAAAACATGACAAGCAATTTAAATcTATTTAAGTAAATCTGCCCAGAAATCGTCGTCAACGTCGTCCAGACTTGGAAGTTGAAGTAGGCTTTGTGGAG1200
ACCCAACGCTGTCCTCATGTTGTGGTTrGCTCTGACTTGAATGTGAAATACCCGGCTGCGTGTGTACATTGGCGTCTCCACTAGCCGTATTTTGAAATAT1300
AGGGGATTTCGAAGCTCCCAGATAAAAACGCCATTCGCTGCTTGAGCTGCAGTGATGGGTACCCCGGTGCGTAAATCCATTGTTAACACAGTTAATATGT
1400
ATATAAATTGAACAGCCGCAAGCGAGATcAATCCTTCTACGTCGTATCTGTCTCTTTGCAAATCTATGGCGAAGTTTGACTTCCGGTGGTGAAGATAGCT1500
TCTTCGATGGTGACGTAGATGGCGTTT1-•TTGGACCCAGTCATTGAGGCTCCTATTTTTCTCTTCGCTGAGGTAGTCTTTATAGGAAGACTGGGGGCCCG 1600
GATTGCAAAGAAAGATAGTGGGTATCCCACCTTTAATTTGAATrGGTTTGCCGTATTTGCAGTTTGA1TGCCAATCCCGTTGGGCCCCCATAAACTCTrT1700
AAAGTGCTTAACATAATGCGGAGGGATGTCATCAATGACGTTATACCATGCATrATTGGAGTAGAT•TTTGGGCTGAGATCCATATGACCACATATGTAA 1800
TTGTGTGGGCCGAGACTTCGGGCCCATAATGT•TTGCCTGTCCGTGAAGGACCCTCGACCACTAATGACAATGGTCTCATTGGCCGCGCAGCGGCATCAC
19~
ATACATTATCAGACACCCATTGTGTCA1-FATTGCAGGCACATTATTAAAGGACGCCTGTTGAAATGGAGGAACCCACGGTTCCGGGGGAGTTTGGAATAT2000
CCGATTAGCGTTTGACACAATGTTATGAAATTGGAGGAAGAAATGCTGAGGTTGTTCTTCCTTTATGATCTGCAGAGCTTCTTCTGCAGATGCTGAATTTZIO0
AACGCCTFAGCATATGTGTCATTAGCAGACTGCTGTCCTCCTCTAGCAGATCTGCCGTCTATTTGGAATTCTCCCCATTCTACGGTATCGCCGTCTTTGTZZ~
CGATGTACGTCTTGACGTCGGAGCTTGATTTAGCTCCCTGAATGTTCGGATGGAAATGTGCTGATCTGGTAGAGGATACGAGGTCAAAGAATCGGTTGTTZ300
CGTGCATFGGTATTTT~CTT~GAA~TGAATAAG~A~GTGCAGATGAGG.~TGCCCATCTTCATGAGATTCTrTGCA/AATTTTGATGTACTTCTTGTTTACC2400
GGCGTCGAGAGG.~TTTGTAGTTGAGCGAGACGCTCTTCT~TGGAAATGGAA~AT~GTGGATAGGTGAGGAAATAATTCTTGGCATTTAAACGAAATCGTT
25~
TAGGTAATGGCATAT-~-FGTAATAAGAGAGGTGTACACCGATTGGAGCTCTTTAACCTGGGCTTATTGTATCGGTGTATTGGTAGCCAATATATAGTATATZ6~
GGGAGTTATCTAGGATCTTCGTACACGTGAG
Z63'I
B
GGCCATCCGTTATAATA`•-•-A•CGGATGG••GA•CGCTT•CACTCTCTTTCCTTTGGGACAGCTGGCGCGCACTATGTA-FrATGTTTACGTGGCATCATGT
100
GGGTCG.i-[GGATGAA~CCAATCGCG~GCCTTCAT-~TCAAATTAAAGTGTGTGT~CATACATCGAGAAATGTGTAATGACGTGGAGCGTTCTCCACCATrC ZOO
CTGAATCGTTAGATAATTGTTTGAC•AGGACCACAGCTGTCAT•TGGGACCACACGTCCTTTGGGACCACCACTATAATGATAATGTTTCCTGTTA•TGC 300
GGTCCACGTGGTCCAATTAAATTGCACCTCGCGAGTCTACATATCCACAATTTTGAATATCCTATTCTATAAAATGGCTTCCAT•TTTATATTCAAAATT400
ATATTCACATCTCTTTTAATATATATTTATCTTTAAGCAATTTAATATGTATTCTACTAGATTTAGACGTGGGTTATCCTATGTTCCACGGCGTTATAAT 500
CCA~GTAATTATGGTTTTAAACGTACATTCGTCGTTAAACGTG~TGATGCTAAA~GACGTCAGACTCAAGTGAAGAAACTAACAGAAGATGTTAAAATGT
600
CATCACAACGCAT•CATGAAAATCAATATGGTCCAGAATTTGTCATGGCGCATAATACAGCAATATCTACATTCATCAATTATCCCCAACTGTGTAAGAC 700
TCAG•CCAATCGTAGTAGGTCATATAT•.AAG-•TAAAATCGTTACATFTTAAGGGAACCTTAAAGATCGAACGTGTTGGGTCTGAGGTAAATATGGCTGGG 800
TTAAATCCGAAGATTGAGGGTGTGTTTACTGTGGT•-TTAGTTGTTGACCGTAAGCCACA.FrTGAATCCTACTGGTAACTTGCTACAGTTTGACGAGTTATO~0
TTGGTGCAAGAATTCACAGTCTAGGGAACTTAGCCGTTACCCCGGCGTTGAAAGAACGGTTCTACATACTGCATGTGTTGAAGCGAGTTATCTCCGTTGA
1000
GAAGGATAGTATGATGCTGGAC
CTAGAAGGATCCAC'I-I'GTCTCTCTAGTCGGCGTTATAATTGTTG
GTCTACATTTAAGGACC'I-I'GATCCTTCGTCATGT"11~
AACGGCGTCTATGATAATATAAGCAAAAACGCCATA-~TAGTTTATTATTGTTGGATGTCGGATGCTATGTCTAAGGC~TCCACAT-~TGTATCA-~--~TGATT1200
TGGACTATTTTGGTTAAGAAATAATTGACTTGC
GTAGTTTGCTCATATTTGTATTTTGTCACAAAATAAAATATTATTATCTTAGCGACTTCGGTTGTGT
1300
CGGATTACAATTACTGTTAATACATTCATGGACCGTAGTCCTTACAAGCTCATTCAACTGGGCCAAG
GACATAGTTATATTTGATTGAGAGC
GTGTTAGA1400
CCCACTTGTGATGCTGAATCACCTGGGTCCAAAA
CACTTCCGCCTAACTGATGAAGATCTTTATACGGATGTAATGCGCTATGTCCTTGGTTGTCAG
CAT 1500
CTGTGTGAGTGGTTCCTATGGTGCTTCTACAAGCCCAGGATTCACCTGGTTTTAATTCAATTGGGCCTGTAATGCCGAACC-•TGACATGGATGCTGACCT 1600
CAATGG'ITITCTCTCCCACCTGCCGTAGTCCACATGTGTAAAGTCCACATCGTTATGGGTGAACTGTTTCGATAAAATCTTCACCGTC
GGAGCCCGGAAA1700
GGTATATCCACGGAGTG.•-••TAGCTGTGGACAACTTCAATTTCCCTTTGAACTTGGCAAAATGGGTGTTCTGATGTACGTTAGTATCGGAGACTCTGTAAT 181~
ATAGCTTCCAGGGTATGGGGTCCTTCAAGGAGAAGAAGGATGCTGAGAAATAATGGAGATCGATGTTACATCTTAGTGGAAATGTCCAAGAAGCTTGTAA1900
TGATTCATTGTCTGTCATTCGTTTGTCATGGATTTCCACTATGACCGACCCAGTGGCGT.•TATCGGAACTTGCTGTCTATACTCGATAACG
CAATGGTCA20~
ATTTTCATACAGCTACGACTAAGTCTGGCAGCGTACTGCGACGCCGTTGACGGAAATTGAAGTATTATCTCCGTTAAGTCATGAGAGAGCTGATATTCAT
2100
CTCTATGTGACTCTATATAA-•-•.GAATGCGCTAGGAGGATTCGCCAACCATGAATCCATATATGAAAATTTGGCAGCGCACGTGAAGGCTTACGGAGTCTG ?_.2~
AATCTGGTAATAAGAAGCTATACCTAACAATGTTAATGGTAATGAAAATGACAAATTACTATrTGCTGAAAGAGTTCAAAAATAAATGCTTACTTAGTTA
2300
TTAAGATATTGCTATTAGCAGCAACAATATATGAGGAAACCGGTGAGGATGAAAGCAAAAGCGTCTTCAGAAGACAGAGCAGAAAGAATTGGTATGAATA2400
ATr'AAATGAACAGGCAGTGTCGTTATATAGAAGATCATTGTGTTTTAGAGAGAGAAAATITTGCAGTGG
CATTI'GTGTAATATGGAGGGGTACACCGATTZ5~
GGAGCTCTTT~d~CCTGGGCTTA-~TGTAT~GGTGTATTGGTAGCCAATATATAGTATATGGGAGTTATCTAGGATCTTCGTACACGTGGA
2589
Fig. 1. Nucleotide sequence of the viral sense strand of both components of PHV (A and B). The underlined regions (30 bases)
correspond to the consensus found in all bipartite geminiviruses. In both cases, nucleotide 1 corresponds to the first base of the 30-mer
consensus.
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I. Torres-Pacheco and others
2228
(a)
ALl ~
L
TYLCV-S
TYLCV-I
ACMV
5
~
ARll
~" ~
BGMV-PR
BGMV-GA
SqLCV
TGMV
BDMV
PYMV
BGMV-BZ
PHV
AbMV
R1
AL3
Fig. 2. Proposed genomic organization for PHV. Arrows represent
ORFs in both orientations. CR represents the 180 base common region
for both components which contains the 30 base stem-loop structure
found in all bipartite geminiviruses. ORF coordinates are: AR1,230 to
982; ALl, 2513 to 1467; AL2, 1543 to 1130; AL3, 1380 to 985; AL5,
853 to 341; BR1,447 to 1214; BL1, 2158 to 1280.
.[_~~
(b)
T a b l e 1. Amino acid sequence similarities (%) between
putative translation products o f P H V and selected
geminiviruses*
U
I
AbMV
ACMV
BCTV
BDMV
BGMV-PR
BGMV-BZ
BGMV-GA
PYMV
SqLCV
TGMV
TYLCV-I
TYLCV-S
AR1
ALl
AL2
AL3
BR1
BL1
82
68
29
87
88
86
90
87
88
86
68
68
65
65
61
67
64
61
66
63
48
66
68
69
54
50
30
59
48
50
49
52
51
54
50
49
67
48
43
66
67
67
68
66
66
67
50
48
61
37
NA'~
67
68
67
67
65
53
68
NA
NA
63
46
NA
82
82
76
84
81
77
78J;
NA
NA
* All values were calculated using the MacDNAsis program. Other
programs (GeneWorks and UWGCG) gave similar results.
~ NA, Not available; TYLCV and BCTV do not have component B.
;~ For TGMV BL1 the sequence used was as reported by von Arnim
& Stanley (1992) because the sequence originally reported by Hamilton
et al. (1984) contained a mistake that produced a truncated BL1
protein.
o b s e r v a t i o n s w e r e o b t a i n e d ( T a b l e 1 a n d Fig. 3 b). F i r s t ,
PHV presented the highest similarity with both isolates
o f T Y L C V , S a r d i n i a , 6 9 % , a n d Israel, 6 8 % . T h e
similarity to the WH geminivirus cluster was slightly
l o w e r ( 6 1 % to 6 7 % ) . W h e n a n A L l p r o t e i n - b a s e d
phylogeny tree was constructed, PHV clearly clustered
w i t h t h e E H g e m i n i v i r u s e s ( A C M V a n d T Y L C V ) (Fig.
3 b). A l i g n m e n t o f A L 1 p r e d i c t e d p r o t e i n s s u p p o r t e d t h e
c l a s s i f i c a t i o n d i s p l a y e d in t h e p h y l o g e n y tree. F i g . 4
s h o w s a d o m a i n o f 40 a m i n o a c i d s f o r s e v e r a l r e p l i c a s e s
t h a t c o n t a i n s s o m e r e s i d u e s f o u n d in E H g e m i n i v i r u s e s
( A C M V , T Y L C V - I a n d T Y L C V - S ) a n d p r e s e n t also in
P H V , b u t n o t f o u n d in a n y o t h e r W H g e m i n i v i r u s .
I n a d d i t i o n to this d o m a i n , o t h e r r e s i d u e s c o n s e r v e d
b e t w e e n P H V a n d E H g e m i n i v i r u s e s a r e V(218), C(241),
TGMV
BGMV-BZ
BDMV
PYMV
BGMV-GA
AbMV
BCTV
TYLCV-I
ACMV
PHV
SqLCV
Fig. 3. Phylogeny trees for several geminiviruses. (a) Tree based on
pairwise homologies of the coat proteins. (b) Tree based on the ALl
products (replicase). Protein sequences were obtained directly from
databases as described in Methods. The AbMV ALl sequence was
corrected as suggested by Gilbertson et al. (1993). The figures are not
to the same scale. When the standard error value for a branch point
overlaps the standard error value of a point below or above it, it is not
possible to say which branched first. This was expressed as collapsed
nodes that produced tri- and higher -chotomies.
PHV
TYLCV-S
TYLCV-I
ACMV
BGMV-BZ
PYMV
BGMV-GA
BGMYLPR
BDMV
TGMV
SqLCV
AbMV
BCTV
Consensus
(255-294)
(253-294)
(253-292)
(254-293)
(256-297)
(256-297)
(256-297)
(256-297)
(256-297)
(257-297)
(252-293)
(256-297)
(256-297)
AWYNVZDDZPPHYVK--HFK EFMGAQRDWQ SNCKYGKPI( 294
AWYNVZDDVDPHYLK--HFK EFMGAQRDWQ 5NTKYGKPI( 294
AWYNVTDDVDPHYLK--HFK EFMGAQRDWQ SNTKYGKPI( 292
AWYNVZDDVDPHYI.K--HFK EFM6SQRDI~ SNTIOfGKPV( 293
AEYNVZDDZAPHYLKLK}MK ELMGAQKDWQ SNCKYgKPV( 297
VEYNVTDDVAPQYLKLKHWK ELLGAQRDI~ SNCKYGKPV( 297
VEYNVZDDZS PNYLKLKI'~K ELIGAQY,DIVQ SNCXYGKPV( 297
VEYNVZDDZSPiCYLKLKHWK ELIGAQKDM~ SNCKYGKPV( 29 ?
VEYNVZODVA PHYLKLKHWK ELZGAqK~ SNCK~GKtW( 297
VEYNVIDOV'r PQYLKLKHWK EL'rGAQRDWQ TNCKYGKPV( 297
VKYNVZDDVAPHYLKLK~K ELZGAQRDWQ SNCKYGKPV( 293
VEYNVIDDVA PHYLKLK~K ELLGAQKDWQ SNCKLAKPV( 297
VEYNVZDDVDPTYLK~KFmK HLIGAQKEWQ TNLKYGKPRV 297
**YNVIDD-- P-YLK**H*K E**GAQRDWQ SN*KYGKP*Q 48
Fig. 4. Alignment of a 40 amino acid domain of selected ALl proteins.
A 40 amino acid domain from several ALl proteins was selected and
aligned by computer. The consensus sequence is shown in the bottom
line. The asterisks (*) mark residues conserved among PHV, ACMV
and TYLCV and different to the WH consensus. Dashes (-) mark
positions where more than three different amino acids can be found
(highly variable). Letters indicate residues conserved in all geminiviruses.
T(299), F(301) a n d P(307). A m o r e d e t a i l e d analysis,
however, showed that the ALl protein also of PHV
has s o m e r e s i d u e s f o u n d o n l y in W H g e m i n i v i r u s e s
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Complete nucleotide sequence of P H V
[PEPW(177 to 180), N(191), C(275)]. Although the ALl
protein comparison suggests a close relationship between
PHV and EH geminiviruses, the analyses of the other
ORFs as well as the fact that PHV ALl also possesses
some WH characteristics do not allow a definitive overall
clustering of PHV. These results propose PHV as a
hybrid or transition geminivirus between the WH and
EH types.
Trees obtained using other software packages (e.g.
PCGene CLUSTAL program) showed minor variations
for the position of some geminiviruses inside the main
clusters. However in all cases the clustering of PHV
remained identical. In any case, the main purpose of the
tree is to illustrate similarities between sequences encoded
by a specific ORF. Implications for an evolutionary path
will require more rigorous analysis.
In contrast to the results found with the AR1 protein
where the similarity between PHV and SqLCV was one
of the highest (88 %), the similarity between products
of the PHV ALl and SqLCV ALl ORFs was the
lowest (48 %), even lower than the value obtained with
equivalent proteins from BCTV (61%) (Stanley et al.,
1986). When SqLCV was included in the ALl phylogeny
tree it was found to stand by itself forming a separate
branch, whereas in the AR1 phylogeny tree it was clearly
clustered with the WH geminiviruses. BCTV, which is a
subgroup II geminivirus transmitted by leafhoppers, was
included in the comparisons used for the ALl tree as an
internal control of the method, and clustered with the
WH bipartite subgroup as reported previously (Howarth
& Vandemark, 1989). Phylogeny trees based on similarities between products of ORFs AL2 and AL3
displayed similar results to the AR1 phylogeny tree. The
clustering of PHV was similar in all three cases and the
only difference was the degree of similarity that PHV
showed with the rest (Table 1).
Computer analysis revealed two possible initiation
codons, in the same reading phase, for the PHV AL2
ORF. The first one generates a protein of 208 amino
acids. The second initiation codon is found 210 bases
downstream producing a protein with only 138 amino
acids. Sequence and size comparisons with AL2 proteins
from other geminiviruses suggested that the smaller
protein is the actual product. Point mutation experiments
are being carried out to dismiss the possibility of any
initiation of translation at the first ATG codon.
An unexpected fifth ORF (ORF AL5) was detected
in component A. The nomenclature of AL5 (instead
of AL4) was suggested to avoid confusion with the
previously described A L 4 0 R F for other geminiviruses.
This ORF is located entirely inside ORF AR1 (coat
protein) but in the opposite orientation. At present it
is not known whether this ORF is transcribed and
translated. To verify whether this situation is unique for
2229
PHV or whether similar proteins could be found in other
geminiviruses, the sequences of all previously reported
geminiviruses were analysed. The survey included ORFs
producing proteins smaller than 10000 Mr which has
been a common size limit for geminiviral proteins.
Several viruses (BGMV, BDMV and PYMV) had similar
but smaller ORFs. Although the overall pairwise
similarity was relatively low in all cases (50 %), computer
alignments of the predicted AL5 proteins displayed some
conserved regions. A genetic study is also underway to
determine the importance of AL5.
For the B L 1 0 R F , PHV showed the largest degree of
similarity (84%) to BGMV-GA and the lowest to
ACMV (42%). A similar situation, although with a
lower degree of similarity, was found for BR1 where
BGMV-PR, TGMV (68 %) and BDMV, BGMV-BZ,
BGMV-GA (67 %) showed the higher degree of similarity to PHV and the lowest to ACMV (37%).
Phylogeny trees placed all WH geminiviruses in one
homogeneous cluster leaving only ACMV on an independent branch (data not shown).
The AR1, ALl, BR1 and B L 1 0 R F s have typical
eukaryotic promoters with CAAT and TATA boxes and
a cap signal. The distances between TATA boxes and the
cap signal are similar although not identical. The position
of the CAAT box showed more divergence. Perhaps the
nearby stem-loop structure (e.g. for ALl) could have
some effect on the structure of the promoter. The
position of the poly(A) signal was also different since the
one for AR1 is virtually on the stop codon whereas BRI
has its signal a few bases downstream. This might be a
reflection of the fact that the AR1 stop codon overlaps
with that of AL3 resulting in the absence of an intergenic
region (see Fig. 2). Component B, on the other hand,
presents an intergenic region of 59 bases. The poly(A)
signal for ALl was not clearly identified. In addition, no
typical regulatory sequences could be found for ORFs
AL2, AL3 and AL5. The expression of these overlapping
ORFs has yet to be determined.
The overall analysis of the PHV genome showed it to
be a WH geminivirus. However, the fact that the PHV
ALl protein is more closely related to the equivalent
proteins from ACMV and TYLCV suggests the possibility of some type of recombination between geminiviruses.
There are several non-characterized geminiviruses in the
same geographical area infecting pepper and tomato
crops (Brown et al., 1986; Brown & Nelson, 1988, 1989;
Brown & Poulos, 1990). It is possible that a virus similar
to TYLCV, at the molecular level, might be present in
the area. Mixed infections are common and several
diseases have been reported to be caused by a mixture of
geminiviruses (Brown et al., 1989; Brown & Nelson,
1989). The presence of several geminiviruses in the same
plant provides the opportunity for recombination. This
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2230
L Torres-Pacheco and others
opportunity is increased in this area since possible hosts
for the viruses are found in all seasons. The relatively low
degree of similarity (68 %) between PHV and TYLCV
replicases suggests that this possible recombination event
is not a recent one, and that the viruses have had enough
time to diverge to the present level. Several experiments
in which components of bipartite geminiviruses were
exchanged (pseudorecombination) have been recently
reported (Gilbertson et al., 1993; yon Arnim & Stanley,
1992). This pseudorecombination mechanism can now
be added to the intermolecular recombination reported
for ACMV (Etessami et al., 1989; Klinkenberg et at.,
1989; Stanley & Townsend, 1986). Both mechanisms,
independently or acting in a concerted manner, might be
important in nature for the generation of new viruses.
The unexpected clustering of SqLCV also supports a
recombination hypothesis. SqLCV AR1 is highly similar
to those of WH geminiviruses whereas SqLCV ALl is
clustered completely on its own.
An alternative explanation is simply that PHV, ACMV
and TYLCV share a closer common ancestor and have
diverged in adapting themselves to the surrounding
environment (e.g. vectors and host plants in the
respective geographical area). The characterization of
more geminiviruses from the same area where PHV was
isolated could provide new information about their
phylogenetic relationships.
That Mexico is a centre of diversity for both pepper
and tomatoes might be of importance for the number of
geminiviruses being found in this area. Nevertheless, the
sudden outbreak of diseases caused by geminiviruses in
the late 1980s still remains unexplained.
We thank B. Jimenez for her generous help during the sequencing of
PHV and A. Alvarez for critical reading of the manuscript. We would
also like to thank the anonymous referees for their critical comments
towards improvement of the manuscript. This research was supported
by a grant from CONACYT-M6xico (1150-N9202) to R.F.R.-B.
I.T.-P. also acknowledges fellowship support from CONACYT.
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