Download Lethal mutations within the conserved stem

Journal of General Virology (1994), 75, 3203 3209. Printedin Great Britain
3203
Lethal mutations within the conserved stem-loop of African cassava
mosaic virus DNA are rapidly corrected by genomic recombination
Sign Roberts and John Stanley*
Department o f Virus Research, John Innes Institute, John Innes Centre for Plant Science Research,
Norwich N R 4 7UH, U.K.
The nonanucleotide motif TAATATTAC occurs in the
intergenic region of all geminiviruses that have been
examined to date. The motif is invariably located within
the loop of a potential stem-loop structure that has been
implicated in viral DNA replication. To investigate the
contribution of these sequences to virus proliferation,
African cassava mosaic virus (ACMV) DNA B mutants
have been screened for their ability to infect Nicotiana
benthamiana when co-inoculated with DNA A. Mutants
in which the putative stem structure was altered by the
introduction of single nucleotide mismatches remained
as infectious as the wild-type virus and the mutations
were retained in the progeny. Mutants containing
nucleotide substitutions within the loop sequences were
similarly infectious but analysis of progeny showed that
in most cases wild-type sequences were restored by
recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious
when co-inoculated, although they were once again
efficiently rescued by recombination when inoculated
with the wild-type components. Co-inoculation of
genomic components containing the motif TAGTATTAC did not result in a systemic infection while mutants
containing the motif TAATACTAC were infectious and
the mutation was stable. The results demonstrate that
ACMV will tolerate some modification to this highly
conserved region of the genome that might allow more
precise mapping of the position at which the viral DNA
is nicked during replication.
African cassava mosaic virus (ACMV) is typical of the
majority of geminiviruses that infect dicotyledonous
plants. Two genomic components (DNAs A and B) are
essential for systemic infection (Stanley, 1983; Stanley &
Gay, 1983) and genetic analysis has revealed that DNA
A is responsible for viral DNA replication, coat protein
synthesis and the production of virus particles (Townsend
et al., 1985, 1986; Klinkenberg & Stanley, 1990) while
D N A B is required for cell-to-cell and systemic movement of virus (von Arnim et al., 1993). The genomic
components have a highly conserved sequence of
approximately 200 nucleotides (common region) that is
located within their intergenic regions (Stanley & Gay,
1983). Within the common region is a sequence that can
potentially form a stem-loop structure in single-stranded
DNA or a cruciform structure in double-stranded DNA
(nucleotides 133 to 165, Fig. 1; here referred to as the
stem-loop sequence), and at the apex of this structure is
the nonanucleotide sequence TAATATTAC that has
been found in all geminiviruses characterized to date.
Characterization of replicative intermediates of
ACMV has suggested that double-stranded DNA amplification and the accumulation of single-stranded DNA
proceeds by a rolling circle mechanism (Saunders et al.,
1991), resembling the strategies adopted by bacteriophages and some prokaryotic plasmids (reviewed by
Baas & Jansz, 1988; Gruss & Ehrlich, 1989). The product
of gene AC1 is the only virus-encoded protein essential
for ACMV DNA replication (Etessami et al., 1991). The
protein from the related virus tomato golden mosaic
virus (TGMV) binds in vitro to TGMV sequences located
upstream of the stem-loop, but not to the stem-loop
itself (Fontes et al., 1992). However, the analysis of
TGMV and squash leaf curl virus (SqLCV) recombinants
containing chimeric common region sequences has
demonstrated that the stem loop structure is also an
integral part of the replication origin (Lazarowitz et al.,
1992). AC1 might function in association with host
proteins by binding to the double-stranded DNA and
destabilizing the adjacent stem-loop sequences to allow
the introduction of a nick in the virion-sense strand prior
to the initiation of rolling circle replication.
Although the stem-loop sequence probably plays an
important role in replication, no attempts have been
made to introduce subtle changes into this region. For
dicot-infecting geminiviruses, analysis is currently confined to the insertion of an 8 bp fragment into the
nonanucleotide of TGMV, which served to abolish
0001-2686 © 1994SGM
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(Apa I )
i00
DNA
A
T TTGAAT
•
DNA
II0
TAAAAGGC
130
SspI
140
160
170
180
TCAAAAGGCTCAGAACACCCAAGGGGCCAACCGTATAATATTACCGGTTGGC•CCGCCC•CTTT-AA-TGTGGTCCCCGC
:
TCAGAACACCCAAGGGGCCAACCGTATAATATTACCGGT
:
:
:
TGGCCCCGCCCCTTTTGAAGTGTGGACCCCGC
MUTANTS
...........................................................
..................................................................
A155
G162
(ApaI)
150
.
T T TCAAATAAAAGGCTCAAAAGGC
B
120
A ...................................
G ............................
C136/G162
........................................
C .........................
G ............................
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C151
G145/C151
.......................................................
.................................................
A146/C151
G148
..................................................
....................................................
A155
G162
PROGENY
...........................................................
..................................................................
C136/G162
A
C151
........................................
G .....
C .......................................
C .......................................
A .... C .......................................
G ..........................................
A ...................................
G ............................
/ ...............................................................................................
20
3
I
1
G
C .........................
T
I .......................................................
G145/C151|
---G--T
G ............................
i0
I0
C .......................................
........................................................................................
I
A146/C151{ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G T
9
1
14
10
12
3
Fig. 1. Nucleotide sequences of A C M V D N A B stem-loop m u t a n t s produced in vitro and their progeny detected in infected N.
benthamiana. Part of the common region sequencesof DNA A and DNA B have been aligned to indicated nucleotidedifferencesC)Gaps have been introduced into the DNA A sequenceto maximizethe alignment. The positions of stem sequences(underlined) and
the conservednonanucleotide(bold) located within the loop sequenceare shown in relation to Ssp! and ApaI sites (the latter have been
introduced into mutants BG162and Bc136/G162). The number of examples of each progeny type, determined by sequence analysis of
cloned PCR fragments, is indicated on the right.
infectivity in Nicot•na benthamiana (Revington et al.,
1989)• Here, we examine the contribution of stem-loop
sequences by introducing nucleotide substitutions in this
region and screening mutants for infectivity in N.
benthamiana.
The construction in M13 vectors of infectious cloned
ACMV components used in this investigation (DNA A,
MluI clone pJS092; D N A B, PstI clone pJS094) has been
described previously (Stanley, 1983). Because modification of D N A A common region sequences could
prevent viral D N A replication by disrupting AC1
expression, the stem-loop sequences of D N A B were
targeted. The mutations summarized in Fig. 1 were
introduced by oligonucleotide-directed second strand
synthesis (Curtis & James, 1991). A transversion, from C
to G, was introduced at position 162, using a primer
corresponding to nucleotides 153 to 164 with a single
mismatch, to produce mutant B QIG2. The mutation also
served to introduce a unique ApaI site into D N A B.
Using mutant B ~1n2 as a template and a primer
corresponding to nucleotides 126 to 143 with a single
mismatch at position 136, a compensating G to C
transversion and a second ApaI site was introduced on
the opposite side of the stem (mutant Bc13G/Q~2).
Digestion of the mutant with ApaI and religation of the
construct removed nucleotides 135 to 162, corresponding
to the top half of the stem and the entire loop, to produce
mutant BA. A similar D N A A mutant (mutant A~), in
which the entire stem-loop sequence (nucleotides 133 to
165) was removed, was constructed using a primer
corresponding to nucleotides 117 to 132/166 to 173.
Additional mutations were introduced into D N A B
using a single primer corresponding to nucleotides 118 to
180, encompassing the entire stem-loop structure and 15
nucleotides on either side, and incorporating 20%
nucleotide degeneracies at positions 145 (G), 146 (A),
149 (A), 151 (C), 154 (G) and 155 (A). Using this primer,
mutations within the stem (mutant B A15~) and loop
(mutants B c15~, B m~/cl~ and BA146/c") were produced.
Mutants were identified by sequence analysis using the
T7 sequencing kit (Pharmacia).
To investigate mutant viability, wild-type D N A A and
D N A B mutants were excised from their cloning vectors
by digestion with MluI and PstI, respectively, and
mechanically co-inoculated onto N. benthamiana as
described previously (Stanley, 1983). Somewhat surprisingly, all D N A B mutants tested retained the ability
to produce a systemic infection and, with the exception
of mutant B~, their infectivity was comparable to wildtype D N A B (Table 1, experiment I). Furthermore, in all
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Table 1. Infectivity of A C M V stem-loop mutants
following mechanical inoculation onto N. benthamiana
Experiment
Inoculum
Plants infected/inoculated
I
A+ B
A + B A155
A + B G162
A + Bc136/°I~
A + Ba
A + B caS1
A + B °14~/c151
A + B A146/C151
27/28
22/26
21/26
20/24
4/25
22/26
27/28
24/28
II
A+B
AA+B
Aa+B A
9/10
2/10
0/10
III
A+B
A G14s+ B G14s
A clSl + B c151
7/10
0/10
4/10
cases symptom development was neither delayed nor
attenuated when compared with wild-type infection.
Total nucleic acids were extracted from systemically
infected leaves (Etessami et al., 1991), avoiding the lower
inoculated leaves that might have retained residual
inoculum DNA. Viral DNA was analysed by agarose gel
electrophoresis, Southern blotting onto Hybond-N membranes (Amersham) and hybridization to probes specific
to DNA A or B (Etessami et al., 1991). The results
indicated that levels of both genomic components in all
mutant-inoculated plants were similar to those associated
with wild-type infection (data not shown).
The nucleotide substitutions in mutants B~IG~ and
BAa~5were designed to alter the stability of the putative
stem-loop structure by introducing mismatches within
the stem (Fig. 2a). The two nucleotide substitutions in
m u t a n t Bc136/G162 serve to restore the stability of the
stem-loop structure to the wild-type level by creating a
C-G pair (inverted with respect to the wild-type
sequence) within the stem. Our initial premise, that
decreasing the amount of base-pairing within the stem
would affect the ability of the mutants to infect N.
benthamiana, is clearly invalid. To investigate whether
the mutant progeny had retained the mutations, a
1' 1 kbp fragment of DNA B (nucleotides 2232 to 2724/1
to 620) was amplified by PCR using a Perkin-Elmer
thermocycler (100 ng total nucleic acid in a 50 gl
reaction, amplified using the following conditions: 95 °C
for 7 min; 30 cycles of 95 °C for 30 s, 42 °C for 30 s,
72 °C for 60 s; 72 °C for 10 min). For sequence analysis,
fragments were cloned into M13mpl9 after digestion
with XbaI and HindIII (unique sites are present in DNA
B at positions 2281 and 608, respectively). Digestion of
the PCR fragments derived from the progeny of mutants
BG162 and Bcla6/al~2 with ApaI produced fragments of
0.6 kbp and 0-5 kbp, indicating that the mutations had
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been maintained during infection. This was confirmed by
sequence analysis of 10 B~162 clones and 20 Bc136/m6~
clones (Fig. 1), all of which had retained the mutation.
Although a diagnostic restriction site was not present in
mutant BA155, sequence analysis of 10 clones showed that
this mutation was also stable. The stem sequences are
highly conserved between dicot-infecting geminiviruses
and there has been only a single report of a mismatch
occurring in a natural virus isolate, for bean dwarf
mosaic virus (BDMV), in which a T-G pair is substituted
for the usual C G pair (Fig. 2b; Hidayat et al., 1993).
While this suggests that the maintenance of complementary sequences confers a selective advantage on the
virus, our results demonstrate that modifications introduced at the otherwise invariant positions 136 (G), 155
(C) and 162 (C) are tolerated.
Removal of most of the DNA B stem loop sequences
did not prevent infection when mutant B6 was coinoculated with DNA A. Analysis of the 1.1 kbp
fragment amplified by PCR showed that it had lost the
introduced ApaI site and regained an SspI site, suggesting
that the stem-loop sequences had been restored by
recombination with DNA A. This was subsequently
confirmed by sequence analysis of four clones (Fig. 1). In
three of these clones, DNA B-specific nucleotides were
found upstream at position 102 and downstream at
position 171, implying that recombination must have
occurred somewhere between these nucleotides. In the
reciprocal experiment, a DNA A mutant containing a
stem-loop deletion (mutant A A) also retained a low level
of infectivity (Table 1, experiment II) and produced wildtype levels of viral DNA in systemically infected leaves.
Restriction analysis of a 1'8 kbp fragment of DNA A
(nucleotides 2685 to 2779/1 to 1711) produced by PCR
amplification indicated that the progeny of mutant A A
had regained an SspI site, implying that the mutation
had once again been corrected by recombination between
the genomic components. Because the stem-loop sequences participate in viral DNA replication (Lazarowitz et
at., 1992), recombination in these instances must have
occurred by a non-replicative mechanism involving
inoculum DNA, a fact that might account for the
decrease in infectivity of these particular mutants. Coinoculation of mutants A A and BA did not produce a
systemic infection (Table 1, experiment II) because the
absence of identical stem-loop sequences from both
genomic components precludes recombinational rescue
of this essential region.
A DNA B loop mutant in which the nonanucleotide
motif had been changed to TAATACTAC (mutant
Bc1~1) was infectious when co-inoculated with DNA A.
The mutation was retained in nine clones analysed after
PCR amplification of progeny viral DNA and a tenth
clone showed reversion to the wild-type sequence
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Short
communication
(a)
A
T
A
A
A
T
A
A
T
A
T
T
T
A
T
CCAA
C
G'C
C-G
C'G
A-T
A-T
C-G
C'G
G-C
G-C
G'C
G-C
CCAA
wild-type
A
A
T
A
A
T
T
A
A
~C
A~
C-G
C'G
A "T
A "T
C'G
C'G
G'C
G-C
G'C
G-C
T
A
T
T
A
G
GCCC
T
C
G'C
C'G
C'G
A "T
A "T
C'G
C'G
G
@
G'C
G'C
G'C
CCAA
GCCC
A155
A
A
T
A
T
T
A
T
T
GCCC
T
C
G-C
C-G
C-G
A-T
A-T
C-G
C-G
CCAA
G'C
G'C
G-C
GCCC
0136/G162
G162
(b)
C
ACMV
ICMV
TLCV
TYLCV
TGMV
A/B
A/B
A
B
A/B
TMoV
A/B
AbMV
B G M V - P R A/B
BGMV-BZ A
B
A
BDMV
B
A/B
SqLCV
A/B
PYMV
A/B
PHV
BCTV
consensus
C
A
G
GqGGCCAaCCG-TATAATATTACCGGtTGGCCcC
GCGGCCATCCG-TATAATATTACCGGATGGCCGC
GCGGCCATCCG-TcTAATATTACCGGATGGCCGC
GCGGCCATCCG-TATAATATTACCGGATGGCCGC
GCGGCCATCCG-TtTAATATTACCGGATGGCCGC
GCGGCCATCCGtTtTAATATTACCGGATGGCCGC
GCGGCCATCCGaTATAATATTACCGGATGGCCGC
GCGGCCATCCGcTATAATATTACCGGATGGCCGC
GGCGGCCATCCGaTATAATATTACCGGATGGCCGCC
GCGGCCATCCGcTATAATATTACCGGATGGCCGC
GCGGCCATCCGtTATAATATTACCGGATGGCCGC
GCGGCCATCCG-TATAATATTACCGGATGGCCGC
GCGGtCATCCG-TATAATATTACCGGATGGCCGC
CGGCCATCCGcaATAATATTACCGGATGGCCG
GCGGCCATCCGtTATAATATTACCGGATGGCCGC
GGCCATCCGtTATAATATTACCGGATGGCC
qGGCCATCCGtTATAATATTACCGGATGGCCc
GCGGCCATCCG-TATAATATTACCGGATGGCCGC
Fig. 2. (a) Possible stem loop structures for wild-type and mutant single-stranded DNAs. The positions of the conserved
nonanucleotide (bold) and altered nucleotides (circled) are shown. (b) Comparison of stem loop sequences of dicot-infecting
geminiviruses. Sequences are from the following sources: African cassava mosaic virus (ACMV) (Stanley & Gay, 1983); Indian cassava
mosaic virus (ICMV) (Hong et al., 1993); tomato leaf curl virus (TLCV) (Dry et al., 1993); tomato yellow leaf curl virus (TYLCV)
(Navot et al., 1991); tomato golden mosaic virus (TGMV) (Hamilton et al., 1984); tomato mottle virus (TMoV) (Abouzid et al., 1992);
abutilon mosaic virus (AbMV) (Frischmuth et al., 1990); bean golden mosaic virus (strains BGMV-PR and BGMV-BZ) (Howarth et
al., 1985; Gilbertson et al., 1993); bean dwarf mosaic virus (BDMV) (Hidayat et al., 1993); squash leaf curl virus (SqLCV) (Lazarowitz
& Lazdins, 1991); potato yellow mosaic virus (PYMV) (Coutts et al., 1991); pepper huasteco virus (PHV) (Torres-Pacheco et al., 1993);
beet curly top virus (BCTV) (Stanley et al., 1986). TLCV, TYLCV and BCTV are all single component viruses. With the exception of
TGMV, BGMV-BZ and BDMV, sequences are identical between genomic components of individual viruses. Nucleotides that differ
from the consensus sequence are shown in lower case. Stem (underlined) and loop sequences (bold) are indicated. Modifications to the
ACMV sequence that are tolerated are shown at the top.
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(Fig. 1). Having demonstrated that modification of the
nonanucleotide in DNA B is tolerated, an identical
mutation was introduced into DNA A, using a primer
corresponding to nucleotides 133 to 165 with a single
mismatch at position 151, to produce mutant A c1~1. Coinoculation of mutants A c15a and Bcl'~x produced an
infection in N. benthamiana (Table 1, experiment III),
confirming that the mutation at nucleotide 151 is not
lethal to the virus. Chlorotic lesions developed at the
same time on leaves inoculated with either mutant or
wild-type virus, although the appearance of systemic
symptoms associated with the mutant was delayed by 1
to 2 days. As a consequence, plants became slightly less
severely infected. This suggests that although conservation of the motif at this position is not essential for
replication it is advantageous to the virus. A similar
conclusion has been reached concerning an identical
nonanucleotide mutant of the monocot-infecting geminivirus maize streak virus (MSV) that was infectious but
exhibited a slight delay in symptom appearance (Schneider et al., 1992). In this instance, the mutation arose
spontaneously in vivo when maize was inoculated with a
mutant containing a 40 nucleotide insert within the
nonanucleotide motif.
When co-inoculated with DNA A, DNA B mutants
containing modifications to the loop sequences in
addition to the tolerated mutation at position 151, either
external to the nonanucleotide (mutant BG14~/c151) or
within the motif itself (mutant BAa4G/cI51), were as
infectious as wild-type virus (Table 1). Sequence analysis
of 24 BG~45/c~5~ clones and 15 BA146/c151 clones derived
from the mutant progeny once again demonstrated that
the mutations had been corrected by recombination with
DNA A (Fig. 1). Comparison with other geminiviruses
shows that while there is a strong preference for A at the
position preceding the nonanucleotide (equivalent to
ACMV position 145), variation is tolerated in TGMV
and tomato yellow leaf curl virus (TYLCV) (Fig. 2 b). In
the absence of mutants containing single nucleotide
substitutions at positions 145 and 146, we are unable to
determine if the lack of mutant stability is due to the
disruption of these specific nucleotides or to the
cumulative effect of the double mutations.
Having demonstrated that a mutant modified at
position 151 remained viable, we were interested to know
whether the nonanucleotide motif could be altered
elsewhere without adversely affecting virus infectivity.
For this purpose, A to G transitions were introduced
into both genomic components, using a primer corresponding to nucleotides 133 to 165 with a single
mismatch at position 148, to produce mutants A °~48 and
B G14s containing the altered nonanucleotide TAGTATTAC. This position was targeted because nonanucleotide
variants TAGTATTAC and TATTATTAC, also located
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at the apex of comparable hairpin-loop structures, have
been described for the monocot-infecting viruses coconut
foliar decay virus (CFDV) (Rohde et al., 1990) and
banana bunchy top virus (BBTV) (Harding et al.,
1993), respectively. Co-inoculation of mutants A m~8 and
B°1~8 failed to produce either chlorotic lesions on the
inoculated leaves or systemic symptoms in N. benthamiana (Table 1, experiment III), demonstrating that
the mutations in ACMV are lethal. Although both CFDV
and BBTV have circular single-stranded DNA genomes,
neither has been assigned to the geminivirus group
because of fundamental differences in particle structure,
genome size and organization. However, in view of their
overall similarities, it is likely that the CFDV and BBTV
motifs play the same role as their geminivirus counterparts but have become adapted for specific interactions
with virus- and/or host-encoded proteins. In comparison, all naturally occurring monocot-infecting geminiviruses characterized to date have retained the conserved
TAATATTAC motif (Mullineaux et al., 1984; MacDowell et al., 1985; Donson et al., 1987; Andersen et al.,
1988 ; Chatani et al., 1991 ; Briddon et al., 1992) and none
are known to infect coconut or banana, all having host
ranges confined to Gramineae.
Our results demonstrate two novel characteristics
associated with ACMV. Firstly, stem-loop nucleotide
substitutions and deletions that are presumed to incapacitate the virus are corrected by homologous
recombination between common region sequences. This
contrasts with a previous report in which a TGMV DNA
B loop mutant containing an 8 bp insert was unable to
infect N. benthamiana when co-agroinoculated with wildtype DNA A (Revington et al., 1989). The different
behaviour of ACMV and TGMV mutants may be
attributable to the nature of the mutation (nucleotide
substitutions and deletions in ACMV, an insertion in
TGMV), the method of introduction of the viral DNA
into plants (mechanical inoculation of ACMV linearized
double-stranded DNAs, agroinoculation of partial repeats of the TGMV genomic DNAs), variation in their
host adaptation, or a combination of these factors.
Interestingly, ACMV DNA A deletion mutants rapidly
reverted to wild-type size during infection of N. benthamiana by processes involving either intramolecular or
intermolecular recombination (Etessami et al., 1989;
Klinkenberg et al., 1989) while a comparable TGMV
deletion mutant remained unaffected during infection of
this host (Gardiner et al., 1988), suggesting that ACMV
stem-loop mutants are infectious because the virus has a
greater propensity for recombination than TGMV. The
efficient rescue of ACMV stem loop mutants, as judged
by the rapid onset of infection, indicates that recombination occurs frequently during virus proliferation.
The virus will undoubedly benefit from this process
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because it ensures that advantageous common region
sequences are selectively maintained on both components. Furthermore, recombination events of this
nature could promote the exchange of genetic material
between distinct viruses, encouraging diversity and
adaptation within geminivirus populations. Secondly,
the virus can tolerate nucleotide substitutions within the
stem-loop sequences that reduce the extent of basepairing within the stem and, in one case, alters the
otherwise ubiquitous nonanucleotide motif located within the loop. The fact that stem mutants remain viable
indicates that conservation of the stem sequences per se
is not critical for replication and suggests that the
residual base-pairing remains sufficient to promote
destabilization of the double-stranded DNA in this
region, if this is indeed its function. Additional mutants
might be used to investigate the contribution of stem
sequences to virus replication although further modification could encourage recombinational rescue, making
it necessary to introduce mutations into both genomic
components.
The analysis of ACMV size revertants produced
during infection ofN. benthamiana with deletion mutants
identified a recombinational hot-spot at the 3' terminus
of the conserved nonanucleotide, within the tetranucleotide TACC (positions 152 to 155; Fig. 1) (Etessami
et al., 1989). Because origin-nicking enzymes are known
to mediate recombination during rolling circle replication
of bacteriophages and plasmids (Michel & Ehrlich,
1986a, b), the result suggested that ACMV DNA
replication was initiated by nicking the virion-sense
strand at this position. The analysis of wheat dwarf virus
(WDV) recombinants produced in vivo has recently
allowed the nick site to be mapped to the equivalent
region of the genome within the pentanucleotide sequence TACCC (Heyraud et al., 1993), consistent with
this proposal. Using genotypically distinct beet curly top
virus (BCTV) strains, the nick site has been mapped to 20
nucleotides within the stem-loop, including the tetranucleotide sequence (Stenger et al., 1991). Having
identified viable mutants containing nucleotide substitutions within this region, we are now in a position to
apply a similar approach to map the initiation site of
ACMV DNA replication.
We are grateful to Professor J. W. Davies for helpful discussion.
S.R. was supported by a studentship from the Gatsby Charitable
Foundation.
References
ABOUZID, A. M., POLSTON, J. E. & HIEBERT, E. (1992). The nucleotide
sequence of tomato mottle virus, a new geminivirus isolated from
tomatoes in Florida. Journal of General Virology 73, 3225-3229.
ANDERSEN, M.T., RICHARDSON,K.A., HARBISON, S.-A. & MORRIS,
B. A. M. (1988). Nucleotide sequence of the geminivirus chloris striate
mosaic virus. Virology 164, 443-449.
BAAS,P. D. & JANSZ, H. S. (1988). Single-stranded DNA phage origins.
Current Topics in Microbiology and Immunology 136, 31-69.
BRIDDON, R. W., LUNNESS, P., CHAMBERLIN, L. C. L., PINNER, M. S.,
BRUNDISH, H. & MARKHAM,P. G. (1992). The nucleotide sequence of
an infectious insect-transmissible clone of the geminivirus Panicum
streak virus. Journal of General Virology 73, 1041-1047.
CHATANI, M., MATSUMOTO, Y., MIZUTA, H., IKEGAMI, M., BOULTON,
M. I. & DAVIES, J. W. (1991). The nucleotide sequence and genome
structure of the geminivirus miscanthus streak virus. Journal of
General Virology 72, 2325-2331.
COUTTS, R. H.A., COFFIN, R.S., ROBERTS, E. J. F. & HAMILTON,
W. D. O. (1991). The nucteotide sequence of the infectious cloned
DNA components of potato yellow mosaic virus. Journal of General
Virology 72, 1515-1520.
CURTIS, M. D. & JAMES,R. ( 1991). Investigation of the specificity of the
interaction between colicin E9 and its immunity protein by sitedirected mutagenesis. Molecular Microbiology 5, 2727 2733.
DONSON, J., ACCOTTO, G. P., BOULTON, M. I., MULLINEAUX,P. M. &
DAVIES, J. W. (1987). The nucleotide sequence of a geminivirus from
Digitaria sanguinalis. Virology 161, 160-169.
DRY, I.B., RIGDEN, J.E., KRAKE, L.R., MULLINEAUX, P.M. &
RrZAIAN, M.A. (1993). Nucleotide sequence and genome organization of tomato leaf curl geminivirus. Journal of General Virology
74, 147-151.
ETESSAMI,P., WATTS, J. & STANLEY,J. (1989). Size reversion of African
cassava mosaic virus coat protein gene deletion mutants during
infection of Nicotiana benthamiana. Journal of General Virology 70,
277-289.
ETESSAM1, P., SAUNDERS, K., WATTS, J. & STANLEY, J. (1991).
Mutational analysis of complementary-sense genes of African
cassava mosaic virus DNA A. Journal of General Virology 72,
1005-1012.
FONTES, E. P. B., LUCKOW, V. A. & HANLEY-BOWDOIN, L. (1992). A
geminivirus replication protein is a sequence specific DNA binding
protein. Plant Cell 4, 597-608.
FRISCHMUTH, T., ZIMMAT, G. & JESICE, H. (1990). The nucleotide
sequence of abutilon mosaic virus reveals prokaryotic as well as
eukaryotic features. Virology 178, 461-468.
GARD1NER, W. E., SUNTER, G., BRAND, L., ELMER, J. S., ROGERS, S. G.
& BISARO, D. M. (1988). Genetic analysis of tomato golden mosaic
virus: the coat protein is not required for systemic spread or
symptom development. EMBO Journal 7, 899-904.
GILBERTSON, R.L., FARIA, J. C., AHLQUIST, P. & MAXWELL, D.P.
(1993). Genetic diversity in geminiviruses causing bean golden
mosaic disease: the nucleotide sequence of the infectious cloned
DNA components of a Brazilian isolate of bean golden mosaic
geminivirus. Phytopathology 83, 709 715.
GRUSS, A. & EHRLICH, S. D. (1989). The family of highly interrelated
single-stranded deoxyribonucleic acid plasmids. Microbiological
Reviews 53, 231 241.
HAMILTON, W. D. O., STEIN,V. E., COUTTS,R. H. A. & BUCK, K. W.
(1984). Complete nucleotide sequence of the infectious cloned DNA
components of tomato golden mosaic virus: potential coding regions
and regulatory sequences. EMBO Journal 3, 2197-2205.
HARDING, R.M., BURNS, T.M., HAFNER, G., DIETZGEN, R.G. &
DALE, J. L. (1993). Nucleotide sequence of one component of the
banana bunchy top virus genome contains a putative replicase gene.
Journal of General Virology 74, 323-328.
HEYRAUD, F., MATZEIT, V., KAMMANN,M., SCHAEFER,S., SCHELL, J. &
GRONENBORN, B. (1993). Identification of the initiation sequence for
viral-strand DNA synthesis of wheat dwarf virus. EMBO Journal 12,
4445-4452.
HIDAYAT, S. H., GILBERTSON, R. L., HANSON, S. F., MORALES, F.J.,
AHLQUIST, P., RUSSELL, D. R. & MAXWELL, D. P. (1993). Complete
nucleotide sequences of the infectious cloned DNAs of bean dwarf
mosaic geminivirus. Phytopathology 83, 181 187.
HONG, Y. G., ROBINSON, D. J. & HARRISON, B. D. (1993). Nucleotide
sequence evidence for the occurrence of three distinct whiteflytransmitted geminiviruses in cassava. Journal of General Virology 74,
2437-2443.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 12:27:22
Short communication
HOWARTH, A. J., CATON, J., BOSSERT, M. & GOODMAN, R. M. (1985).
Nucleotide sequence of bean golden mosaic virus and a model for
gene regulation in geminiviruses. Proceedings of the National
Academy of Sciences, U.S.A. 82, 3572 3576.
KLINKENBERG, F. A. & STANLEY, J. (1990). Encapsidation and spread
of African cassava mosaic virus DNA A in the absence of DNA B
when agroinoculated to Nicotiana benthamiana. Journal of General
Virology 71, 1409-1412.
KLINKENBERG, F.A., ELLWOOD, S. & STANLEY, J. (1989). Fate of
African cassava mosaic virus coat protein deletion mutants after
agroinoculation. Journal of General Virology 70, 1837-1844.
LAZAROWITZ, S. G. ~z LAZDINS, I. B. (1991). Infectivity and complete
nucleotide sequence of the cloned genomic components of a bipartite
squash leaf curl geminivirus with a broad host range phenotype.
Virology 180, 58-69.
LAZAROWITZ, S. G., Wu, L. C., ROGERS, S. G. & ELMER, J. S. (1992).
Sequence-specific interaction with the viral ALl protein identifies a
geminivirus DNA replication origin. Plant Cell 4, 799-809.
MACDOWELL, S. W., MACDONALD, H., HAMILTON,W. D. O., COUTTS,
R. H. A. & BUCK, K. W. (1985). The nucleotide sequence of cloned
wheat dwarf virus DNA. EMBO Journal 4, 2173-2180.
MICHEL, B. & EHRLICH, S. D. (1986a). Illegitimate recombination at
the replication origin of bacteriophage MI3. Proceedings of the
National Academy of Sciences, U.S.A. 83, 3386-3390.
MICHEL, B. & EHRLICH, S.D. (1986b). Illegitimate recombination
occurs between the replication origin of the plasmid pC194 and a
progressing replication fork. EMBO Journal 5, 3691-3696.
MULLINEAUX, P. M., DONSON, J., MORRIS-KRSINICH, B. A. M., BOULTON, M. I. & DAVIES,J. W. (1984). The nucleotide sequence of maize
streak virus DNA. EMBO Journal 3, 3063 3068.
NAVOT, N., PICHERSKY, E., ZEIDAN, M., ZAMIR, D. 8¢~ CZOSNEK, H.
(1991). Tomato yellow leaf curl virus: a whitefly-transmitted
geminivirus with a single genomic component. Virology 185,
151 161.
REVINGTON, G.N., SUNTER, G. & BISARO, D.M. (•989). DNA
sequences essential for replication of the B genome component of
tomato golden mosaic virus. Plant Cell 1,985-992.
ROHDE, W., RANDLES, J.W., LANGRIDGE, P. & HANOLD, D. (1990).
3209
Nucleotide sequence of a circular single-stranded DNA associated
with coconut foliar decay virus. Virology 176, 648 651.
SAUNDERS, K., LUCY, A. & STANLEY, J. (1991). DNA forms of the
geminivirus African cassava mosaic virus consistent with a rolling
circle mechanism of replication. Nucleic Acids Research 19, 23252330.
SCHNEIDER,M., JARCHOW, E. & HOHN,B. (1992). Mutational analysis
of the 'conserved region' of maize streak virus suggests its
involvement in replication. Plant Molecular Biology 19, 601 610.
STANLEY, J. (1983). Infectivity of the cloned geminivirus genome
requires sequences from both DNAs. Nature, London 305, 643-645.
STANLEY, J. & GAY, M. R. (1983). Nucleotide sequence of cassava
latent virus DNA. Nature, London 301,260-262.
STANLEY, J., MARKHAM, P. G., CALLIS, R. J. & PINNER, M. S. (1986).
The nucleotide sequence of an infectious clone of the geminivirus
beet curly top virus. EMBO Journal 5, 1761-1767.
STENGER,D. C., REVINGTON,G. N., STEVENSON,M. C. & BISARO,D.
M. (1991). Replicational release of geminivirus genomes from
tandemly repeated copies : evidence for rolling circle replication of a
plant viral DNA. Proceedings of the National Academy of Sciences,
U.S.A. 88, 8029-8033.
TORRES-PACHECO, I., GARZON-TIZNADO, J. A., HERRERA-ESTRELLA, L.
& RIVERA-BUSTAMANTE,R. F. (1993). Complete nucleotide sequence
of pepper huasteco virus: analysis and comparison with bipartite
geminiviruses. Journal of General Virology 74, 2225 2231.
TOWNSEND, R., STANLEY, J., CURSON, S. J. & SHORT, M . N . (1985).
Major polyadenylated transcripts of cassava latent virus and location
of the gene encoding coat protein. EMBO Journal 4, 33 37.
TOWNSEND, R., WATTS, J. & STANLEY, J. (1986). Synthesis of viral
DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with
cassava latent virus (CLV); evidence for the independent replication
of one component of the CLV genome. Nucleic Acids Research 14,
1253 1265.
VON ARNIM, A., FRISCHMUTH, T. & STANLEY, J. (1993). Detection and
possible functions of African cassava mosaic virus DNA B gene
products. Virology 192, 264-272.
(Received 3 June 1994; Accepted 22 July 1994)
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