Journal of General Virology (1995), 76, 2415-2422.
Printed in Great Britain
Regulation of African cassava mosaic virus complementary-sense
gene expression by N-terminal sequences of the replication-associated
protein AC1
Yiguo H o n g and John Stanley*
Department o f Virus Research, John Innes Centre, Cohmy Lane, Norwich N R 4 7UH, UK
Fragments of the African cassava mosaic virus (ACMV)
genome, cloned upstream of the fl-glucuronidase (GUS)
reporter gene in an expression cassette, were analysed
for their ability to direct complementary-sense gene
expression in tobacco protoplasts by measuring GUS
activity. Five arbitrary domains (A-E) have been desig-
nated that contribute to the expression of AC1
(replication-associated protein) and AC4. Consistent
with earlier reports, AC1 gene expression was negatively
regulated (80 % reduction in activity) by its own protein
product, and suppression was mimicked by truncated
versions of AC1 comprising the N-terminal 57 amino
Introduction
African cassava mosaic virus (ACMV), a whitefly-
transmitted geminivirus infecting dicotyledonous plants,
has a genome comprising two circular single-stranded
(ss) DNAs (A and B) (Stanley & Gay, 1983;
Stanley, 1983). DNA A contains six putative genes that
are conserved in other group members, distributed
between the virion- and complementary-sense strands of
a double-stranded (ds) DNA intermediate. Virion-sense
gene A V1 encodes the coat protein (Townsend et al.,
1985). A function has yet to be assigned to the
overlapping gene, A V2, and neither virion-sense gene is
required for systemic infection of Nicotiana benthamiana
(Stanley & Townsend, 1986; Etessami et al., 1989). Of
the four overlapping complementary-sense genes, AC1 is
essential for viral DNA replication (Brough et al., 1988;
Elmer et al., 1988; Etessami et al., 1991), A C 2 trans-
activates the expression of coat protein and DNA B
genes (Haley et al., 1992; Sunter & Bisaro, 1992) and
A C 3 is required for normal levels of viral DNA
replication (Sunter et al., 1990; Etessami et al., 1991;
Morris et al., 1991). No function has been assigned to
* Author for correspondence. Fax +44 1603 456844. e-mail
STANLEYJ@BBSRC.AC.UK
0001-3283 © 1995 SGM
2415
acids. AC1 also suppressed AC4 gene expression to a
similar extent. Nucleotide sequences responsible for
suppression were mapped to domain A, a 92 bp fragment
located immediately upstream of the AC1 initiation
codon encompassing the consensus TATA box and
transcription start point. Complementary-sense gene
expression also decreased by 30-40 % in the presence of
AVI (coat protein) although other DNA A-encoded
proteins (AV2, AC2, AC3 and AC4) had no effect. The
results are discussed in the light of recent advances
concerning the initiation of viral DNA replication and
the control of gene expression.
A C 4 and the gene is dispensable for systemic infection of
N. benthamiana, although homologues in beet curly top
virus (BCTV), tomato yellow leaf curl virus (TYLCV)
and tomato leaf curl virus (TLCV) have been implicated
in either symptom development (Stanley & Latham,
1992; Rigden et al., 1994) or virus movement (Jupin et
al., 1994).
The diverging genes AC1 and A V2 are separated by an
intergenic region that contains the common region, a
sequence of approximately 200 nucleotides that is highly
conserved between the genomic components (Stanley &
Gay, 1983). The intergenic region contains cis-acting
elements that participate in the control of viral DNA
replication and gene expression. For example, start sites
for bidirectional transcription have been mapped within
this region to nucleotide 278 (virion-sense) and approxi-
mately nucleotide 20 (complementary-sense) (Townsend
et al., 1985; Ward, 1989). In the related virus tomato
golden mosaic virus (TGMV), ALl (the homologue of
AC1) binds to common region sequences upstream of the
ubiquitous nonanucleotide motif TAATATTAC (Fontes
et al., 1992, 1994a, b) implicated in the initiation of
rolling circle replication (Saunders et al., 1991; Stanley,
1995). Because the ALl binding site is positioned
between the consensus TATA box and the start of the
A L l transcript, binding of the protein during the
initiation of replication would disrupt its own expression,
2416 Y. Hong and J. Stanley

a phenomenon that has been observed for both ACMV
and TGMV (Haley et al., 1992; Sunter et al., 1993; Eagle
et al., 1994; Gr6ning et al., 1994). TGMV AL4 (the
homologue of AC4) also appears to suppress A L l
expression by an unknown mechanism (Gr6ning et al.,
1994). In view of the small size of geminivirus genomes
and the overlapping arrangement of viral genes, it is
likely that processes occurring during the course of
infection are highly co-ordinated by a complex in-
teraction of viral and host components, and that the
intergenic region plays a major role in the regulatory
process. Here, we investigate in detail the contribution
of ACMV sequences to the expression of the
complementary-sense genes A C 1 and A C 4 by analysing
their ability to direct the transient expression of fl-
glucuronidase (GUS) reporter gene in tobacco proto-
plasts.
CACCAGTGGtaCCCACATTG (nucleotides 303-284), GATTGGgg-
tACcTAAGTAGTG (nucleotides 21 0-190), GGTTGGtaCCTTGGG-
TGTTC (nucleotides 142-123) or CAAGggTGCCATTTAGAGACA-
C (nucleotides 77-56). For the analysis of AC4 expression, fragments
were amplified using the virion-sense primer GTGAGAAccAtggTC-
TTGGC (nucleotides 2710-2729) in combination with the
complementary-sense primers described above. Lower-case letters
indicate nucleotide changes introduced to create NcoI (virion-sense
primer) and KpnI (complementary-sense primers) sites. The expression
cassette pJIT166 contains the GUS reporter gene cloned between a
tandem repeat of the cauliflower mosaic virus (CaMV) 35S promoter
and polyadenylation sequences (Guerineau et al., 1992). To analyse
AC 1 expression, fragments were cloned into pJIT166 after removal of
the 35S promoter sequence by digestion with Ncol and Kpnl, to
produce GUS expression cassettes pAC1Prol-pAC1Pro5 (see Fig. 1).
The expression cassettes pAC4Prol-pAC4Pro3 resemble pAClProl
pAC1Pro3, respectively, except that the 3' co-terminal fragments
extend to the putative AC4 coding sequences. The control plasmid
pKH 166, without an inserted fragment, was constructed by removal of
the 35S promoter from pJIT166 using KpnI and H#ldIII and
recircularization of the plasmid after Klenow infill of DNA termini.

Construction of DNA A gene expression cassettes. Fragments

Methods
Source of A C M V clones. The construction of a full-length infectious
clone of ACMV DNA A (pJS092) and mutant derivatives (pCLVAC 1-
1, pCLVAC 1-2, pCLVAC2-1, pCLVAC2-2, pCLVAC3-1, pCLVAC3-
2 and pCLVAC4-1) has been described (Stanley 1983; Etessami et al.,
1991).
Construction o f GUS expression cas~'ettes. Nucleotide numbering of
DNA A is according to Stanley & Gay (1983). For the analysis of ACI
expression, DNA A fragments were PCR-amplified from pJS092 using
a combination of the virion-sense primer CGAGGAGTTCcatgGTT-
GACC (nucleotides 2743-2763) together with the compleme~tary-
sense primer GGTCGgTaCcACATAATTAC (nucleotides 459--440),
encompassing DNA A coding sequences were synthesized by PCR
amplification from wild-type or mutant clones as summarized in Table
l. Fragments were cloned into the expression cassette pJIT163
(Guerineau et al., 1992) between a tandem repeat of the 35S promoter
and polyadenylation sequences, to produce the DNA A gene expression
cassettes shown in Fig. 2. To ensure that no errors had been introduced
during PCR amplification, the integrity of viral DNA inserts in the
GUS expression cassettes and DNA A gene expression cassettes was
verified by sequence analysis using a T7 sequencing kit (Pharmacia)
and DNA A-specific primers.
Protoplast preparation and transfection. Suspension cultures of the
tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2)

Table 1. A C M V DNA A gene expression plasmids

Genes and Cloning

Plasmid mutants Template Primers* Co-ordinates sites

pAV l AV1 pCLV1.3A CAGTTATCAaGetTCGTAATTATG 425~448 HindIII

GAGTGCAAGTcGACTCATGA 1260-1241 SalI
pAV2 AV2 pJS092 CGAAGTTGaaGCTtGTGCGCA(ATG) 265-285 HindIII
TGTCTGGtCgaCTATACATC 638-619 SalI
pAC 1 AC 1 pJS092 CTTGGTCAtCATGAGAACTCC 2766-2746 B~pHI
CCACAGACAgGATCCACTCTC 1659-1679 BamHI
pMACI-1 mACI-I pCLVACI-1 Primers as for pACI
pMACI-2 mACI-2 pCLVAC1-2 Primers as for pAC1
pMAC1-3 mAC1-3 pCLVACI-1 CTTGGTCAtCATGAGAtagCCTCG 27662743 BspHI
CCACAGACAgGATCCACTCTC 1659 1679 BamHI
pAC2 AC2 pJS092 GGCATTAAAeeATGgAATCTTC 1782 1761 NcoI
CATCTATAggATCCAAGTATT 1329-1349 BamHI
pMAC2-1 mAC2-1 pCLVAC2-1 Primers as for pAC2
pMAC2-2 mAC2-2 pCLVAC2-2 Primers as for pAC2
pAC3 AC3 pJS092 GCTCCAAcCATGGATTTACGC 1632-1612 NcoI
TCCTGTATAgtCGACGTTGAA 1165-1185 Sa~
pMAC3-1 mAC3-1 pCLVAC3-1 Primers as for pAC3
pMAC3-2 mAC3-2 pCLVAC3-2 Primers as for pAC3
pAC4 AC4 pJS092 CGAATTCAAGCttAGAATGTC 2738 2718 HindllI
AGTCCTTgteGACTAATTCC 2288-2307 Sail
pMAC4-1 mAC4-1 pCLVAC4-1 Primers as for pAC4
* Restriction sites are shown in bold type and initiation codons (ATG) and an in-phase stop codon (TAG) are
underlined. The position of the AV2 initiation codon has been indicated but was not included in the primer.
Lower-case letters indicate nucleotide changes introduced to create restriction sites.
 ACMV c o m p l e m e n t a r y - s e n s e gene expression 2417

. D

< AV2 ] I AC4 _

domain I E I D [ C I B I A

69 2757
pAC1Pro5 I
134 2757
pAC1Pro4 I

198 2757
pAC1Pro3 I

292 2757
pAC1Pro2 [

449 2757

Kpnl Ncol

GUS

ori

polyA

Fig. 1. GUS expression cassettes used for the analysis of complementary-sense gene expression. ACMV DNA A fragments originating
from the region between the diverging virion- and complementary-sense genes A V1 and A C1 were cloned upstream of the GUS coding
sequence such that the AC1 initiation codon replaced that of GUS. The nucleotide co-ordinates of the 3' co-terminal fragments that
define the arbitrary domains (A-E) are indicated, as are the positions of the conserved stem loop ( I ) within the common region
(stippled box) and consensus TATA sequence (V) located upstream of the complementary-sense transcript start. Expression cassettes
pAC4Pro I~AC4Pro3, used to analyse AC4 expression, are similar to pAC 1Pro I~AC1Pro3 except that the 3' co-terminal fragments
extend to the first in-frame ATG of the AC4 coding region (nucleotide 2723).

(Nagata et al., 1992) were maintained at 25 °C in semi-dark conditions
with shaking at 125 r.p.m. Cells were subcultured on a weekly basis by
50-fold dilution in MS medium (Murashige and Skoog plant salt
mixture; ICN Biomedicals) supplemented with 3 % sucrose, 100 lag/ml
inositol, 1 lag/ml thiamin, 0.2 lag/ml 2,4-dichlorophenoxyacetic acid,
200 lag/ml KH2PO~). Four days after subculturing, 10 ml of cells were
harvested by centrifugation at 60 g for 5 min to obtain a packed cell
volume of 4-5 ml. The cells were resuspended in 50 ml 0.4 M-mannitol
(pH 5.5) containing 1% cellulase (Sigma), and incubated at 30 °C for
1.5 h. Protoplasts were isolated and resuspended in W5 medium as
described by Brough et al. (1992).
Protoplast transfection was carried out essentially as described
(Negrutiu et al., 1987; Brough et al., 1992). For the analysis of gene
expression, 5 lag of each expression cassette and 12.5 lag of carrier DNA
(Sigma) were introduced into 105-106 protoplasts. After incubation at
25 °C in the dark for 36 h in 5 ml of PCM (protoplast culture medium
comprising Murashige and Skoog plant salt mixture supplemented
with 3% sucrose, 3 lag/mI naphthaleneacetic acid, 1 lag/ml 6-
benzylaminopurine and 0-4 n-mannitol, pH 5-8), the protoplasts were
harvested and assayed for GUS activity. For each experiment, positive
and negative controls were carried out using pJIT166 and pKH166,
respectively.
Determination of GUS activity. GUS activity was determined
according to Jefferson (1987). For each experiment, background GUS
activity associated with non-transfected protoplasts (6.4 pmol/min/mg
protein) was subtracted throughout. Protein concentration was
estimated using a Bio-Rad protein assay kit based on the method of
Bradford (1976).
Results
A n a l y s i s o f c o m p l e m e n t a r y - s e n s e gene expression
T o i n v e s t i g a t e A C 1 e x p r e s s i o n , five e x p r e s s i o n c a s s e t t e s
w e r e c o n s t r u c t e d b y r e p l a c i n g t h e C a M V 35S p r o m o t e r
i n p J I T 1 6 6 w i t h A C M V D N A A f r a g m e n t s ( F i g . 1). T h e
2418 Y. Hong and J. Stanley

Table 2. Analysis of GUS activity from ACMV (a)

complementary-sense promoter fragments in tobacco
B Y-2 cells
GUS expression RelativeGUS activity (%)*
cassette (Mean ± sE) No. of expts
pAC1Prol 100 13
pAC 1Pro2 74-8± 7.8 10
pAC 1Pro3 62-3+ 66 10
pAC 1Pro4 48.4 4-_59 7
pAC 1Pro5 16.04-7-4 7
pKH 166~ 2.74- 1-9 13
pJIT166~ 1002.3 +_247.0 13
pAC4Pro 1 3.6 ± 3.7 8
pAC4Pro2 100 8
pAC4Pro3 89.1 ± 2"5 4 (b)
pJIT166t 1991.3 ± 689.9 8
[ 35s I ORF/mutant ..~ polyA
* GUS activity from pAC1Prol (92-9pmol/min/mg protein) and
pAC4Pro2 (53.5 pmol/min/mg protein) was given an arbitrary value
of 100 % in each set of experiments. pAVl ~- "~
To compare relative levels of expression, protoplasts were v2
transformed with pJIT166 containing GUS downstream of a tandem
repeat of the CaMV 35S promoter. Background activitywas monitored pAC1 ~ ,,,,: ii,i:: :...... I
using pKH 166, derived from pJIT 166 by removal of the 35S promoter. pMACl-1 ~ ]
pMAC1-2 ~ ]
pMAC1-3 I - - ]
largest fragment (nucleotides 2757-2779/1-449) covered pAc2
the region between AV1 and AC1 coding sequences pMAC2-1 ~---~.--]
(pAC1Prol) and the smaller fragments were 3' co- pMAC2-2 L-~'--I
terminal with this sequence (pAC1Pro2-pAC1Pro5). In pAc3
each case, the G U S coding region was fused in-frame to pMAC3-1 [[--]
the initiation codon of AC1. Each of the five fragments pMAC3-2
influenced G U S expression when introduced into pAC4
tobacco BY-2 cells (Table 2), and the level of expression
progressively and significantly decreased (Student's t-
Fig. 2. DNA A gene expression cassettes used to analyse the control of
test, P ~< 0"0013) as the length of the viral D N A fragment complementary-sense gene expression. (a) Genome map of ACMV
decreased. On the basis of this result, we arbitrarily DNA A. (b) Intact viral coding sequences were cloned between the
subdivided the entire region into five domains (A-E, Fig. CaMV 35S promoter and polyadenylation signal. In each case, the first
1). D o m a i n A (nucleotides 2757-2779/1-69), located ATG downstream of the transcript start corresponds to the initiation
immediately upstream of the AC1 coding sequence and codon of the viral gene. Mutations introduced into the genes either
disrupted the coding sequences (open boxes) or fused overlapping
containing the putative T A T A box for complementary- coding sequences to produce potential chimeric proteins (combined
sense transcription (Ward, 1989), is responsible for only stippled and cross-hatched boxes; mutant pMAC 1-1).
16% of the total G U S expression. The progressive
addition of domains B-D (nucleotides 70-134, 135-198
and 199-292, respectively) increased expression to 48 %, domain D sequences (pAC4Pro3) served to significantly
62 % and 75 %, respectively, of the entire region. reduce G U S expression by 11% (Student's t-test, P =
In a similar set of experiments, AC4 expression was 0'0033), indicating that this domain made a similar,
investigated using the expression cassettes p A C 4 P r o l - relatively low, contribution to expression from both
pAC4Pro3 (Table 2). The cassettes resemble p A C 1Pro 1- pAC1Pro3 and pAC4Pro3.
pAC1Pro3, respectively, except that the A C M V 3' co-
terminal fragments extend downstream to the first in-
Regulation of complementary-sense gene expression
frame A T G of the AC4 coding sequence. Unlike its AC 1
counterpart, pAC1Prol, GUS expression from To investigate the effect of A C M V D N A A genes on the
p A C 4 P r o l (containing the entire region encompassing control of complementary-sense gene expression, virion-
domains A - E ) was reduced almost to background level. and complementary-sense genes and their mutant
Maximal G U S expression occurred from pAC4Pro2 at derivatives were inserted downstream of the C a M V 35S
68 % of the level from its counterpart pAC1Pro2 (after promoter in the expression cassette pJIT163 (Fig. 2).
normalization against the pJIT166 control). Removal of Fragments containing mutated genes were the same size
 A C M V complementary-sense gene expression 2419

Table 3. Regulation of GUS expression from pAC1Prol Table 4. Regulation of GUS expression .from pAC4Pro2
by DNA A genes and their mutants in tobacco BY-2 by DNA A genes and their mutants in tobacco BY-2
cells cells
Relative GUS activity (%)* Relative GUS activity (%)*
Construct (MeanisE) No. of expts Construct (Mean___SE) No. of expts

pAC1 17-8 ±4.3 12 pAC1 12.3 ± 7.7 8

pMACI-1 14-4_+2-1 8 pMACI-1 10.2_+6.6 4
pMAC1-2 18-7_+ 8.2 8 pMAC 1-2 10.7 ___4.5 4
pMAC1-3 104-3 _+8.9 8 pMAC 1-3 102.4 ± 20.2 4
pAC2 91 "7 _+8.9 8 pAC2 103.4_+ 10-3 8
pMAC2-1 90.4 _+6.2 5 pMAC2-1 92.8 ± 3-9 4
pMAC2-2 91"9 ± 7.4 6 pMAC2-2 93.6_+ 3-3 2
pAC3 93-1 ± 8-3 8 pAC3 97.3 ± 11-9 8
pMAC3-1 91.3 ±4.1 6 pMAC3-1 90-3 _+5.9 4
pMAC3-2 91.5 _+1.9 2 pMAC3-2 91-6 _+4.9 4
pAC4 94-9 _+4.8 6 pAC4 92.3 _+5.3 4
pMAC4-1 96.2 _+2.8 6 pMAC4-1 93.8 _+4.1 4
pAVI 70.9 _+ 14.4 8 pAV1 62.8 ± 3-1 4
pAV2 90.9 _+3.2 5 pAV2 90.9 _+8.3 4
pJIT163t 94.8 _+6.4 8 pJIT163t 95.7_+4.2 4
* GUS activity from pAC1Prol was given an arbitrary value of * GUS activity from pAC4Pro2 was given an arbitrary value of
100%. Background level of GUS activity from pKH166 is given in 100%. Background level of GUS activity from pKHI66 is given in
Table 2. Table 2.
t pJIT 163 has no DNA fragment inserted downstream of the CaMV t pJIT163 has no DNA fragment inserted downstream of the CaMV
35S promoter. 35S promoter.

as those containing wild-type genes but had either
reduced coding capacity or the potential to encode a
chimeric protein (pMACI-1). In pMACI-1, 57 N-
terminal amino acids of AC1 are fused to 93 C-terminal
amino acids of AC4. The AC1 coding region is
terminated after 57 amino acids in pMAC1-2 and
immediately in pMAC1-3. The coding capacity of both
pMAC2-1 and pMAC2-2 is reduced to 39 N-terminal
amino acids of AC2, pMAC3-1 and pMAC3-2 retain 20
and 114 N-terminal amino acids of AC3, respectively,
and pMAC4-1 retains 48 N-terminal amino acids of
AC4.
GUS expression was approximately 10 times stronger
from the duplicated 35S promoter in pJIT166 than from
the ACMV fragment in pAC1Prol (Table 2) and, hence,
the viral proteins and their mutant derivatives should be
highly expressed relative to the reporter gene. Each
expression cassette was introduced into tobacco proto-
plasts in the presence ofpAC1Prol, and its effect on AC1
expression was monitored by measuring GUS activity
(Table 3). GUS expression was reduced to 18 % by AC1
(pAC1), and a similar level of suppression was achieved
by the ACI/AC4 chimera (pMACI-1) and truncated
AC1 (pMAC1-2), suggesting a regulatory role for the N-
terminal amino acids of AC1. GUS expression was fully
restored when AC 1 expression was completely disrupted
(pMAC1-3), confirming that AC1 expression per se is
responsible for down-regulation. Neither pAC4 nor
pMAC4-1 had an effect on GUS activity, demonstrating
that AC4 coding sequences that overlap the N-terminus
of AC1 do not participate in the regulatory effect. Of the
remaining viral gene products (AC2, AC3, AV1 and
AV2), only the coat protein (AV1) had a significant effect
(Student's t-test, P = 0'0002), reducing GUS expression
by approximately 30 %.
Similar results were obtained when the effect of viral
genes on AC4 expression was investigated using
pAC4Pro2 (Table 4). GUS expression was reduced to
12% in the presence of AC1 (pAC1), comparable
suppression was obtained using AC1 mutants produced
by pMACI-1 and pMAC1-2, and expression was
unaffected in the presence of pMAC 1-3. Of the remaining
viral gene products, once again only the coat protein had
a significant effect, reducing GUS expression by approxi-
mately 40%. It should be noted that, because the
upstream AC1 initiation codon has been left intact in
pAC4Prol-pAC4Pro3 in order to resemble as closely as
possible the wild-type juxtaposition of translational
elements, it is possible that expression of the residual 12
N-terminal amino acids of AC1 may have an adverse
effect on GUS expression from these cassettes.
Identification of nucleotide sequences responsible for
down-regulation of complementary-sense gene expression
Having demonstrated that AC1 can suppress
complementary-sense expression in our protoplast assay,
we investigated the contribution of ACMV sequences to
2420 Y. Hong and J. Stanley
Table 5. Suppression of GUS activity from A C M V complementary-sense
promoter fragments by AC1 protein and mutant derivatives
Relative GUS activity [Mean±sE (no. of expts)]*
Plasmid pAC1Prol pAC1Pro2 pAC1Pro3
None 100 (12) 100 (6) 100 (6)
pAC1 17-8+_4-3(12) 20-8±8.0(6) 20.2±2.3(6)
pMACI-1 14.4±2.1(8) 18.3±4.2 (4) 16.7±3.1(4)
pMAC1-2 18.7±8.2(8) 16.4±3.2(4) 21.5_+3.2(3)
pMAC1-3 104.3±8-9(8) 97"6_+1.9 (4) 98.2_+4.7(4)
* For each cassette (pAC1Pro1-pACIPro5) the levelof GUS expressionin the absence of AC 1
or mutant derivativehas been assigned a value of 100%. Background level of GUS activity from
pKH166 is given in Table 2.
this phenomenon by comparing GUS expression from
p A C 1 P r o l - p A C 1 P r o 5 in the presence of AC1 or mutant
derivatives (Table 5). The relative reduction in the level
of GUS expression by AC1 was similar for each
expression cassette (79-85 %), and Student's t-tests failed
to show any statistical differences in the level of
suppression (P > 0.1). GUS expression from each cas-
sette was largely unaffected in the presence of pMAC 1-3,
but both the AC1/AC4 chimera (pMACI-1) and the
truncated version of AC 1 (pMAC1-2) caused a reduction
in GUS expression comparable to that seen for AC1. The
data indicate that the N-terminal 57 amino acids of AC1
can suppress GUS expression from the complementary-
sense promoter, and suggest that domain A (Fig. 1) plays
an important role in the regulatory process.
Discussion
It has become increasingly clear that the intergenic
region of the geminivirus genome, located between the
diverging virion- and complementary-sense genes, con-
tains a complex arrangement of cis-acting elements
necessary for the co-ordinated control of gene expression
and viral D N A replication. Here, we have investigated
the expression of ACMV overlapping complementary-
sense genes AC1 and AC4 in tobacco BY-2 cells by fusing
the GUS reporter gene downstream of fragments derived
from this region of D N A A. In this host background,
expression from the entire region (nucleotides
2757-2779/1-449 in pAC1Prol) is approximately 10%
of that for a tandemly" repeated CaMV 35S promoter
fragment (Guerineau et al., 1992). Deletion of upstream
sequences progressively reduced the level of GUS
expression, indicating that each of the arbitrary domains
A - E (Fig. 1) contributes to complementary-sense gene
expression. Although domain A includes the consensus
T A T A box and transcription start point, it makes a
relatively small contribution to complementary-sense
gene expression, implying that upstream sequences play
an important regulatory role. The regulatory mechanism
pAC1Pro4 pAC1Pro5
I00 (4) I00 (6)
15.3±1.4 (4) 15.7±4.7(6)
10.9±5.9 (4) 16.6±1.6 (6)
17.3± 2.0 (4) 19.5±1.5 (6)
113.0±3.0(4) 102.3±13.3(6)
is not known but the sequences may interact with host
factors, for example, as observed for the maize streak
virus repl motif responsible for virion-sense trans-
criptional activation (Fenoll et al., 1990).
No role could be assigned to ACMV AC4 based on the
analysis of mutants in N. benthamiana (Etessami et al.,
1991), although its counterparts in the monopartite
viruses BCTV and TLCV have been shown to be
important symptom determinants (Stanley & Latham,
1992; Rigden et al., 1994). It is not known if ACMV AC4
is functional in less permissive hosts than N. benthamiana
or if it represents a vestigial gene that no longer has a
biological role in a bipartite virus. Our data suggest that
little or no AC4 expression will occur when the entire
region under investigation (domains A E ) is present,
which could be taken to imply that expression of this
gene is silenced in ACMV. Surprisingly, however,
removal of upstream sequences within domain E
increased expression to a level only slightly less than that
of AC1. The data suggest that if AC4 is functional,
domain E could participate in the differential regulation
of the overlapping genes AC1 and AC4. Apart from the
contribution of domain E, the similar influence of AC1
on GUS expression from AC1 and AC4 initiation codons
suggests that both genes may be expressed from the same
transcriptional unit via a polycistronic message. Con-
sistent with this, our results using pAC4Pro2 and
pAC4Pro3 indicate that a relatively high level of
expression can occur from the internal AC4 initiation
codon located downstream of the intact AC1 initiation
codon. As the inclusion of domain E could allow the
expression of the N-terminal half of AV2, it is possible
that the latter acts as a translational repressor of AC4
without affecting ACI expression.
Our results demonstrate that AC1 down-regulates its
own expression by a factor of more than 80 %. A similar
conclusion was reached in an earlier study on A C M V
(Haley et al., 1992), although the effect on expression
that we observe is much greater than the 50 % reduction
previously reported. It is worth noting that our strategy

for constructing GUS expression cassettes differs from
that of Haley et al. (1992). Unlike the earlier work that
used transcriptional fusion, we joined the GUS coding
region in-frame to the AC1 initiation codon. We consider
that GUS expression from such a translational fusion
construct will more accurately reflect AC1 expression
from the viral genome. This, in combination with a
different host (N. clevelandii mesophyll protoplasts were
used in the earlier work), could account for the observed
differences in expression. Although AC1 negatively
regulated its own expression, a small but significant level
of activity remained, contrasting with the situation for
TGMV in which ALl effectively silenced its own
expression (Sunter et al., 1993; Gr6ning et al., 1994).
Also, we found that AC4 had no effect on AC1
expression, contrasting with the significant suppression
of TGMV ALl expression by AL4 (Gr6ning et al., 1994).
The coat protein caused a significant (30-40 %) reduction
in complementary-sense gene expression. Although it is
known that the coat protein affects the relative levels of
ssDNA and dsDNA (Stanley & Townsend, 1986; Sunter
et al., 1990), its influence on gene expression has not been
reported previously. While it is possible that the coat
protein has a specific effect on complementary-sense
expression, it could cause a general reduction in the level
of gene expression simply as a result of its ability to bind
to dsDNA (Ingham et al., 1995), in this instance to the
GUS expression cassettes. As AC2 is a transcriptional
transactivator of coat protein expression (Sunter &
Bisaro, 1992), it is surprising that it had no indirect
adverse effect on complementary-sense expression from
the entire region (domains A-E). However, because the
relative level of ACMV virion-sense expression is low,
even in the presence of AC2 (unpublished data), any
influence of AC2 on complementary-sense expression
may remain undetected in our assay.
We have demonstrated that the N-terminal 57 amino
acids of AC1 have the same effect on complementary-
sense expression as intact AC1, suggesting that this part
of the protein is solely responsible for the regulatory
activity. In addition, expression levels were reduced to
the same extent regardless of the length of the fragment
used in the assay, implying that at least some cis-acting
sequences responsible for down-regulation are located
within domain A. However, since domain A is re-
sponsible for only 16 % of the total GUS expression, the
possibility that other domains participate in down-
regulation cannot be ruled out. Although ACMV AC1
binding has not yet been investigated, the simplest
interpretation of these data is that the N-terminal portion
of AC1 interacts with sequences within domain A to
suppress AC1 expression. The idea is consistent with
reports describing the specific binding of ALl of TGMV
and bean golden mosaic virus to sequences within the
A C M V complementary-sense gene expression 2421
common region equivalent to domain A (Fontes et al.,
1992, 1994a; Th6mmes et al., 1993). A more detailed
analysis of the TGMV ALl binding site and its
involvement in transcriptional repression has identified a
13 bp element as a high affinity binding site, located nine
nucleotides upstream of the ALl transcription start
point (Fontes et aI., 1994b; Eagle et al., 1994). Since the
analogous region in ACMV is also positioned between
the consensus TATA box and transcript start point (Fig.
1), AC1 binding during the initiation of ACMV DNA
replication could readily account for its adverse affect on
complementary-sense gene expression. The fact that the
binding site motif is present as a tandem repeat in
TGMV but apparently not in ACMV (Fontes et al.,
1994b) could contribute to the different levels of
AC1/ALl-mediated suppression observed for these
viruses. ACMV DNA is believed to replicate by a rolling
circle mechanism (Saunders et al., 1991), a process
initiated by ACl-mediated nicking within a highly
conserved motif (TAATATTAC) prior to virion-sense
strand displacement (Stanley, 1995). Sequence com-
parisons have suggested a relationship between gemini-
virus replication-associated proteins and prokaryotic
plasmid rolling circle DNA replication initiator proteins
(Gorbalenya et al., 1990; Koonin & Ilyina, 1992), and N-
terminal initiator and C-terminal helicase domains have
been proposed. Our data suggest that the N-terminus of
AC1, encompassing conserved motif 1 and part of motif
2 of the initiator domain, but lacking the tyrosine residue
(motif 3) that may covalently link to the nicked DNA
(Koonin & Ilyina, 1992; Stanley, 1995) and the putative
NTP binding domain (Gorbalenya et al., 1990), is
sufficient for disruption of complementary-sense gene
expression.
We thank Dr Phil Mullineaux for helpful comments on the use of
pJIT expressioncassettesand ProfessorT. Nagata for kindlyproviding
the tobacco BY-2cell line, Y.H. was supported by the BBSRCPlant
Molecular BiologyProgramme.
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(Received 20 March 1995; Accepted 26 June 1995)