Báo cáo khoa học: Multi-tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation potx

Multi-tasking of nonstructural gene products is required
for bean yellow dwarf geminivirus transcriptional
regulation
Kathleen L. Hefferon
1
, Yong-Sun Moon
2
and Ying Fan
3
1 Cornell University, Cornell Research Foundation, Ithaca, NY, USA
2 Yeungnam University, Department of Horticulture, Gyeongsan-si, Gyeongsangbuk-do, Korea
3 Cornell University, Cornell School of Veterinary Medicine, Ithaca, NY, USA
Geminiviruses belong to a family of plant viruses that
can be classified into four distinct genera on the basis
of genomic organization, vector transmissibility and
host range. These include the mastreviruses, which
possess monopartite genomes, are transmitted by leaf-
hoppers and infect monocotyledonous plants. Excep-
tions to the rule are the Australian-derived tobacco
yellow dwarf virus and South African-derived bean
yellow dwarf virus (BeYDV), two distantly related
mastreviruses that infect dicotyledenous plants [1].
BeYDV consists of a single-stranded circular DNA
molecule of 2.6 kb in length, and contains four ORFs
encoding three different genes. The coding region is
divided bidirectionally by long intergenic regions
(LIR) and short intergenic regions (SIR). The MP
and CP genes are expressed from the virion sense-
strand, while the replication-associated protein (Rep)
is produced from overlapping ORFs C1 and C2 from
the complementary sense-strand. An intron spans the

region overlapping C1 and C2 and this is spliced dur-
ing Rep expression. Both Rep, which functions as the
replication-associated protein, and RepA, the gene
product of ORF C1, are produced during virus infec-
tion [1–3].
Keywords
geminivirus; gene expression; promoter
control; transactivation
Correspondence
K. L. Hefferon, University of Toronto, Center
for Virology, 25 Willcocks St., Toronto, ON,
Canada M5J3B2
Fax: +1 607 254 1015
Tel: +1 607 257 1081
E-mail:
(Received 21 June 2006, accepted 7 August
2006)
doi:10.1111/j.1742-4658.2006.05454.x
Mastreviridae, of the family geminiviridae, possess a monopartite genome
and are transmitted by leafhoppers. Bean yellow dwarf dirus (BeYDV) is a
mastrevirus which originated from South Africa and infects dicoyledenous
plants, a feature unusual for mastreviridae. Previously, the nonstructural
proteins Rep and RepA were examined with respect to their independent
roles in BeYDV replication. This was achieved by placing both gene pro-
ducts under independent constitutive promoter control and examining their
effects on replication-competent constructs. In the current study, Rep and
RepA are examined further for their roles in regulating BeYDV gene
expression using a series of replication-incompetent constructs. While both
Rep and RepA are found to behave as equally potent inhibitors of comple-
mentary-sense gene expression, they differ considerably with respect to

their abilities to transactivate virion-sense gene expression. Furthermore,
RepA is identified as playing more than one role in this transactivation
process. A nuclear localization domain is identified in Rep which is absent
in RepA, and Rep–RepA interactions are examined under in vivo condi-
tions. The study concludes with an investigation into the expression strate-
gies of the BeYDV capsid protein.
Abbreviations
BeYDV, bean yellow dwarf virus; CLE, conserved late element; GFP, green fluorescent protein; HA, hemagglutinin; LIR, long intergenic
regions; MSV, maize streak virus; NLS, nuclear localization site; RBR, retinoblastoma-binding protein; Rep, replication-associated protein;
SIR, short intergenic regions; WDV, wheat dwarf virus.
4482 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
BeYDV, like other geminiviruses, replicates via a
rolling circle mechanism. First, the host cell replication
machinery synthesizes a complementary sense-strand
from a primer located within the SIR to form a dou-
ble-stranded intermediate. Next, Rep binds to the hair-
pin structure located within the LIR, nicks the virion
sense-strand and initiates DNA synthesis from the
5¢-terminus. As DNA synthesis progresses, the virion
sense-strand is displaced and eventually is recircular-
ized and religated by Rep [1,4–6].
The LIR contains sequences responsible for tran-
scription of genes in both genome senses, as well as an
inverted repeat sequence that forms the hair–loop
structure required for replication [7]. A conserved non-
anucleotide sequence, located within the loop of the
hairpin structure, contains the origin of replication.
Cis-acting elements, responsible for both complement-
ary and virion sense gene expression, are also located
within the LIR. An iteron, which contains the Rep-

binding site, is located between the TATAA sequence
and the transcriptional start site of the Rep gene. This
enables Rep to mediate repression of its own promoter
by interfering with initiation of transcription of the
Rep gene. RepA, on the other hand, has been shown
to function as a retinoblastoma-binding protein
(RBR). RepA is involved in controlling the cell cycle,
but is not required for virus replication [8]. RepA of
wheat dwarf virus (WDV), a related mastrevirus, has
been shown to bind to the LIR in addition to Rep,
and may play a role in regulating both complementary
and virion sense gene expression [5,9–11].
A number of studies have suggested that in Mastre-
viridae, the virion sense promoter is transactivated by
Rep gene products. Hofer et al. showed that no activ-
ity was detectable from the virion sense promoter of
WDV in the absence of Rep expression [12]. Further-
more, a replication-deficient mutant, which still pro-
duced Rep, was able to transactivate virion sense
gene expression. Similarly, Zhan et al. found that
Rep could enhance virion sense gene expression of
chloris striate mosaic virus [13]. Further studies, in
which constructs containing a frameshift mutation in
ORF C2 had lost their ability to activate virion sense
expression, suggested that Rep, not RepA (C1), is the
transactivator. Conversely, Collin et al. showed that a
cDNA form of Rep, which lacks the intron and thus
could not produce RepA, was unable to promote viri-
on sense gene expression from a replicating WDV
construct, whereas the full-length Rep gene, with the

intron intact, produced high levels, suggesting that
RepA (C1) alone is required for virion sense expres-
sion [14]. More recently, using maize, WDV RepA
was shown to activate virion-sense gene expression in
maize streak virus (MSV) and WDV, with the
RBR-binding domain of RepA being essential for
activation in MSV but nonessential in WDV [15].
Using RepA RBR-binding mutants, the authors of
this study suggested that the interference of RepA
with an RBR-dependent cellular pathway for gene
expression in one virus, but not in the other, indicates
that two alternative means of activating virion-sense
gene expression may exist.
In order to elucidate further the roles of Rep and
RepA in BeYDV replication and regulation of gene
expression, we separated Rep and RepA activities by
individually placing them under constitutive promoter
control. We cobombarded these Rep constructs, inde-
pendently of one another, along with replication-
incompetent BeYDV-based constructs containing the
viral elements required for both virion and comple-
mentary sense gene expression. We found that Rep A
(C1) acts as a potent transactivator of virion sense
gene expression and inhibitor of complementary sense
gene expression. Rep, on the other hand, while also an
inhibitor of complementary sense gene expression, had
a much weaker effect on virion sense gene expression.
Further studies, using RBR mutant RepA constructs,
indicated that in the BeYDV system, RepA transacti-
vation is still able to take place (albeit to a lesser

degree) in the absence of an intact RBR-binding
domain. We also demonstrated that Rep possesses a
nuclear localization site that is absent from RepA, and
that Rep and RepA are able to interact with each
other under in vivo conditions. Finally, regulation of
BeYDV CP expression was examined. The discovery
of an unconventional mechanism of translational initi-
ation is discussed.
Results
Comparison of Pc and Pv promoter strengths
in NT-1 cells
Previously, we had examined the effects of Rep and
RepA on BeYDV replication by placing various Rep
constructs individually under 35S promoter control.
These constructs were then cobombarded into NT-1
cells along with a replication- competent reporter con-
struct containing both BeYDV LIR and SIR sequences
[8]. In the current study, we wished to examine, in
greater detail, the roles of the Rep gene products in
regulating BeYDV gene expression. We designed a
replication-incompetent construct, pBYD–LIR, which
lacks the SIR required for replication but retains the
LIR from which the promoters Pc and Pv are derived
(Figs 1 and 2). The GUS gene was inserted into the
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4483
EcoRI site of pBYD–LIR and pBYD–LIRSIR, as
described in Hefferon & Dugdale [8]. NT-1 cells were
bombarded with pBYD–LIR or pBYD–LIRSIR and
either pBYSK1.4 or p35SRep (Fig. 1). A Southern blot

was performed to demonstrate that pBYD–LIR was
replication-incompetent (Fig. 2A). Replication prod-
ucts were detected from extracts of NT-1 cells cobom-
barded with the reporter construct pBYD–LIRSIR
and either pBYSK1.4 or p35SRep (expressing the Rep
gene products Rep and RepA) (Fig. 2A, lanes 1 and 2)
but not when the pBYD–LIR reporter construct was
used with these Rep-expressing constructs (Fig. 2A,
lanes 3 and 4). Similar results were achieved when lar-
ger constructs, containing BeYDV sequence beyond
the boundaries of the LIR, were used. In addition, fur-
ther truncation of the constructs containing the LIR
or SIR elements did not change the efficiency of the
Pv1 promoter (data not shown).
BeYDV promoter strengths were compared by gener-
ating various constructs containing the BeYDV LIR in
which the virion sense or complementary sense genes
were replaced with the GUS ORF. pPcGUS was con-
structed by creating an NcoI site at the initiation codon
of the ORF C1 (RepA) and by substituting a GUS
ORF, as well as a termination signal, in place of the
C1:C2 ORF. Similarly, pPvGUS was constructed by
creating an NcoI site at the initiation codon for ORF
V1 (MP) and inserting the GUS ORF and termination
signal in place of ORFs V1 and V2 (CP) (Fig. 2B).
These reporter constructs were bombarded into NT-1
cells and the relative GUS activities were determined
(Fig. 2C). In these assays, luciferase expression from
pLUC was included as an internal control to normalize
DNA delivery for GUS expression [16,17]. GUS under

35S promoter control (p35SGUS) was included as a
positive control, and GUS in the absence of a promoter
Fig. 1. Schematic diagram of the constructs used in this work. (A) Genomic organization of pSKBYD1.4. P, PstI; Xb, XbaI; S, SacI; B, BamHI;
E, EcoRI; C, ClaI; Bg, BglII; C1, C2, V1 and V2 represent complementary and virion sense ORFs, respectively. The bar represents 500 bp. The
intron is represented by an open box. Promoters are indicated by arrows. (B) BeYDV-derived plasmids containing various forms of Rep ORFs.
Rep constructs under 35S promoter control were constructed by PCR amplification of the Rep ORFs. Portions of the Rep gene removed for
35SDintron and 35SDBRep are indicated by a ‘v’. The boxed arrow refers to the cauliflower mosaic virus (CaMV) 35S promoter. The small
rectangle represents TEV leader sequences at the 5¢-end of the constitutively expressed Rep constructs. The VSP termination sequence
(Tvsp) is depicted by an open rectangle at the 3¢ end of the constitutively expressed Rep constructs. (C) BeYDV-derived reporter cassettes
were constructed. The solid line represents the portion corresponding to the BeYDV genome used to construct reporter cassettes.
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4484 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
(pGUS) was included as a negative control. GUS activ-
ities were determined at 6, 12, 24, 36, 48 and 72 h post-
bombardment (Fig. 2C). The highest level of GUS
activity was determined for p35SGUS; less than half of
this level of activity was achieved from the construct,
pPcGUS. While cells bombarded with p35SGUS did
not reach their maximum level of GUS activity until
48 h postbombardment, NT-1 cells bombarded with
the pPcGUS construct reached maximal levels of GUS
activity within 12 h of expression, suggesting that this
promoter is active early on in the infection cycle. GUS
activities generated by the pPvGUS construct were only
slightly higher than activities observed for the control
construct pGUS in the absence of a promoter. The
results of this study indicate that while BeYDV comple-
mentary sense genes appear to be active in NT-1 cells
in the absence of any additional virus-derived or
virus-activated cellular factors, virion sense gene

expression is minimal under these conditions. The relat-
ive promoter strengths did not change significantly over
a time course of 72 h, suggesting that any temporal
changes in relative promoter activity may require
the presence of additional factors. GUS activity from
NT-1 cells bombarded with pGUS was negligible at all
time points.
Effect of Rep gene products on BeYDV gene
expression
To determine the respective roles of various BeYDV
gene products in the regulation of complementary sense
gene expression, we cobombarded Rep gene products
independently, and in a number of combinations, with
these replication-incompetent reporter constructs. The
construct pPcGUS was cobombarded into NT-1 cells
along with various constructs expressing BeYDV gene
products, and complementary gene expression was
quantified by assay for GUS activity (Fig. 3A). Con-
struct p35SDBRep, containing a large deletion within
the Rep ORF, was included in this study as a negative
control (Fig. 3, lane 8) [8]. Cobombardment of either
p35SDintron or p35SRepA with the expression cassette,
Fig. 2. Comparison of Pv and Pc promoter strengths in NT-1 cells. (A) Southern blot depicting replication products observed when reporter
plasmid pBYDLIR–SIR (lanes 1 and 2) and pBYD–LIR (lanes 3 and 4) are cobombarded along with pSKBYD1.4 (lanes 1 and 3) or p35SRep (lanes
2 and 4).
32
P-labelled cDNA, corresponding to the GUS ORF, was used as a probe. Double- and single-stranded DNA replication products are
labelled on the left hand side. Molecular weight markers are labelled on the right. (B) Schematic diagram of pPcGUS and pPVGUS replication-
incompetent constructs. Details are provided in Experimental procedures. LIR refers to the long intergenic region within the genome of BeY-
DV. V1 and C1 refer to the virion-sense and complementary-sense ORFs adjacent to the LIR, respectively. NcoI refers to the restriction site,

inserted, via site-directed mutagenesis, at the ATG initiation codons for C1 and V1, repectively. T35S refers to the 35S terminator. Arrows refer
to the direction of transcription for both constructs. (C) Relative GUS activity (lgÆmg
)1
Æmin
)1
) over a time course for the following constructs
bombarded into NT-1 cells; p35SGUS, pPcGUS, pPvGUS and pGUS. Luciferase was used as an internal control in this assay and all experi-
ments were repeated in triplicate. p35SGUS activity was standardized to a value of 1 and relative GUS activities were determined.
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4485
pPcGUS, revealed that reporter gene expression was
significantly inhibited by either gene product. Inhibition
remained consistant, regardless of whether RepA, Rep
or both gene products were simultaneously present
(Fig. 3A, compare lanes 1–4 with lane 8). No difference
in the inhibition of Pc was observed when p35SRep or
p35SRepA were substituted with their respective RBR
mutants (Fig. 3A, compare lanes 5 and 6 with lane 8).
Cobombardment of the expression cassette, pPcGUS,
with the p35SCP construct had no effect on comple-
mentary sense gene expression (Fig. 3A, compare lane
7 with lane 8). Cobombardment of cells containing the
reporter construct, pPvGUS, with p35SRepA revealed
that RepA was capable of strongly transactivating the
virion sense promoter, whereas cobombardment of
pPvGUS with p35SDintron had little effect on transac-
tivation (Fig. 3B, compare lanes 1 and 2 with lane 8).
Cobombardment of p35SRep (from which both Rep
and RepA gene products are produced) and pPvGUS
into NT-1 cells also resulted in a great amount of trans-

activation (Fig. 3, lane 3). However, simultaneous co-
bombardment of p35SDintron and p35SRepA, along
with pPvGUS, did not enhance GUS activity further
(Fig. 3B, lane 4).
Transactivation of virion sense gene expression was
also examined when p35SRep and p35SRepA were
replaced with their RBR mutant counterparts. Replace-
ment of p35SDintron with p35SDintron
RBR–
resulted in
no significant change in GUS activity (Fig. 3B, com-
pare lane 1 with lane 5). However, a significant
decrease in Pv activation was observed when
RepA
RBR–
was substituted for RepA (Fig. 3B, compare
lane 2 with lane 6).
The effect of p35SCP on virion-sense gene expres-
sion was also examined in this study. No increase in
GUS activity was observed when constructs expressing
either the CP from BeYDV (p35SCP) or the CP from
a nonrelated plant virus (p35SPVXCP) were included
(Fig. 3B, compare lane 7 with lane 8, data not shown).
Transcript stability and expression levels support the
GUS assay results (data not shown).
Subcellular localization of Rep gene products
Examination of Rep and RepA nucleotide sequences
revealed that Rep, but not RepA, possesses a putative
nuclear localization site (NLS) within the C-terminal
half of the molecule (Fig. 4A). To determine whether

this site is functional, constructs p35SDintron–green
fluorescent protein (GFP) and p35SRepA–GFP were
designed, creating Rep–GFP fusion products. To
ensure that these fusion products were still biologically
active, p35SDintron–GFP was shown (by Southern blot
analysis) to promote BeYDV replication, and RepA–
GFP was shown (by assay for GUS activity) to transac-
tivate virion sense gene expression, (data not shown).
Tobacco protoplasts were electroporated with these
constructs and visualized under UV light (Fig. 4B–E).
The results of this study indicated that the Rep–GFP
fusion product localized exclusively to the nucleus
(Fig. 4B,C), whereas the RepA–GFP fusion product
was found to be distributed equally throughout both
the nucleus and the cytosol (Fig. 4D,E).
Interaction of BeYDV Rep and RepA in vivo
Horvath et al. and Missich et al. have published con-
flicting data regarding the interactions between Rep
and RepA of WDV and MDV, using the two-hybrid
yeast system [18,19]. To examine, in greater detail, the
hetero-oligomerization properties of BeYDV Rep and
Fig. 3. Effect of Rep gene products on BeYDV gene expression.
Relative GUS activities are shown for constructs (A) pPvGUS and
(B) pPcGUS cobombarded into NT-1 cells along with the following
BeYDV-encoded gene products: lane 1, p35SDintron; lane 2,
p35SRepA; lane 3, p35Srep; lane 4, p35Dintron + p35SRepA; lane
5, p35SDintron
RBR–
; lane 6, p35DRepA
RBR–

; lane 7, p35SCP; lane 8,
p35SDBRep. Samples were collected 24 h after cobombardment.
Luciferase was used as an internal control in this assay. The experi-
ments were repeated in triplicate.
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4486 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
RepA under in vivo conditions, NT-1 cells were
cobombarded with both p35SHA6HISRep and
p35SHARepA. p35SHA6HISRep was collected on a
Ni
2+
column and removed by washing the column,
then collecting the eluate into 100 lL fractions. Frac-
tions were subjected to electrophoresis on a gradient
gel, and western blot analysis was performed using
antisera to hemagglutinin (HA) (Fig. 5). Rep and
RepA were easily detected from cells bombarded with
p35SHA6HISRep or p35SHARepA alone (Fig. 5,
lanes 1 and 2). While RepA was detected from the
first of several washed fractions derived from samples
of NT-1 cells bombarded with both constructs (Fig. 5,
lanes 3–5), both Rep and RepA were found in the
final eluate, indicating that these gene products can
interact with each other in vivo (Fig. 5, lane 6). Detec-
tion of Rep and RepA in the final eluate was con-
firmed by immunoprecipitation, indicating that the
presence of RepA in this fraction was not the result of
an artifact (Fig. 5, lanes 7 and 8).
Regulation of expression of BeYDV CP
While the MP of BeYDV appears to be expressed

from the V1 promoter, the manner by which the coat
protein (V2) is expressed is less clear. As CP expression
is known to bring about an increase in single-stranded
DNA replication products, replication experiments,
using constructs containing the virion sense half of the
BeYDV genome, were performed to examine the effect
of CP expression on the replication product profile by
Southern blot analysis (Fig. 6A,B) [8]. NT-1 cells were
cobombarded with the replication-competent expres-
sion cassette, pBYDLIR–SIR [8], p35SRep and one of
several constructs that contain functional MP or CP
genes. When cells were bombarded with a construct
that contains both functionally active MP and CP
genes (pBYV1V2), a single-stranded replication prod-
uct was observed (Fig. 6B, lane 1). Previous studies
have indicated that accumulation of single-stranded
DNA is probably a consequence of CP accumulation
[8] and, because a similar pattern of replication prod-
ucts was observed when the CP was placed under 35S
promoter control, the results presented in this study
are suggestive of CP expression [8]. When a deletion
was placed within the MP gene to prevent a functional
protein (pBYXV2) from being expressed, and this con-
struct was cobombarded along with the replication cas-
sette, a single-stranded gene product was still observed,
again suggesting that CP accumulation has taken place
(Fig. 6B, lane 2). Destruction of the CP gene in con-
struct pBYV1X, or elimination of it entirely from this
Fig. 5. Interaction of BeYDV Rep and RepA under in vivo condi-
tions. p35SHA6HISRep and p35SHARepA were cobombarded into

NT-1 cells. Extracts prepared from these cells were then loaded
onto a Ni+ column and p35SHA6HISRep was purified according to
the protocol of Hefferon & Fan [46]. Lane 1, extracts from cells
bombarded with p35SHA6HISRep alone; lane 2, extracts from
cells bombarded with p35SHARepA alone; lane 3, extracts from
cells bombarded with both p35SHA6HISRep and p35SHARepA
after the first wash; lane 4, after the second wash; lane 5, after the
third wash; and lane 6, after the elution buffer. Location of Rep and
RepA are indicated by arrows. Lanes 7 and 8, Immunoprecipitation
of Rep products. Lane 7, extracts of cells after elution buffer; lane
8, nonbombarded cells.
E
B
C
D
PPLKKKKLKDD
A
p35S
intron/GFP
p35SRepA/GFP
35S
35S
T
T
G
FP
GFP
Rep
A
Rep

Fig. 4. Subcellular localization of Rep gene products. (A) Schematic
diagram of constructs p35SRep–GFP and p35SRepA–GFP. Location
of the putative nuclear localization site is indicated above the Rep–
GFP fusion construct. (B–E) Visualization of protoplasts electropo-
rated with BeYDV constructs under either UV (B, D) or visible
(C, E) light. (B, C) Protoplasts electroporated with p35SRep–GFP;
(D, E) protoplasts electroporated with p35SRepA–GFP.
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4487
replication assay, resulted in double-stranded DNA as
the predominant replication product (Fig. 6B, lanes 3
and 4). These experiments suggest indirectly that the
BeYDV CP may be expressed independently from the
downstream cistron of a dicistronic transcript in
the absence of a translatable MP (V1). Examination of
the sequence surrounding the termination codon of V1
and the initiation codon of V2 revealed a short gap of
13 nucleotides. No obvious promoter signature is
apparent within or surrounding this region.
Comparison of sequences of similar regions for rela-
ted geminiviruses indicates that a similar gap of 10
nucleotides also exists for MSV. WDV and tobacco
yellow dwarf virus, on the other hand, possess overlap-
ping V1 and V2 ORFs, suggesting that an alternative
mechanism of translational initiation may exist among
these geminiviruses. Furthermore, the V1 AUG codon
of BeYDV is in a suboptimal context (ttgAUGg), sug-
gesting that leaky scanning may be the favoured
method of translation of the CP. To determine, in
greater detail, whether V2 is expressed from a smaller

monocistronic transcript, or as the downstream cistron
of a larger polycistronic transcription unit, a northern
blot was performed on NT-1 cells bombarded with
pBYSK1.4, using a
32
P-labelled cDNA probe corres-
ponding to the BeYDV CP gene. A 1.4 kb RNA tran-
script, corresponding to the size of a full-length
polycistronic transcription unit, was observed, imply-
ing that CP translation takes place from the down-
stream cistron of a single, dicistronic transcript
(Fig. 6D, lane 2). No transcripts were observed in non-
bombarded NT-1 cells (Fig. 6D, lane 1).
Discussion
Previous experiments, using replication-competent
BeYDV-based constructs, demonstrated that the maxi-
mum rate of reporter gene expression is not achieved
when active replicons are used [8,20]. In this study, to
examine the regulation of gene expression in detail,
transcription was uncoupled from replication by the
creation of replication-incompetent reporter constructs,
and the relative strengths of BeYDV C1 and V1 pro-
moters were examined. A reporter construct (pBYD–
LIR) containing the LIR, but lacking the SIR, of
BeYDV was shown (by Southern blot analysis) to be
unable to support replication (Fig. 2A). The present
work serves to elucidate further the roles of Rep gene
products in BeYDV infection and in transcriptional
regulation in general. In the system described in this
article, weak expression from the virion sense promoter

was attributed to an absence of virus-derived or virus-
activated cellular factors from the system. Previous
experiments performed with WDV and MSV demon-
strated that virion sense promoter activity is greater
in phloem cells, suggesting that phloem-specific
Fig. 6. Regulation of expression of BeYDV
CP. (A) Design of constructs. ‘X’ marks the
site where each ORF was disrupted. (B)
Southern blot illustrating the profile of repli-
cation products collected from NT-1 cells
cobombarded with the following
BeYDV-based constructs. Lane 1,
pBYDLIR-SIR, p35SRep and pBYV1V2; lane
2, pBYDLIR-SIR, p35SRep and pBYXV2;
lane 3, pBYDLIR-SIR, p35SRep and pBYV1X;
lane 4, pBYDLIR-SIR and p35SRep. Double-
stranded (ds) and single-stranded (ss) DNA
species are indicated on the left hand side.
(C) Nucleotide sequence surrounding V1 and
V2 of several mastreviruses. Start and stop
codons for ORFs V1 and V2 are indicated by
arrows and bold text. (D) Northern blot of
total RNA from NT-1 cells cobombarded
with pSKBYD1.4 using a
32
P-cDNA probe
corresponding to the CP ORF of BeYDV.
The RNA ladder is labelled on the left hand
side. The single RNA species is indicated by
an arrow. Lane 1, nonbombarded NT-1 cells;

lane 2, cells bombarded with pSKBYD1.4.
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4488 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
transcription factors play a role in activating virion
sense expression in the infection cycle [21–23]. In
addition to this, a cell cycle specificity has been identi-
fied for both virion sense and complementary sense
promoters of MSV [15]. The differential activity of pro-
moters in developmental or tissue-specific cells suggests
that cellular proteins may modify Rep to modulate
both replication and repression activites [23,24]. The
use of suspension cells in the current study would
explain the low activity of the virion sense promoter
reported here.
Addition of Rep to the BeYDV reporter system
inhibited expression from the complementary sense
promoter. The ability of Rep to down-regulate expres-
sion of its own promoter has been studied previously.
The AL1 protein of tomato golden mosaic virus, for
example, has been shown to play a dual role in tran-
scription and replication and it can inhibit its own
expression by 20-fold [4,22]. It is likely that either the
modification of Rep, or the interaction of Rep with
other viral or cellular proteins, may be involved in
regulating the role of Rep as either a participant in
viral replication or as a repressor of complementary
sense gene expression.
RepA of BeYDV is not required for BeYDV replica-
tion [8,25]; however, the data presented here indicate
that it plays an essential role in transactivating the viri-

on sense promoter. Transactivation may take place by
two mechanisms. The first involves direct binding of
RepA to DNA. Similar modes of transactivation have
been demonstrated in other virus systems [19,26]. For
example, the E1A protein can bind to and transacti-
vate the adenovirus major late promoter, and VP16
can stimulate herpes simplex virus-1 early promoters
[25]. RepA binding may also be mediated through
interactions between RepA and other transcription fac-
tors in a manner analogous to those demonstrated for
adenovirus E1A and herpesvirus VP16 [26–28]. The
second mechanism by which transactivation takes
place may involve the binding of RepA to the RBR
[26,29]. Activation of late gene expression by RBR
binding has also been demonstrated for other DNA
virus systems, such as the E1A protein of adenovirus,
the large T-antigen of Simian virus-40 and the E7 pro-
tein of papillomavirus [26]. In each instance, the viral
transactivator protein possesses an LXCXE motif that
can interact within a subdomain of RBR. This site of
interaction overlaps with the E2F-binding site present
on the RBR protein and forces the release of the tran-
scription factor. E2F. E2F can then bind to, and initi-
ate, transcription from a wide variety of cellular and
viral promoters and control transition from G to S
phase of the cell cycle, therefore promoting cell cycle
progression to one that is more environmentally per-
missive for viral replication [25,30–35].
RepA, which is considered to be a functional ana-
logue of animal virus oncoproteins, also contains an

LXCXE motif [25]. A search revealed two potential
E2F-binding sites within the LIR of BeYDV, each
located on either side of the hairpin structure. The first
site, GTTCCCGC, is located on the virion sense
strand (nucleotides 63–68) and the second, TTG
GCCGC, is located on the complementary sense-
strand (nucleotides 2440–2447). Both have a one-
nucleotide mismatch from the consensus sequence
TTTG ⁄ CG ⁄ CCGC. Two similar binding sites have
been identified within the LIR of WDV, and one of
these sites has been shown to interact with human E2F
[15]. The same authors further showed that when this
sequence was fused as a trimer to a minimal 35S pro-
moter controlling GUS, an enhancement of GUS
activity was observed in the presence of RepA, but not
in the presence of a RepA RBR-binding deficient
mutant, indicating that this viral sequence motif is a
binding site for E2F and is activated by RepA. It was
therefore postulated that RepA can stimulate virion
sense gene expression by interfering with a cellular
pathway involving both cellular RBR and E2F. The
results of work presented in the present article suggest
that a similar pathway of gene regulation may occur
for BeYDV. However, the fact that transactivation of
gene expression could still be observed, although at a
lower level, when wild-type RepA was substituted with
an RBR-binding mutant, suggests that the RBR-bind-
ing pathway is not the exclusive means by which trans-
activation occurs. It is more likely that direct RepA
binding also plays a role in BeYDV virion-sense gene

expression.
Besides the E2F-binding sites, two additional con-
served late elements (CLEs), each deviating from the
consensus GTGGTCCC in one position, were also
found to lie 123 and 88 nucleotides upstream of the
V1 initiation codon within the BeYDV LIR, respect-
ively. CLEs, which had originally been identified as
evolutionally conserved DNA sequences present in
several different Geminivirus and Nanovirus species,
have been shown to have intrinsic enhancer activity in
the absence of viral gene products. In begomoviruses,
the CLE has been implicated in AC2-mediated trans-
activation of the rightward promoter [36,37]. It is
possible that the CLEs identified in the current study
may contribute, in some way, to transactivation of
virion sense gene expression.
Our studies indicate that while RepA activates virion
sense gene expression, Rep has little effect [37–40]. As
both Rep and RepA contain the same LXCXE motif
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4489
for RBR binding, the question of how each performs
such different functions in transcriptional activation
arises. Secondary structural predictions of WDV Rep
and RepA have been made to analyze in detail the
region around the LXCXE motif of both gene prod-
ucts. Different hydrophobicity patterns, and a differen-
tial distribution of L-helices and M-strands between
the two proteins, suggest a difference in predicted sec-
ondary structures within the same area of the two pro-

teins [1,11,19]. The fact that Rep does not interact
with RBR suggests that the C-terminus of Rep hinders
its ability to bind its LXCXE motif to the appropriate
site in RBR. These steric differences between Rep and
RepA may also explain how each have overlapping,
but different, binding sites within the LIR of WDV
[19,22]. While differential binding may also play a role
in the inability of Rep to transactivate virion sense
gene expression, the altered binding site of RepA may
still have the same effects as Rep binding to inhibit
complementary sense gene expression. Therefore, in
the BeYDV system, inhibition of the complementary
sense promoter by either Rep or RepA may differ ster-
ically, but the overall effects are similar.
The greatest transactivation levels for virion sense
gene expression were found when construct p35SRep,
which expresses both Rep and RepA gene products,
was used in this study. However, placing Rep and
RepA each independently under 35S promoter control
resulted in a decrease of gene expression. These results
are in agreement with earlier experiments using a repli-
cation-competent construct [8]. It is possible that alter-
ations in the ratios of Rep ⁄ RepA affects the ability of
RepA to bind to the LIR, once again suggesting that
binding of RepA to the LIR is, at least partially,
responsible for transactivation of virion sense gene
expression.
To understand, in greater detail, the roles of Rep
and RepA in regulating BeYDV gene expression, we
explored the subcellular localization properties of

these gene products by constructing Rep and RepA–
GFP fusion proteins and electroporating them into
tobacco protoplasts. The exclusivity of Rep in the
nucleus, and diffuse pattern of RepA throughout
both the nucleus and cytoplasm, support the hypothe-
sis that the NLS identified within the Rep ORF is
indeed functional. Using AC1 of the begomovirus
African cassava mosaic virus in a PVX expression
vector, Hong et al. found that mutant AC1–GFP
fusion proteins, with an altered nuclear localization
site, were also not particularly restricted to the nuclei
of cells, but occurred in equal proportions throughout
the cytoplasm in a pattern resembling the results des-
cribed in the present article [41,42]. It is possible that
RepA is small enough to passively enter the nucleus
in the absence of an NLS.
It has been suggested previously that Rep may inter-
act to form hetero-oligomers with RepA to assist in its
entry into the nucleus [11]. Indeed, Rep–RepA interac-
tions have been observed, with varying degrees of suc-
cess, by using the two-hybrid yeast system [18,19,43].
As an alternative to the two-hybrid yeast system, we
further examined the ability of these two proteins to
interact by copurification of RepA with 6His-tagged
Rep from plant extracts from a Ni
2+
column. The
strength of the interactions found in this study add
another layer of complexity to the roles of Rep and
RepA in transcription and replication.

The expression of BeYDV CP from the downstream
cistron of a single transcript is not unique [44]. A num-
ber of plant viruses use unconventional translational
initiation mechanisms to express proteins [45]. These
mechanisms include leaky scanning, ribosomal frame-
shifting, ribosomal shunting, transactivation and cap-
independent ribosome binding at internal ribosome
entry sites. Future research should shed some light
regarding the underlying molecular mechanisms behind
CP expression of BeYDV. From a biotechnology per-
spective, such knowledge may serve as a powerful tool
to enhance or direct the translation of foreign proteins
in plants to more desirable levels within BeYDV-based
expression vector systems [46,47]. Such a system is cur-
rently being used to produce foreign proteins from a
plant virus expression vector [48].
The results described in the present article assist in
completing a general picture of the multiple roles of
Rep and RepA during the BeYDV life cycle. We have
demonstrated that Rep and RepA perform different
functions with respect to regulating BeYDV bidirec-
tional promoter activity. In the early stages of BeYDV
infection, both gene products are expressed from pro-
moter Pc, apparently in the absence of other virus gene
products. While high levels of Rep and RepA result in
a shut-off of promoter Pc, RepA alone is responsible
for transactivating late genes V1 and V2 as a single
dicistronic transcription unit from promoter Pv. This
transactivation takes place at least partially via a dis-
tinct RBR-binding pathway. The identification of an

NLS that resides within Rep, but not RepA, further
defines the different roles of these two gene products.
Furthermore, their ability to form hetero-oligomers
with one another illustrates the intimate associations
which exist between Rep and RepA during BeYDV
infection.
We have suggested, in the current study, that CP
expression may take place by a mechanism alternative
to conventional scanning. It is thought that the
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4490 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
Mastrevirus CP may sequester single-stranded DNA
molecules for assembly and encapsidation into nascent
virus particles, and therefore dictates the ratio of sin-
gle-stranded DNA to double-stranded DNA produced
during replication [49]. Therefore, transactivation of
the virion-sense genes V1 and V2 by RepA during the
later stages of BeYDV infection ultimately results in
the formation of a pool of virus particles that are
ready to be transported to neighbouring cells. The
work presented here, in combination with the results
of other studies, will assist in the future design and
improvement of Geminivirus vectors for expression of
foreign proteins in plants and will provide valuable
information regarding the biology of this virus.
Experimental procedures
Cells and viruses
NT-1 tobacco cell suspensions were maintained in NT-1
liquid medium as shaker cultures, as described previously
[8,50]. The NT-1 suspensions were prepared for biolistic

DNA delivery by pipetting a 10-day-old culture onto NT-1
agar plates and preincubating the cells for 3–4 days prior to
bombardment. pBYD1.4mer and pDintron were generously
provided by J. Stanley (John Innes Centre, Norwich, UK).
For bombardments, one micron gold particles (Bio-Rad,
Hercules, CA) were used at 800 psi ( 5.52 Mpa) with the
Bio-Rad Model PDS-10000 ⁄ He Bioloistic Particle Delivery
System, to deliver 2 lg of plasmid DNA prepared according
to the Qiagen maxiprep kit protocol (Qiagen, Valencia, CA).
Construction of plasmids
A schematic diagram of the constructs made is shown in
Fig. 1. pSKBYD1.4 contains 1.4 copies of the BeYDV gen-
ome cloned into pSK and was generously provided by
J. Stanley (John Innes Center) [2]. Construction of p35SRep,
p35SDintron, p35SDBRep, p35SRepA and p35SCP are des-
cribed by Hefferon & Dugdale [8]. pBYD–LIRSIR was pre-
pared by removing the XbaI–SacI fragment of pSKBYD1.4
(encompassing the Rep gene, LIR and SIR) and subcloning
the fragment into pBluescriptSKII+. The construct was ren-
dered replication deficient by BamHI digestion to release a
727 bp Bam HI fragment within the Rep gene, followed by
religation, as in the construction of p35SDBRep [8]. pBYD–
LIR was constructed by digestion of pSKBYD1.4 with XbaI
and BamHI and subcloning the released fragment, contain-
ing the LIR only, into pBluescriptSKII+. The p35SGUS
reporter cassette was inserted into the EcoRI site, as des-
cribed by Hefferon & Dugdale [8].
pPcGUS and pPvGUS were constructed by introducing
an NcoI site at the ATG initiation codon, corresponding to
C1 or V1 ORFs of pBYD–LIR, by site-directed mutagen-

esis (BRL, Nimes – Cedex 5, France) using primers NcoC1
(CAACACCATGGCTTCTGC) or NcoV1 (GGTATTC
CATGGAGCG). An NcoI–HindIII fragment, isolated from
the plasmid pGUS2 and containing the GUS gene and 35S
terminator, was then inserted into these constructs to gener-
ate pPcGUS and pPvGUS reporter constructs, respectively
[48]. To create the Rep and RepA–GFP fusion constructs,
fragments containing Rep and RepA ORFs were PCR
amplified using primers 5¢-NcoRep (GGGCCCCCATGG
CTTCTGC) and 3¢-SacRepA (GCAGGTATATGAGCT
CCCCGGG), and subcloned into pXbaGFP [49]. pLUC,
the luciferase vector, was kindly provided by T. Delaney
(Cornell University, Ithaca, NY).
Construction of plasmids p35SHA6HISRep and
p35SHARepA are described in Hefferon & Dugdale [8] and
Hefferon et al. [51], respectively. pBYV1V2 was generated
by a BamHI digest of pBYD1.4mer to release a 2.5 kb frag-
ment containing the virion-sense genes of the BeYDV
genome and subcloned into the plasmid vector pBluescript-
SKII+. pBYXV2 was generated by PstI digestion, blunt-
ended with mung bean exonuclease (New England Biolabs,
Ipswich, MA) to disrupt the V1 ORF and religated with T4
ligase (New England Biolabs). pBYV1X was generated by
SalI digestion, blunt-ended with mung bean exonuclease
(New England Biolabs) and religated with T4 ligase (New
England Biolabs) [52].
Southern blot analysis
Two micrograms of each plasmid DNA was cobombarded
into a thick slurry of NT-1 cells that had been slowly pipetted
onto Petri dishes containing NT-1 cells media plus 8 g of

agar-1 (Sigma, St Louis, MO). Plates were then incubated for
up to 8 days at 28 °C, depending on the experiment per-
formed, and DNA was extracted from cells using the proce-
dure described by Wilke [53]. Ten micrograms of total DNA
of each sample was digested with HindIII (for replication
competency studies) or BamHI (for CP studies) and loaded
onto a 1% agarose gel. DNA was transferred onto nitrocellu-
lose by capillary action [19]. A 0.5 kb fragment containing
the LIR of BeYDV was labelled with
32
P by random priming,
according to the conditions recommended by the manufac-
turer (Life Technologies, Invitrogen, Carlsbad, CA) and used
as a probe for hybridization in 25 nm Tris ⁄ HCl, pH 7.2,
1mm EDTA and 5% SDS at 65 °C, and the signal was
detected and quantified by the STORM Optical Scanner sys-
tem (Molecular Dynamics, Sunnyvale, CA).
GUS assays
NT-1 cells, cobombarded with pBYGUS constructs, were
analyzed for GUS activity using the protocol of Jefferson
[54]. Briefly, 1 g of NT-1 cells was crushed using a micro-
pestle, resuspended in GUS extraction buffer (50 nm
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4491
NaPO
4
, pH 7.0, 0.1% SDS, 10 mm Na
2
EDTA and 10 mm
2-mercaptoethanol) and total protein concentrations were

determined by the Bradford assay (Bio-Rad) using BSA as
a standard. GUS activities were determined by fluorometric
assay using 1 mm 4-methylumbelliferyl B-d-glucuronide as
substrate and 4-methylumbelliferone as standard, and
assays were monitored by fluorimetry (DYNEX technol-
ogies Fluorolite 1000, Chantilly, VA). Data have been pre-
sented here as lmolÆmin
)1
(per mg of total protein).
Luciferase from plasmid pLUC was included as an internal
control in this assay, and statistical analysis was performed
according to Breyne et al. [55]. All GUS experiments were
repeated in triplicate.
Northern blot analysis
Plant total RNA was isolated by the method of Chomczyn-
ski & Sacchi, and 10 lg of purified total RNA per lane was
loaded onto a 1% agarose denaturing gel [56]. Electropho-
retic separation of RNA, membrane transfer and detection
were performed as described for Southern blots. A
32
P-spe-
cific probe was generated by random priming a cDNA frag-
ment corresponding to the CP ORF of BeYDV. Primers
used are described in Hefferon & Dugdale [8].
GFP experiments
Tobacco protoplasts were prepared as described by Hefferon
& Fan [47]. Protoplasts were electroporated with cons-
tructs p35SGFP, p35SRep–GFP, p35SRepA–GFP and
pSKBYD1.4, and visualized under a Bio-Rad MRC 600 con-
focal microscope adapted to a Nikon Optiphat microscope,

with a · 40 Fluor. N.A. 1.30 oil immersion objective lens
(both from Mel Sobel Microscopes, Hicksville, NJ).
Rep interaction experiments
6xHis-tagged Rep was purified from tobacco NT-1 cells
using the Geminivirus purification system described by Hef-
feron et al. [45]. For immunoblot analysis,  50 lg of pro-
tein was extracted from NT-1 cells and ground in sample
loading buffer (100 mm Tris ⁄ HCl, pH 6.8, 1% SDS, 20%
glycerol). HA6HIS-tagged Rep was purified on Ni-NTP
agarose resin from NT-1 cells, as described in the
QiaExpressionist protocol (Qiagen). Washes and extractions
were performed according to the same protocol. Each sam-
ple was boiled for 10 min and loaded onto a gradient gel.
Protein was electrotransferred onto nitrocellulose mem-
branes. Blots were incubated overnight with antibody to
HA (at a concentration of 0.1 lg Æ mL
)1
) and proteins were
visualized using an enhanced chemiluminescence kit (Amer-
sham Biosciences, Piscataway, NJ). Immunoprecipitation
was performed according to Sojikul et al., using anti-HA
specific sera [57].
Acknowledgements
The authors would like to thank Dr Mounir AbouHai-
dar, who provided the p35S PVX CP construct. This
work was funded by grant #N65236-98-1-5411 from
the Defence Advanced Research Projects Agency.
References
1 Gutierrez C, Ramirez-Parra E, Castellano MM, Sanz-
Burgos AP, Luque A & Missich R (2004) Geminivirus

DNA replication and cell cycle interactions. Vet Micro-
biol 98, 111–119.
2 Liu LTT, Pietersen G, Davies JW & Stanley J (1997)
Molecular characterization of a subgroup I geminivirus
from a legume in South Africa. J Gen Virol 78, 2113–
2117.
3 Palmer KE, Rybicki EP, Maramorosch K, Murphy FA
& Shatkin AJ (1998) The molecular biology of mastre-
viruses. Adv Virus Res 50, 183–234.
4 Settlage SB, Miller AB & Hanley BL (1996) Interactions
between geminivirus replication proteins. J Virol 70,
6790–6795.
5 Eagle PA, Orozco BM & Hanley-Bowdoin L (1994) A
DNA sequence required for geminivirus replication also
mediates transcriptional regulation. Plant Cell 6, 1157–
1170.
6 Moon Y-S & Hefferon KL (2006) Geminivirus replica-
tion. In Recent advances in DNA virus replication, pp.
321–334. Transworld Sciences International, Research
Signpost, Kerala.
7 Heyraud NF, Schumacher S, Laufs J, Schaefer S, Schell
J & Gronenborn B (1995) Determination of the origin,
cleavage and joining domain of geminivirus Rep
proteins. Nucleic Acids Res 23, 910–916.
8 Hefferon KL & Dugdale B (2003) Independent expres-
sion of Rep and RepA and their roles in regulating bean
yellow dwarf virus replication. J Gen Virol 84, 3465–
3472.
9 Sunter G, Hartitz MD & Bisaro DM (1993) Tomato
golden mosaic virus leftward gene expression: autoregu-

lation of geminivirus replication protein. Virology 195,
275–280.
10 Sanz-Burgos A & Gutierrez C (1998) Organization
of the cis-acting element required for wheat dwarf
geminivirus DNA replication and visualization of a Rep
protein-DNA complex. Virology 243, 119–129.
11 Gutierrez C (2000) Geminiviruses and the plant cell
cycle. Plant Mol Biol 43, 763–772.
12 Hofer JM, Dekker EL, Reynolds HV, Woolston CJ,
Cox BS & Mullineaux PM (1992) Coordinate regulation
of replication and virion sense gene expression in wheat
dwarf virus. Plant Cell 4, 213–223.
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4492 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS
13 Zhan X, Richardson KA, Haley A & Morris BAM
(1993) The activity of the coat protein promoter of
Chloris Striate Mosaic Virus is enhanced by its own and
C1–C2 gene products. Virology 193, 498–502.
14 Collin S, Fernandez-Lobato M, Gooding PS, Mulli-
neaux PM & Fenoll C (1996) The two nonstructural
proteins from wheat dwarf virus involved in viral gene
expression and replication are retinoblastoma-binding
proteins. Virology 219, 324–329.
15 Munoz-Martin A, Collin S, Herreros E, Mullineaux
PM, Fernandez-Lobato M & Fenoll C (2003) Regula-
tion of MSV and WDV virion-sense promoters by
WDV nonstructural proteins: a role for their retinoblas-
toma protein-binding motifs. Virology 306, 313–323.
16 Alam J & Cook JL (1990) Reporter genes; applications
to the study of mammalian gene transcription. Anal

Biochem 188, 245.
17 Wood KV (1990) Firefly luciferase: a new tool for
molecular biologists. Promega Notes 28,1.
18 Horvath GV, Pettko-Szandmer A, Nikocics K, Bilgin
M, Boulton M, Davies JW, Gutierrez C & Dudits D
(1998) Prediction of functional proteins of the maize
streak virus replication-associated proteins by protein–
protein interaction analysis. Plant Mol Biol 38, 699–712.
19 Missich R, Ramirez-Parra E & Gutierrez C (2000) Rela-
tionship of oligomerization to DNA binding of wheat
dwarf virus RepA and Rep proteins. Virology 273, 178–
188.
20 Gooding PS, Batty NP, Goldsbrough AP & Mullineaux
PM (1999) Plant cell-directed control of virion sense
gene expression in wheat dwarf virus. Nucleic Acids Res
27, 1709–1718.
21 Dinant S, Ripoll C, Pieper M & David C (2004) Phloem
specific expression driven by wheat dwarf geminivirus
V-sense promoter in transgenic dicotyledenous species.
Physiol Plantarum 121, 108–116.
22 Eagle PA & Hanley-Bowdoin L (1997) Cis elements that
contribute to geminivirus transcriptional regulation and
the efficency of DNA replication. J Virol 71, 6947–6955.
23 Ramos PL, Fuentes AD, Quintana Q, Castrillo G,
Guevara-Gonzalez RG, Peral R, Rivera-Bustamante RF
& Pujol M (2004) Identification of the minimal sequence
required for vascular-specific activity of the Tomato
mottle Taino virus replication-associated protein promo-
ter in transgenic plants. Virus Res 102, 125–132.
24 Frey PM, Scharer-Hernandez NG, Futterer J, Potrykus

I & Puonti-Kaerlas J (2001) Simultaneous analysis of
the bidirectional African cassava mosaic virus promoter-
activity using two different lucifierase genes. Virus Genes
22, 231–242.
25 Liu L, Saunders K, Thomas CL, Davies JW & Stanley
J (1999) Bean yellow dwarf virus RepA, but not Rep,
binds to maize retinoblastoma protein, and the virus
tolerates mutations in the consensus binding motif.
Virology 256, 270–279.
26 Flint J & Shenk T (1997) Viral transactivating proteins.
Annu Rev Genet 31, 177–212.
27 Ben-Israel H & Kleinberger T (2002) Adenovirus and
cell cycle control. Front Biosci 7, 1369–1395.
28 McMurray H, Nguyen D, Westbrook TF & McAnce
DJ (2001) Biology of human papillomaviruses. J Exp
Pathol 82, 15–33.
29 Kaelin G Jr (1999) Functions of the retinoblastoma
protein. Bioessays 21, 950–958.
30 Egelkrout EM, Robertson D & Hanley-Bowdoin L
(2001) Proliferating cell nuclear antigen transcription is
repressed through an E2F consensus element and acti-
vated by geminivirus infection in mature leaves. Plant
Cell 13, 1437–1452.
31 Egelkrout EM, Mariconti L, Settlage SB, Cella R,
Robertson D & Hanley-Bowdoin L (2002) Two E2F
elements regulate the proliferating cell nuclear antigen
promoter differently during leaf development. Plant Cell
14, 3225–3236.
32 Bagewadi B, Chen S, Lal SK, Choudhury NR &
Mukherjee SK (2004) PCNA interacts with Indian

mung bean yellow mosaic virus Rep and downregulates
Rep activity. J Virol 78, 11890–11903.
33 Castillo AG, Collinet D, Deret S, Kashoggi A &
Bejarano ER (2003) Dual interaction of plant PCNA
with geminivirus replication accessory protein (Ren)
and viral replication protein (Rep). Virology 312,
381–394.
34 Liu L, Davies JW & Stanley J (1998) Mutational analysis
of bean yellow dwarf virus, a Mastrevirus that is adapted
to dicotyledonous plants. J Gen Virol 79, 2265–2274.
35 Cazzonelli CI, Burke J & Velten J (2005) Functional
characterization of the geminiviral conserved late ele-
ment (CLE) in uninfected tobacco. Plant Mol Biol 58,
465–481.
36 Velten J, Morey KJ & Cazzonelli CI (2005) Plant viral
intergenic DNA sequence repeats with transcription
enhancing activity. Virol J 2, 16.
37 Ruiz-Medrano R, Guevara-Gonzalez RG, Arguello-
Astorga GR, Monsalve-Fonnegra Z, Herrera-Estrella
LR & Rivera-Bustamante RF (1999) Identification of a
sequence element involved in C2-mediated transactiva-
tion of the pepper huasteco virus coat protein gene.
Virology 253, 162–169.
38 Sunter G & Bisaro DM (2003) Identification of a mini-
mal sequence required for activation of the tomato
golden mosaic virus coat protein promoter in proto-
plasts. Virology 305, 452–462.
39 Hung H-C & Petty ITD (2001) Functional equivalence
of late gene promoters in bean golden mosaic virus with
those in tomato golden mosaic virus. J Gen Virol 82,

667–672.
40 Shivaprasad PV, Akbergenov R, Trinks D, Rajeswaran
R, Veluthambi K, Hohn T & Pooggin MM (2005)
Promoters, transcripts and regulatory proteins of
K. L. Hefferon et al. Geminivirus transcriptional regulation requirements
FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS 4493
Mungbean Yellow Mosaic Geminivirus. J Virol 79,
8149–8163.
41 Hong Y, Stanley J & van Wezel R (2003) Novel system
for the simultaneous analysis of geminivirus DNA repli-
cation and plant interactions in Nicotiana benthamiana.
J Virol 77, 13315–13322.
42 Dong Y, van Wezel R, Stanley J & Hong Y (2003)
Functional characterization ofthe nuclear localization
signal for a suppressor of posttranscriptional gene silen-
cing. J Virol 77, 7026–7033.
43 Orozco BM, Kong L-J, Batts LA, Elledge S & Hanley-
Bowdoin L (2000) The multifunctional character of a
geminivirus replication protein is reflected by its com-
plex oligomerization properties. J Biol Chem 275, 6114–
6122.
44 Dekker EL, Woolston CJ, Xue Y, Cox B & Mullineaux
PM (1991) Transcript mapping reveals different expres-
sion strategies for the bicistronic RNAs of the gemini-
virus wheat dwarf virus. Nucleic Acids Res 19, 4075–
4081.
45 Hefferon KL, Golshani A & AbouHaidar MG (2002)
Translational control of plant virus RNAs. In Recent
Research Developments in Virology, Transworld Research
Network, pp. 1–12. Research Signpost, Kerala.

46 Hefferon KL & Fan Y (2004) Expression of a vaccine
protein in a plant cell line using a geminivirus-based
replicon system. Vaccine 23, 404–410.
47 Hefferon KL, Kipp P & Moon YS (2004) Expression
and purification of heterologous proteins in plant tissue
using a geminivirus vector system. J Mol Microbiol
Biotechnol 7, 109–114.
48 Toth RL, Chapman S, Carr F & Santa Cruz S (2001) A
novel strategy for the expression of foreign genes from
plant virus vectors. FEBS Lett 489, 215–219.
49 Liu H, Boulton MI, Oparka KJ & Davies JW (2001)
Interaction of the movement and coat proteins of Maize
streak virus: implications for the transport of viral
DNA. J Gen Virol 82, 35–44.
50 Paszty C & Luiquin PF (1987) Improved plant proto-
plast plating ⁄ selection technique for quantitation of
transformation frequencies. Biotechniques 5, 716–718.
51 Dugdale B, Bewetham PR, Becker DK, Harding RM &
Dale JC (1998) Promoter activity associated with the
intergenic regions of banana bunchy top virus DNA-1
to 6 in transgenic tobacco and banana cells. J Gen Virol
79, 2301–2311.
52 Huang Z, Andrianov VM, Han Y & Howell SH (2001)
Identification of arabidopsis proteins that interact with
the cauliflower mosaic virus (CaMV) movement protein.
Plant Mol Biol 47, 663–675.
53 Wilke S (1996) Isolation of total genomic DNA. In
Plant Molecular Biology (Clark MS, ed.), p. 315. Sprin-
ger, Berlin.
54 Jefferson GA (1987) Assaying chimeric genes in plants:

the GUS gene fusion system. Plant Mol Biol Report 5,
387–405.
55 Chomezynski P & Sacchi N (1987) Single-step method
of RNA isolation by acidguanidinium thiocyanate-
phenol-chloroform extraction. Anal Biochem 162,
156–159.
56 Breyne PM, De Loose A, Dedonder M, Van Montagu
M & Depicker A (1993) Quantitiative kinetic analysis of
Beta-Glucuronidase activities using a computer-directed
microtiter plate reader. Plant Mol Biol Report 11, 21–31.
57 Sojikul P, Buehner N, Mason HS (2003) A plant signal-
peptide-hepatitis B surface antigen fusion protein with
enhanced stability and immunogenicity expressed in
plant cells. Proc Natl Acad Sci USA 100, 2209–2214.
Geminivirus transcriptional regulation requirements K. L. Hefferon et al.
4494 FEBS Journal 273 (2006) 4482–4494 ª 2006 The Authors Journal compilation ª 2006 FEBS