Journal of General Virology (1993), 74, 323-328. Printedin Great Britain
323
Nucleotide sequence of one component of the banana bunchy top
virus genome contains a putative replicase gene
Robert M . Harding, 1 T h o m a s M . Burns, 1 G r e g o r y H a f n e r , ~ R a l f G. D i e t z g e n 2 and J a m e s L. D a l e 1.
1Centre for Molecular Biotechnology, Queensland University of Technology, GPO Box 2434, Brisbane,
Queensland 4001 and 2Plant Protection Division, Queensland Department of Primary Industries, Indooroopilly,
Queensland, Australia
One DNA component of the banana bunchy top virus
(BBTV) genome was cloned and sequenced. This
component is present as a circular, ssDNA in the virions
and consists of 1111 nucleotides. It contains one large
open reading frame (ORF) of 858 nucleotides in the
virion sense; this ORF encodes a putative replicase
based on the presence of a dNTP-binding motif
(GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be
assigned any obvious function. Neither of these ORFs
had significant sequence homology with any known
DNA plant virus gene or gene product. Computer
analysis of this component-predicted a strong stem-loop
structure in the virion sense putative untranslated
region; a nonanucleotide sequence in the loop was
nearly identical to the nonanucleotide invariant loop
sequence of geminiviruses and coconut foliar decay
virus. There is strong evidence that the genome of BBTV
consists of more than one component because no ORF
was found that would encode a protein the size of the
BBTV coat protein. BBTV has some characteristics in
common with geminiviruses but cannot be classified as
one. Rather, BBTV probably belongs to an undescribed
plant virus group which could also include subterranean
clover stunt virus and coconut foliar decay virus.
Introduction
particles are usually geminate and they are transmitted
by leafhoppers or whiteflies. However, subterranean
clover stunt virus (SCSV) (Chu & Helms, 1988) and to a
lesser extent coconut foliar decay virus (CFDV) (Randles
et al., 1987) have characteristics very similar to BBTV.
Both of these viruses have 18 to 20 nm isometric particles
which contain circular ssDNA of approximately 1 kb.
SCSV is persistently transmitted by aphids, whereas
CFDV is transmitted by a planthopper. Furthermore,
the genome of SCSV is composed of at least seven
distinct ssDNA molecules each containing one large
open reading frame (ORF) (Chu et al., 1990). The
sequence of one ssDNA molecule of CFDV has been
determined (Rohde et al., 1990) and one of the ORFs
encodes a putative replicase. There are also three animalinfecting viruses that have similar characteristics to
BBTV, SCSV and CFDV, namely porcine circovirus
(PCV), chicken anaemia virus (CAV) and psittacine beak
and feather disease virus (PBFDV) (Todd et al., 1991).
In this paper, we report the cloning, sequencing and
analysis of one component of BBTV DNA.
Banana bunchy top disease is the most serious virus
disease affecting bananas (Musa spp.). Originally it was
assumed to be caused by a luteovirus on the basis of the
biological characteristics of the disease: it is persistently
transmitted by aphids~ causes a yellows type disease and
infected plants have damaged phloem (Dale, 1987).
Further evidence for the involvement of a luteovirus
included (i) association of dsRNA with the disease (Dale
et al., 1986), (ii) purification of 28 nm isometric virus-like
particles (VLPs) from infected plants (Iskra et al., 1989)
and (iii) purification of 20 to 22 nm isometric VLPs
containing ssRNA with an Mr of 2.0 x 106 from infected
plants (Wu & Su, 1990). However, Harding et al. (1991)
and Thomas & Dietzgen (1991) purified 18 to 20 nm
isometric VLPs from infected plants and these particles
contained ssDNA of about 1 kb. Furthermore, Harding
et al. (1991) demonstrated the transmission of this
ssDNA with the disease using a probe cloned from the
ssDNA. These 18 to 20 nm VLPs are now assumed to be
the particles of banana bunchy top virus (BBTV).
BBTV has characteristics different from those of any
described plant virus group; it is most similar to the
geminiviruses, which have ssDNA as their genomic
nucleic acid but their DNA is about 2"7 to 3'0 kb, their
0001-1321 © 1993 SGM
Methods
Sequencing. Mini-preparations of pBT338 (Harding et al., 1991)
were prepared by alkaline lysis followed by polyethylene glycol
precipitation (Hattori & Sakaki, 1986). Sequencingwas done using
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324
R. M. Harding and others
-...-
in 2 x SSC at 60 °C. The dried membranes were exposed to Agfa Curix
RP1 film at - 8 0 °C using intensifying screens.
Results
0
0-25
0.5
0.75
I
I
I
I
1.0
I I11kb
Fig. 1. Strategy for sequencing clones of BBTV component 1. The
genome is represented by the black rectangle and the plasmid vector is
represented by the grey rectangle. The arrows indicate the direction and
extent of sequencing.
[32P]dCTP and a Sequenase kit (US Biochemicals)as recommended by
the manufacturer. Reaction products were analysed on an 8 % (w/v)
polyacrylamide gel containing 7 M-urea. Gels were fixed, dried and
exposed to Agfa Curix RPI film. The primers used for sequencingwere
either universal sequencing primers (US Biochemical) or 17 to 30
nucleotide primers complementary to appropriate regions of the cloned
viral DNA (Fig. 1). The latter primers were synthesized using an
Applied Biosystems 391 DNA synthesizer.
PCR: analysis and cloning. From the nucleotide sequenceof pBT338,
two oligonucleotides (primer A: 5' GGAAGAAGCCTCTCATCTGCTTCAGAGAGC 3'; primer B: 5' CAGGCGCACACCTTGAGAAACGAAAGGGAAY) were synthesizedand were used as primers in
a PCR with purified BBTV DNA. The reaction mix (50 gl) contained
10 mM-Tris-HCl pH 8.3, 50 mM-KCI, 1.5 mM-MgC12, 200 gM each
dNTP, 50 pmol each primer and 0.6 units Taq polymerase (Cetus).
Following the addition of template DNA (approximately 0.1 ng), the
mix was subjected to one cycle consisting of denaturation at 94 °C for
5 min, annealing at 55 °C for 2 rain and extension at 72 °C for 3 min ;
30 cycles consisting of denaturation at 94 °C for 1 min, annealing at
55 °C for 2 min and extension at 72 °C for 3 min; and finally one cycle
consisting of denaturation at 94 °C for 1 rain, annealing at 55 °C for 2
min and extension at 72 °C for 10 min. The amplified product was
analysed by electrophoresis in either 1% agarose gels in Trisacetate-EDTA buffer, pH 7-8 (Maniatis et aL, 1982)or in discontinuous
polyacrylamide gels (5% stacking gel/10% resolving gel) using the
buffer system of Laemmli (1970) without SDS. Nucleic acids were
visualized with ethidium bromide and the size of the amplified product
was estimated by comparison witk a BglI/Hinfl digest of pBR328
(Boehringer Mannheim).
The amplified product was cloned directly into the plasmid vector,
pCR2000, using a TA cloning kit (Invitrogen) as recommended by the
manufacturer. Potential recombinant cloneswere identifiedby screening
on X-gal substrate, and virus-specific clones were subsequently
identified by screening purified plasmids with 32p-labelledinsert from
pBT338 (Harding et al., 1991). Plasmids that hybridized with the
pBT338 insert and contained the largest inserts were selected for
sequencing.
Polarity of virion ssDNA. Purified viral nucleic acid (Harding et al.,
1991) was electrophoresed in 1% agarose gels and capillary blotted on
Hybond-N + (Amersham). A DNA 3' end-labelling kit (Boehringer
Mannheim) was used to prepare 32P-labelled strand-specific oligonucleotide hybridization probes (primers A and B). Membranes were
prehybridized for 3 h at 50 °C in a solution containing 1 M-NaC1 and
1% SDS. Membranes were then incubated for 16 h at 60 °C in a
solution containing 10% dextran sulphate, 1 M-NaC1, 1% SDS,
100 Ixg/mldenatured herring sperm DNA and the 3' end-labelled oligonucleotide probe. The membranes were washed once at room
temperature in 1% SDS and 2 x SSC followed by four 15 min washes
T h e insert from pBT338 ( H a r d i n g et al., 1991) was
completely sequenced in b o t h directions a n d was f o u n d
to c o n t a i n 980 bp. F r o m this, we developed a strategy for
the synthesis o f d o u b l e - s t r a n d e d full-length BBTV D N A
which at the same time w o u l d d e m o n s t r a t e that BBTV
virion s s D N A was circular. U s i n g the sequence inf o r m a t i o n o b t a i n e d from pBT338, two primers (primers
A a n d B, each 30 nucleotides in length) were synthesized,
which hybridized i m m e d i a t e l y adjacent to one a n o t h e r
b u t were reversed in their o r i e n t a t i o n . These primers
were used in a P C R with Taq D N A polymerase. I n this
reaction, p r i m e r A hybridized to the v i r i o n s s D N A a n d
c D N A was synthesized a n d primer B hybridized to the 3'
end o f the r e s u l t a n t c D N A a n d a copy o f the template
D N A was synthesized. This reaction was analysed b y
P A G E ; a single amplified p r o d u c t was evident with a size
o f a b o u t 1.1 kb which a p p e a r e d to be a full-length
d o u b l e - s t r a n d e d copy o f the template D N A (Fig. 2). This
result p r o v i d e d strong evidence that the virion s s D N A of
BBTV was circular. F u r t h e r m o r e it was a s s u m e d that
this p r o d u c t represented the full sequence o f one D N A
c o m p o n e n t o f the BBTV g e n o m e for the following
reasons: (i) it was the only amplification p r o d u c t o f a
P C R that should yield full-length p r o d u c t f r o m a circular
template a n d (ii) it was a p p r o x i m a t e l y the size o f the
s s D N A extracted f r o m purified BBTV virions.
T h e amplified p r o d u c t was ligated into a ' T - t a i l e d '
p l a s m i d (pCR2000) a n d this p l a s m i d was t r a n s f o r m e d
into Escherichia coli. A small sample o f this transf o r m a t i o n was analysed a n d p o t e n t i a l r e c o m b i n a n t s
were screened using pBT338 as a probe. Five p o t e n t i a l
r e c o m b i n a n t s hybridized with the pBT338 insert a n d
c o n t a i n e d inserts of a p p r o x i m a t e l y 1.1 kb. Three of these
1
2
I
bp
2167
1766
1230
--,,-----
1033
-
-
653
517
Fig. 2. PAGE of the DNA product generated using PCR from the
BBTV virion ssDNA template. Lane 1, size markers (BglI/IlinfI digest
of pBR328); lane 2, BBTV PCR product.
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B B T V putative replicase gene
(b)
(a)
AGATGT CCC GAGT TAG T GCGC CACGTA~GCTGGGGCTTAT
TATTACCCCCAC, CGCTCG
GGACGGGACATTTGCAT~AGACCTCCCCCCTCTCCATTACAAGATCATCATCG
ACGACAC~T
M
60
325
1
2
3
1
2
3
120
GGC G C GATAT GT GGTAT GC TGGATGT TCACCAT CAACAAT CCCACAACAC
180
A
R
Y
V
V
C
W
M
F
T
I
N
N
P
T
T
L
T A C C A G T G A T C~AGGGAT G A G A T A A A A T A T A T G G T A T A T C A A G T G
P
V
M
R
D
E
I
K
Y
M
V
Y
Q
V
GAGAGGGGACAG
E
R
G
Q
GAGG 240
G
E
300
G TACTCGTCAT GTGCAAGG TTATG TCGAGATGAAGAGACGAAGCTC
TCTGAAGCAGATGA
T
R
H
V
Q
G
Y
V
E
M
K
R
R
S
$
L
K
Q
M
GAGGCT TCT T CCCAGGC G CACACC T T GA~CGAAAGGGAAGC
G
F
F
P
G
A
H
L
E
K
R
K
G
CATACTGTATGAAGGAAGATACAAGAATCGAAGGTCCCT
Y
C M
K
E
D
T
R
I
E
G
P
TGTCATGTAATGATAATTTAT
S
C
N
D
N
L
F
F
T TGATGTCATACAGGATAT
D
V
I
Q
D
M
S
C A A G A A G A A G C G C G G T 360
Q
E
E
A
R
S
T C G A G T T T G G T T C A T T T A A A T 420
E
F
G
S
F
K
L
GCGTGAAACGCACAAAAGGC
R
E
T
H
K
R
480
P
C T T T G G A G T A T T T A T A T G A T T G T CC T A A C A C C T T C G A T A G A A G T A A G G A T A C A T T A T A C A
L
E
Y
L
Y
D
C
P
N
T F
D
R
S
K
D
T
L
Y
E
540
GAGTACAAGCAGAGAT
GAATAAAAC GAAGGC GAT GAATAGC T GGAGAACT T C T T T CAG T G
V
Q
A
E
M
N
K
T
K
A
M
N
S
W
R
T
S
F
S
A
600
CT TGGACATCAGAGGT GGAGAATATCATGGCGCAGCCATGTCATCGGAGAATAATT
W
T
S
E
V
E
N
I
M
A
Q
P
C
H
R
R
I
T C T A T G GC CCA-AAT G G A G G A G A A G G A A A G A C A A C G T A T
Y
G
P
N
G
G
E
G
K
T T
Y
I
TGGG 660
V
W
@CAAAACAT C TAAT GAAGAC GA
A
K
H
L. M
K
T
K
GAAATGCGTTTTATTCTCCAGGAGGAAAATCATTGGATATAT
N
A
F
Y
S
P
G
G
K
S
L
D
I
C
AGGATATTGT
D
I V
TATTTA.KATTATGGGT TAT
Y
L
N
Y
G
L
L
TATAT T TGATATTCCAAGATGCAAAGAGGAT
I
F
D
I
P
R
C
K
E
D
780
G TAGACTGTATAATTACG
R
L
Y
N
Y
E
TAGAGGAATTTAAGAATGGAATAAT
TCAAAGCGGGAAATATGAACCCGTTTTGAAGATAG
E
E
F
K
N
G
I
I
Q
S G
K
Y
E
P
V
L
K
I
TAGAATATGTCGAAGTCATTGTAATGGCTAACTTCCTTCCGAAGGAAGGAAT
E
Y
V
E
~; I V
M
A
N
F
L
P
K
E
G
AAGATCGAATAAAGTTGGTTTCT
D
R
I
K
L
V
S
TGCTGAACAAGTAATGACT
C
C~GCACACTATGACAAAAGTACGGGTATC
720
900
V
CTTT TCTG
I
F
S
TTACAGCGCACGC
840
960
TCCGA
1020
TGATTGGG TTAT C T TAACGATC TAGGGC
1080
C G T A G G C C C G T G A G C A A T G A A C G G C G A G A T C 1111
Fig. 3. Nucleotide sequence of BBTV component l ssDNA (virion
orientation). Only the predicted amino acid sequence of ORF-V1 is
represented. The potential stem-loop sequence is in bold and
underlined; the potential TATA box is in bold and boxed; the potential
poly(A) signal is in bold and double-underlined; and the putative
dNTP-binding motif (GGEGKT) is in bold italics and underlined.
recombinants (pBTPCR7, -11 and -12) were selected and
the inserts sequenced in both orientations. Each insert
was 1111 bp and contained the 980 bp sequence from
pBT338 as well as an additional 131 bp not present in
pBT338. The sequences, however, were not identical.
There were five nucleotide differences between the four
clones. Two of these five differences resulted in a
potential amino acid change in ORF-V1. At nucleotide
16, the G in pBT338 was relaced by an A in pBTPCR7,
-11 and -12; at nucleotide 256, the A in pBT338,
pBTPCR7 and -11 was replaced by a G in pBTPCR12
resulting in the replacement of a histidine residue by an
arginine residue; at nucleotide 508, the A in pBT338,
pBTPCR7 and -11 was replaced by a T in pBTPCR12
resulting in the replacement of an asparagine residue by
an isoleucine residue; at nucleotide 701, A occurred in
pBT338 and pBTPCRll whereas a T occurred in
pBTPCR7 and -12; at nucleotide 1045, C occurred in
pBT338 and pBTPCRll whereas A occurred in
pBTPCR7 and -12. It is not known whether these
sequence variations were due to the fidelity of the DNA
Fig. 4. Southern blot analysis to determine the orientation of BBTV
component 1 ssDNA. BBTV virion ssDNA hybridized with 3~p endlabelled primer A (a) and primer B (b). Lanes 1, pBT338; lanes 2,
pBT338 insert (EcoRI/Pstl digest); lanes 3, BBTV virion ssDNA.
polymerases used to generate the clones, or reflected
genuine sequence variations in the viral genome. In most
instances, the sequence obtained from pBT338 was the
most common and was used to derive the final sequence
for BBTV DNA component 1 (Fig. 3).
To determine which sequence orientation of the
ssDNA was present in virions, BBTV ssDNA was
extracted from purified virions, electrophoresed through
agarose and transferred to nylon membranes. These
membranes were incubated with one of two 32p endlabelled oligonucleotides (primer A or B). It was found
that primer A hybridized with virion ssDNA (Fig. 4a)
whereas primer B did not (Fig. 4b). Primer A was
complementary to the sequence presented in Fig. 3
confirming that this was the orientation present in
virions.
The sequence of component 1 was analysed using the
GCG program (Devereux et al., 1984). Three ORFs were
found which could encode proteins of approximately
10K or greater (Fig. 5). ORF-V1 occurred in the virion
orientation and had 858 nucleotides. This ORF contained
a start codon at nucleotide 129 and terminated with a
stop codon (TGA) at nucleotide 987. A poly(A) signal
(AATAAA) was present from nucleotides 968 to 973.
When translated, ORF-V1 potentially encoded a protein
of 33'6K (Fig. 3). Upstream from the ORF-V1 start
codon, there was one possible TATA box (TATAAA)
from nucleotides 79 to 84. ORF-C1 (366 nucleotides)
occurred in the complementary orientation from nucleotides 628 to 263 (Fig. 3). It potentially encoded a protein
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326
R. M. Harding and others
0
l
:
A'
•
200
BBTV DNA 1 ~ ~
~
\400
600
Fig. 5. Schematic representation of BBTV component 10RFs
potentiallycodingfor proteinsof approximately10K or greater:ORFVI occursin the virionorientationwhereasORFs C1 and C2 occurin
the complementaryorientation. Positions of the stem-loop sequence
and the potential ORF-V1 TATA box are indicated.
of 10K. No obvious poly(A) signal was associated with
this ORF nor did there appear to be a 5' TATA box.
ORF-C2 (249 nucleotides) was also present in the
complementary orientation from nucleotides 414 to 166.
This ORF potentially encoded a protein of 9.3K, it did
have a possible 5' TATA box (from nucleotides 496 to
491) but there was no poly(A) signal associated with it.
The sequence was also analysed for possible stem-loop
structures. A strong potential stem-loop occurred from
nucleotides 28 to 58 containing a 10 bp stem and an 11
nucleotide loop (Fig. 3).
Discussion
We have demonstrated that the virions of BBTV contain
circular ssDNA of approximately 1.1 kb using a PCR
strategy that would amplify dsDNA from BBTV virion
ssDNA only if this template were circular. The advantages of this strategy were (i) it was an efficient method for
generating linear, full-length dsDNA using BBTV virion
ssDNA as a template in a form suitable for cloning, (ii)
only a small amount of sequence information was
required from anywhere within the BBTV component
and (iii) if the resultant amplified product was of the
expected BBTV component size, the presence of this
product established the circularity of that component.
This method should be useful for generating full-length
dsDNA copies of other potential BBTV components,
other ssDNA viruses such as SCSV and CFDV,
geminiviruses and PCV and related viruses and perhaps
other small circular DNA molecules, the size being
limited by the efficiency of the PCR amplification.
We have sequenced one component, component 1, of
the BBTV DNA genome from both the original cDNA
clone (pBT338) and three full-length PCR clones. There
was a strong stem-loop structure predicted in the virion
orientation of component 1; the loop sequence of 11
nucleotides contained a nine nucleotide sequence
(TATTATTAC) which was almost identical to the
invariant loop sequence present in nine geminiviruses
(TAATATTAC) (Lazarowitz, 1987) and also CFDV
(TAGTATTAC) (Rohde et al., 1990), with only one
nucleotide difference in each case. Evidence from the
study of geminiviruses indicates that this sequence is
involved in DNA replication (Revington et at., 1989).
The stem sequence of BBTV component 1 varied from
that of CFDV and the geminiviruses.
Component 1 contained three ORFs that potentially
encoded proteins of approximately 10K or greater. The
largest ORF (ORF-VI) occurred in the virion sense and
potentially encoded a replicase as it contained the
dNTP-binding motif G(GE)GKT. The G(X)GKT motif
has been shown to be associated with both RNA and
DNA virus replicases (Gorbalenya et al., 1990). BBTV
ORF-V1 was similar to the largest ORF (ORF1) in the
component of CFDV that has been sequenced (Rohde et
al., 1990). Both ORFs were in the virion orientation with
start codons 3' of the predicted stem-loop sequence;
both ORFs had poly(A) signals starting 19 nucleotides 5'
of the stop codon and possible TATA boxes 5' of the
start codon (BBTV, TATAAA; CFDV, TATAAG);
both ORFs potentially encoded proteins about 33K
(BBTV ORF-V1, 33.6K; CFDV ORF1, 33.4K) and both
these proteins had dNTP-binding motifs (BBTV,
GGEGKT; CFDV, GGDGKS) starting at amino acid
positions 183 and 184 respectively (Fig. 6). The two
derived amino acid sequences were compared after
alignment with the GCG PileUp program: there was
33 % sequence similarity over the 286 amino acids of
BBTV ORF-V1 with 47 % sequence similarity over the
104 carboxy-terminal amino acids from the dNTPbinding motif. Conversely, there were no ORFs in the
CFDV sequence that corresponded to BBTV ORF-C1
and ORF-C2 and no significant sequence similarity
could be detected between these two BBTV ORFs and
any CFDV ORF either at the nucleotide or amino acid
level. Furthermore, a computer search failed to reveal
any significant sequence similarity between these two
BBTV ORFs and any published nucleotide or protein
sequence. This would suggest that either BBTV component 1 and the CFDV component have different
genome organizations or that BBTV component 1 and
the CFDV component contain only one gene.
Only one recognized plant virus group has ssDNA as
its genomic material, the geminiviruses. However, BBTV
differs from the geminiviruses in a number of important
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B B T V putative replicase gene
BBTV
CFDV
1
.... M A R Y V V C W M F T I N N P T T L P V S ~ D E I K
M G S S IRR_WCF T L N Y E T E E E A ~ I E _ S L
5O
Y M V Y Q V . , .Ig RGQ.E~TP,~lV
NLV'Z___AIVGDE_V A P S T G Q R B L
51
I00
BBTV
QGXVEMKrU~_ S LKQMRGFFV GA..HL_~EKP,K GSQE_EARSYC M K E D T R I E G P
CFDV
QGF IHLKTGR
BBTV
CFDV
101
150
FE_F_GSFKLSC NDNLFDVI~D M R E T H K R P _ L E YLYDCP_NTFD __RSKDTLYRVQ
LE_HG. V P T R P G V K R p R L A Q R FAE_E... P_DE LRLEDP_GGYR __RC..V V H G A S
RL_QGLKTVLG NDRIHL____EEPTR GSDEQNIRDYC SKE.. RV.. L
151
20O
~ r ~
S~TSrS~WT
CrDV v~wrRm~_- ~ PrvrPYm~/_Q L E V L S A I GEP__ A D D R T I L W I C
BB~
201
250
BBTV
CFDV
y2LKHLMKTRN A F Y S P G G K S L
FAKYLGLKPD WFYTCGGTRK
BBTV
CFDV
251
GLLEEFKNGI
ALLECVKNRA
BBTV
CFDV
301
SC
NI
D__ICRL~y.E_ DI...VIFDI P R C K E D Y L N Y
DV..LYQ_YIE_ D_PKRNLILDV P R C N L E Y L N Y
IQSGKYEPVL KI.VEYVEVI VMANFLPKEG
VLPDYL
F S S_DKYEPLS YLGFDHVIIVL V F
3OO
IFSEDIIIKLV
KISRDKIKLW
Fig. 6. A comparison of the potential translation products of BBTV
component 1 ORF-V1 and CFDV ORF1 after alignment with the
GCG PileUp program. Identical amino acids are underlined and the
putative dNTP-binding motif is boxed.
characteristics: BBTV has isometric virions (geminiviruses have geminate virions), BBTV is transmitted by
aphids (geminiviruses are transmitted by leafhoppers or
whiteflies), the unit size of the BBTV genome is about 1.1
kb (geminivirus virion DNA is about 2.7 kb) and BBTV
has a coat protein of about 20K (geminivirus coat
proteins are 26K to 34K). BBTV is more similar to CFDV
and SCSV. Both CFDV and SCSC have small isometric
virions containing circular ssDNA of about 1 kb. SCSV
is also transmitted by aphids, but CFDV is transmitted
by Myndus taffini, a planthopper. Furthermore SCSV
has a coat protein of about 19K; the size of CFDV coat
protein has not been reported. The sequence of one
component of CFDV has been determined (Rohde et al.,
1990); it is not known at this time whether CFDV has a
mono- or multi-component genome. It has been reported
that the genome of SCSV consists of seven components
of ssDNA (Chu et al., 1990). Almost certainly, the BBTV
genome consists of more than one component: BBTV
has a coat protein with an approximate M r of 20100
(Harding et al., 1991 ; Thomas & Dietzgen, 1991) and no
ORF that could potentially encode a protein of this size
was found in component 1. We are therefore attempting
to clone and sequence further components of the BBTV
genome and investigate whether mRNAs are transcribed
in vivo from one or all of the ORFs of component 1.
From the evidence presented here, it would appear
that BBTV belongs to an undescribed group which could
include SCSV and CFDV. Three animal viruses, CAV,
PBFDV and PCV, also have small isometric virions (17
to 22 nm) that contain circular ssDNA. These three
viruses form the family Circoviridae; BBTV, CFDV and
SCSV are also potentially members of this family. There
327
are however some important differences between these
animal and plant viruses. The virion ssDNA of the
animal viruses is apparently monopartite and 1-7 to 2-3
kb in size (Todd et al., 1991); CAV and PCV have one
virion-associated protein of 50K and 36K respectively
and PBFDV has three virion-associated proteins of
15.9K, 23.7K and 26"3K (Ritchie et al., 1989). Furthermore, there was no significant sequence similarity
between BBTV component 1 and CAV (Noteborn et al,
1991) either at the nucleotide or at the amino acid level.
We thank Mr Eric Gall and Mr Don Andersen of the Queensland
Department of Primary Industries for BBTV-infected banana plants.
This project was supported by the Australian Research Council and the
Rural Industries Research and Development Corporation.
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