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.. RepA of wheat dwarf vir
Trang 1for bean yellow dwarf geminivirus transcriptional
regulation
Kathleen L Hefferon1, Yong-Sun Moon2and Ying Fan3
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: klh22@cornell.edu
(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.
Trang 2BeYDV, 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 expressiviri-on 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
Trang 3EcoRI 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.
Trang 4(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)1Æmin)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.
Trang 5pPcGUS, 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 p35SDintronRBR–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 RepARBR–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.
Trang 6RepA under in vivo conditions, NT-1 cells were
cobombarded with both p35SHA6HISRep and
p35SHARepA p35SHA6HISRep was collected on a
Ni2+ 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
D
PPLKKKKLKDD
A
p35S intron/GFP
p35SRepA/GFP
35S
35S
T
T
GFP
GFP RepA
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.
Trang 7replication 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 32P-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 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-Double-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.
Trang 8transcription 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 Transactivativiri-on 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
Trang 9for 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 Ni2+ 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
Trang 10Mastrevirus 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 BamHI 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 28C, 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 with32P 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,
1 mm EDTA and 5% SDS at 65C, 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