Journal of General Virology (2007), 88, 1831–1841
DOI 10.1099/vir.0.82513-0
Identification of long intergenic region sequences
involved in maize streak virus replication
Janet A. Willment,1 Darrin P. Martin,1 Kenneth E. Palmer,2,3
Wendelin H. Schnippenkoetter,4 Dionne N. Shepherd5
and Edward P. Rybicki1,5
1
Correspondence
Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences,
University of Cape Town, Observatory 7925, South Africa
Edward P. Rybicki
[email protected]
2
James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street,
Louisville, KY 40202, USA
3
Department of Pharmacology and Toxicology, University of Louisville, 570 South Preston Street,
Louisville, KY 40202, USA
4
14 Ashby Drive, Bungendore, NSW 2621, Australia
5
Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch,
Cape Town 7701, South Africa
Received 30 August 2006
Accepted 20 February 2007
The main cis-acting control regions for replication of the single-stranded DNA genome of maize
streak virus (MSV) are believed to reside within an approximately 310 nt long intergenic region
(LIR). However, neither the minimum LIR sequence required nor the sequence determinants of
replication specificity have been determined experimentally. There are iterated sequences, or
iterons, both within the conserved inverted-repeat sequences with the potential to form a
stem–loop structure at the origin of virion-strand replication, and upstream of the rep gene TATA
box (the rep-proximal iteron or RPI). Based on experimental analyses of similar iterons in viruses
from other geminivirus genera and their proximity to known Rep-binding sites in the distantly
related mastrevirus wheat dwarf virus, it has been hypothesized that the iterons may be
Rep-binding and/or -recognition sequences. Here, a series of LIR deletion mutants was used
to define the upper bounds of the LIR sequence required for replication. After identifying MSV
strains and distinct mastreviruses with incompatible replication-specificity determinants (RSDs),
LIR chimaeras were used to map the primary MSV RSD to a 67 nt sequence containing the
RPI. Although the results generally support the prevailing hypothesis that MSV iterons are
functional analogues of those found in other geminivirus genera, it is demonstrated that neither the
inverted-repeat nor RPI sequences are absolute determinants of replication specificity. Moreover,
widely divergent mastreviruses can trans-replicate one another. These results also suggest
that sequences in the 67 nt region surrounding the RPI interact in a sequence-specific manner
with those of the inverted repeat.
INTRODUCTION
The determinants of replication specificity for the plantinfecting single-genome-component DNA viruses in the
genus Mastrevirus (family Geminiviridae) are currently unknown. Rolling-circle replication in geminiviruses involves
an apparently sequence-specific interaction between a virus
replication-associated protein (Rep) and viral DNA in close
proximity to the origin of virion-strand replication (V-ori)
(Fontes et al., 1992, 1994a, b; Lazarowitz et al., 1992;
Argüello-Astorga et al., 1994b; Jupin et al., 1995; Choi &
Supplementary figures are available with the online version of this paper.
0008-2513 G 2007 SGM
Stenger, 1996; Gladfelter et al., 1997; Akbar Behjatnia et al.,
1998; Sanz-Burgos & Gutierrez, 1998; Castellano et al.,
1999; Chatterji et al., 1999, 2000). Sequence analysis of this
genomic region has revealed iterative elements called
iterons, the sequences of which are a major determinant
of Rep-binding specificity for viruses in the related genera
Begomovirus and Curtovirus (Fontes et al., 1992, 1994a, b;
Argüello-Astorga et al., 1994a, b; Choi & Stenger, 1995,
1996; Chatterji et al., 2000; Ramos et al., 2003). Complementation of replication functions by begomo- and
curtoviruses is restricted to individuals that share nearly
identical iteron sequences (Stanley et al., 1985; Lazarowitz
et al., 1992; Gilbertson et al., 1993; Fontes et al., 1994b;
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1831
J. A. Willment and others
Hanley-Bowdoin et al., 1996, 2000; Gladfelter et al., 1997;
Chatterji et al., 2000; Saunders et al., 2002; Ramos et al.,
2003). In addition to the iterons, undefined sequences
located between the iterons and the inverted-repeat
sequence (stem–loop) of begomovirus genomes are potential secondary replication-specificity determinants (RSDs)
(Fontes et al., 1994b; Hou & Gilbertson, 1996; Gladfelter
et al., 1997). Whereas the correct spatial arrangement of
begomovirus iterons, in relation both to one another and
to the inverted repeat, is essential for efficient replication
(Argüello-Astorga et al., 1994b; Gladfelter et al., 1997;
Orozco et al., 1998; Chatterji et al., 2000), the precise
arrangements of iterons and inverted-repeat sequences are
less constrained in the curtoviruses (Choi & Stenger, 1996).
Also, the inverted-repeat sequences of either curtoviruses
or begomoviruses apparently contain no primary RSDs
(Fontes et al., 1994b; Choi & Stenger, 1996).
Unlike the situation with begomoviruses, the mastrevirus
V-ori inverted-repeat sequences are highly diverse and it
has been hypothesized that both the inverted-repeat and
rep-proximal iteron (RPI) sequences might constitute RSDs
in this genus (Argüello-Astorga et al., 1994a, b). Evidence
that both the iterons and the potential stem–loop structure
created by the inverted repeat as a whole are required
during mastrevirus replication has been obtained in mutational studies of the maize streak virus (MSV) long intergenic region (LIR) (Schneider et al., 1992). Conversely, a
wheat dwarf mastrevirus (WDV) mutant with a deletion of
the entire inverted-repeat sequence was able to initiate
replication. There was, however, a defect in termination of
replication, resulting in high-molecular-mass concatemeric
forms of viral DNA (Kammann et al., 1991). Heyraud et al.
(1993) subsequently found that the mutant virus was able
to initiate replication from a second site (TACCC) resembling the nicking site in the inverted-repeat sequence.
This second initiation site is apparently unique to WDV
and it is likely that the intact inverted repeat is required
for replication initiation and termination by all other
geminiviruses.
Both Rep–LIR interactions and the core LIR sequences
required for replication are well defined in the genome of
the mastrevirus WDV (Suarez-Lopez et al., 1995; SuarezLopez & Gutierrez, 1997; Sanz-Burgos & Gutierrez, 1998;
Castellano et al., 1999; Missich et al., 2000). The upper
bounds of the minimal LIR sequence absolutely required to
support WDV replication lie between 188 and 178 nt 59,
and 25 and 28 nt 39, of the V-ori, with an extra approximately 70 nt 59 and 30 nt 39 of this region enabling efficient
replication (Sanz-Burgos & Gutierrez, 1998). Electronmicroscopic visualization of Rep–LIR binding and DNA
footprinting revealed two high-affinity and one low-affinity
Rep-binding sites within the LIR in the vicinity of the
complementary-sense gene TATA box, the virion-sense
gene TATA box and the inverted repeat, respectively (SanzBurgos & Gutierrez, 1998; Castellano et al., 1999). Although
evidence of a Rep–inverted-repeat sequence (stem–loop)
complex is strongly suggestive of Rep-binding signals
1832
required for replication (and hence RSDs) residing here,
the role of the Rep–LIR complexes in replication is less
certain, in that they may also be active in regulation of
complementary- and/or virion-sense gene expression.
Whilst WDV and MSV are both grass-infecting mastreviruses, they are only very distantly related (approx. 48 %
LIR nucleotide sequence identity); this is far lower than for
any other intrageneric comparisons for curto- or begomoviruses (Rybicki, 1994). The WDV LIR is also 101 nt longer
than that of MSV. A degree of caution is therefore needed
when using these excellent WDV studies to infer either the
minimal LIR sequences required for MSV replication or the
locations of Rep binding in the MSV LIR.
In this study, we sought to define features of the MSV LIR
that are functional during replication. First, we examined
LIR deletion mutants by using transient replication assays
to define the upper bounds of the minimal LIR sequence
absolutely required for replication. trans-Replication assays
were then used to demonstrate complementation of replication functions between viruses with divergent LIR and
iteron sequences. Finally, we examined chimaeras of inefficiently trans-replicating viruses by using agroinoculation
and competitive trans-replication assay experiments to
localize the primary MSV RSD to a 67 nt region surrounding the RPI. Our results indicate that sequences in this
region interact cooperatively with those in the inverted
repeat to determine the efficiency of MSV replication.
METHODS
Virus isolates and plasmids. Infectious clones of the MSV isolates
and strains MSV-Kom, MSV-VM, MSV-Tas, MSV-VW, MSV-Set
and Panicum streak virus-Karino (PanSV-Kar) (full genomes with
reiterated LIRs, cloned in pUC19) have been described previously
(Schnippenkoetter et al., 2001a; Willment et al., 2002) and are
referred to here as pMSV-Kom, pMSV-VM, pMSV-Tas, pMSV-VW,
pMSV-Set and pPanSV-Kar, respectively.
pKEP176 (Palmer, 1997) is a pUC19-based construct containing the
MSV-Kom V1 gene followed by the b-glucuronidase (uidA) gene (a
replacement of the V2 gene), and was used as an intermediate
construct in the creation of plasmids pLIR1[290,+148],LIR2[290,+148]
and pLIR1[2106,+148],LIR2[2106,+148] (described below).
pKomPstI [called pMSVPstI by Palmer & Rybicki (2001)] is a
replication-incompetent tandem dimer of MSV-Kom. pKomRep
[called pKEP132 by Palmer (1997)] is an expression cassette
containing the MSV-Kom rep gene cloned between the rice actin 1
promoter and the nopaline synthase (nos) terminator (both from
pCOR113; McElroy et al., 1991), for high-level expression of Rep in
maize suspension cells.
pSekA.2, an agroinfectious, mostly MSV-Kom chimaera containing
MSV-Set LIR sequence from 24 nt 59 of the V-ori to 148 nt 39 of
the V-ori (nt 224 to +148), has been described previously
(Schnippenkoetter et al., 2001b).
LIR deletion constructs. Partially dimerized deletion mutants of the
MSV-Kom LIR (Fig. 1) containing (i) wild-type (wt) 59 LIR (LIR-1)
and truncated 39 LIR (LIR-2) sequences, (ii) wt LIR-2 and truncated
LIR-1 sequences and (iii) truncated LIR-1 and LIR-2 sequences were
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Journal of General Virology 88
MSV sequences involved in replication
Fig. 1. (a) Scheme of the MSV-Kom LIR, with relevant features and restriction enzymes used in making deletion mutants
highlighted on the virion-sense strand. RPI, rep-proximal iteron; V-ori, virion-sense origin of replication flanked by invertedrepeat sequences with the potential of forming a stem–loop structure, marked by an arrow and designated +1. Numbers above
the LIR are nucleotide co-ordinates in relation to the V-ori. The location of iterons in the inverted repeat is represented by
arrows. TATA boxes 59 and 39 of the V-ori that define the respective complementary- and virion-sense transcription-initiation
sites are indicated. (b) Deletion mutants used in this study, showing the portion of the LIR that they contain relative to the V-ori
(+1).
constructed as detailed below and as shown in Supplementary Fig. S1
(available in JGV Online).
pKori0-1 [called pMSV35S-bar by Palmer & Rybicki (2001)] consists
of pMSV-Kom with the virion-sense open reading frames replaced by
the cauliflower mosaic virus (CaMV) 35S promoter and bar gene, and
forms the backbone upon which LIR-1 deletion mutants were constructed (Supplementary Fig. S1).
pKori0-2 (Supplementary Fig. S1) is the backbone upon which LIR-2
deletion mutants were constructed. pKori0-2 was constructed by
using the PCR primers LIR-Kom (59-CTATCTAGACGACGACGGAGGTTGAG-39; XbaI site underlined) and SIR-Kom (59-CCGCTCGAGTAATTCATATAGATC-39; XhoI site underlined) to amplify a
1529 nt fragment containing the MSV-Kom LIR (wt LIR-1), V1, V2
and short intergenic region (SIR) sequences. This fragment was then
inserted into the SalI site of a vector already containing the TaqI
fragment of the chloramphenicol acetyltransferase (cat) gene from
pBSG8-15 (Georges Rapoport, Institut Pasteur, Paris, France) and the
PstI–XbaI fragment containing the CaMV 35S promoter from pMF6
(Callis et al., 1987). Both the XhoI site (compatible with SalI) and the
XbaI site (blunt-ligated with SalI) of the PCR fragment were lost
during the cloning steps.
pKori0-1-based constructs, each of which contains a wt LIR-2 and a
truncated LIR-1 sequence, were named according to the portion of
the LIR-1 that they contained relative to the V-ori (Fig. 1). The 39
LIR-1 truncations, which have a 59 XbaI site, were made by deleting
sequences between the (i) DraI and BamHI sites and (ii) ApaI and
BamHI sites (Fig. 2; Supplementary Fig. S1), inserting the fragments
into pUC18 to create a 39 XbaI site and cloning the truncated LIR-1
sequence into the XbaI site of pKori0-1, to give pLIR1[2163,+54] and
pLIR1[2163,+27], respectively. pLIR1[2163,+148] has no LIR-1 deletion.
pKori0-2-based constructs, each of which contains a wt LIR-1 and a
truncated LIR-2 sequence, were named according to the portion of
the LIR-2 that they contained relative to the V-ori. 59 LIR-2 truncations were made by deleting sequences between the (i) XbaI and AccI
sites and (ii) XbaI and TaqI sites (Fig. 2; Supplementary Fig. S1),
inserting the fragments into pUC18 to create flanking XbaI sites
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and cloning the truncated LIR-2 sequence into the XbaI site of
pKori0-2, to give pLIR2[2106,+148] and pLIR2[290,+148], respectively.
pLIR2[2163,+148] has no LIR-2 deletion.
Two mutants containing 59 deletions in both LIR-1 and LIR-2 were
also constructed. To do this, XhoI–BamHI fragments containing
XbaI–AccI and XbaI–TaqI LIR deletions were inserted into the XhoI/
BamHI sites of pKEP176 immediately upstream of the V1 gene to
yield pKoriA and pKoriB, respectively. These contain a 59-truncated
LIR1, V1 gene and uidA gene. SacI fragments of pLIR2[2106,+148] and
pLIR2[290,+148], containing the SIR, cat gene, CaMV 35S promoter
and 59-truncated LIR2 sequences (Supplementary Fig. S1) were
inserted into the SacI sites of pKoriA and pKoriB 39 of the uidA
and
sequence,
to
yield
pLIR1[290,+148],LIR2[290,+148]
[2106,+148]
[2106,+148]
,LIR2
, respectively. These contain a 59pLIR1
truncated LIR1, V1 gene, uidA gene, SIR, cat gene, CaMV 35S
promoter and a 59-truncated LIR2, with the genes either side of the
SIR in the same orientation, as shown in Supplementary Fig. S1
(available in JGV Online).
The complete LIR sequences of pLIR1[2163,+27], pLIR1[2163,+54],
pLIR2[290,+148], pLIR2[2106,+148], pLIR1[290,+148],LIR2[290,+148]
and pLIR1[2106,+148],LIR2[2106,+148] were confirmed (by using an
ABI Prism automated sequencer; Applied Biosystems) to have the
correct sequence and orientation.
LIR chimaera constructs. The 274 nt BamHI–SacI fragment of
pMSV-Kom was used to replace the corresponding fragment of
pMSV-Set to produce the chimaera pSSBK. An approximately 370 nt
NsiI–ApaI fragment was exchanged between the viruses to obtain the
mostly MSV-Kom chimaera pKNASNsiI and the mostly MSV-Set
chimaera pSNAK. Whereas pSNAK contains a functional rep,
pKNASNsiI is missing 10 nt between two closely situated NsiI sites
in pMSV-Set and therefore has a frameshift mutation within rep. The
complementary-sense strand side of the LIR, up to the rpeI element
(Fenoll et al., 1988, 1990), and the first 185 nt of rep were exchanged
between pMSV-Set and MSV-Kom. The 811 nt XhoI–RsrII fragments
of pKNASNsiI and pMSV-Kom were exchanged between these
constructs to produce the chimaeras pKNRSNsiI and pKRAS. Both
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J. A. Willment and others
Fig. 2. Results of mutant LIR transient replication assays. *Restriction sites used to construct deletion mutants of the MSV-Kom
LIR (see also Supplementary Fig. S1, available in JGV Online). Nucleotide co-ordinates are relative to the virion-sense origin of
replication (V-ori, designated +1) in the MSV-Kom LIR (Fig. 1). Arrows either side of the V-ori represent inverted-repeat
sequences with the potential of forming a stem–loop structure; arrowheads 59 of the V-ori designate the rep-proximal iteron.
SB, Southern blot; +, RF-DNA detected; ”, no RF-DNA detected; +/”, marginal RF-DNA detection.
chimaeras contain mostly MSV-Kom sequences, but whilst KNRSNsiI
contains MSV-Set sequence between the NsiI and RsrII sites, pKRAS
contains MSV-Set sequence between the RsrII and ApaI sites.
pKNRSNsiI has the same 10 nt deletion in rep as does pKNASNsiI.
The 2227 nt RsrII fragments of pSNAK and pMSV-Set were exchanged to produce the chimaeras pSNRK and pSRAK. The approximately 2400 nt SacI fragment of pMSV-Set was used to replace the
corresponding SacI fragments of pSNRK and pSNAK to produce the
chimaeras pSSRK and pSSAK, respectively.
Partially dimeric (1.1mer) agroinfectious clones of pKNRSNsiI,
pKNASNsiI, pKRAS, pSSBK, pSNAK, pSNRK, pSRAK, pSSRK and
pSSAK in the binary cloning vector pBI121 (Clontech) were constructed as described previously (Schnippenkoetter et al., 2001b).
Although no attempt was made to obtain properly agroinfectious
clones of pKNRSNsiI and pKNASNsiI (because both contained a
10 nt deletion in rep), these clones were partially dimerized for use
during replication assays in black Mexican sweetcorn (BMS) cells.
A series of Rep-defective mutants of the LIR chimaeras was constructed for use in transient replication assays. The 10 nt rep NsiI
fragment of pMSV-Set and the mostly MSV-Set chimaeras pSRAK,
pSSRK, pSSAK and pSSBK was deleted to produce a set of selfreplication-incompetent constructs called pSetNsiI, pSRAKNsiI,
pSSRKNsiI, pSSAKNsiI and pSSBKNsiI. Construction of the selfreplication-incompetent clones pKNRSNsiI and pKNASNsiI has been
described above. A pMSV-Kom PstI frameshift mutant, called
pKomPstI, was used as a self-replication-incompetent wt MSV-Kom
LIR control. The XhoI–RsrII fragment of pKomPstI was used to
1834
replace the corresponding fragment of pKRAS to produce the
self-replication-incompetent chimaera pKRASPstI. The PstI mutation
in both pKomPstI and pKRASPstI results in translation of a protein
containing the N-terminal 24 aa of Rep and additional altered amino
acids due to the frameshift mutation.
Transient replication assays. Replication proficiency of LIR dele-
tion mutants and complementation of replication functions between
viruses with divergent LIR and iteron sequences were assayed by a
biolistic bombardment-mediated transient assay system as described
by Shepherd et al. (2005). In each experiment, bombardments with
DNA of individual plasmids or plasmid combinations were performed in duplicate and all experiments were repeated at least twice.
DNA was extracted from BMS (Pioneer Hi-Bred) suspension culture
cells 3 or 4 days after bombardment, as described by Xie et al. (1995),
and digested with DpnI to remove input plasmid DNA (i.e. methylated, bacteria-derived DNA). Electophoresis through 0.8 % 0.56
TBE/agarose gels was used to separate either various rolling-circle
replication DNA intermediates or restriction-enzyme fragments
following digestion. DNA was then capillary-transferred onto nylon
membranes (Hybond-N+; Amersham Biosciences) and detected by
using digoxigenin-labelled (Roche Diagnostics) pMSV-Kom- or
CAT-specific probes, following standard protocols (Sambrook et al.,
1989). Evidence of rolling-circle replication in bombarded cells was
also examined by various replicative form (RF)-specific PCR assays.
These involved different combinations of primers specific for the
CaMV 35S promoter sequence (59-CAACCACGTCTTCAAAGC-39)
and two previously described degenerate primers (Willment et al.,
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Journal of General Virology 88
MSV sequences involved in replication
2001): 215–234 and 1770–1792 (which bind at nucleotide co-ordinates
361–343 and 1746–1764, respectively; numbering starts from the last A
of the conserved V-ori TAATATTAC sequence of MSV-Kom, in the
59A39 direction of the primer). Degenerate-primer RF-specific PCR
assays used were described by Shepherd et al. (2005).
Agroinoculation experiments. Agroinfectious clones of the LIR
chimaeras were used to transform Agrobacterium tumefaciens C58C1
(pMP90) (Koncz & Schell, 1986) by using the freeze–thaw method of
Holsters et al. (1978). Agroinoculation of 3-day-old sweetcorn
‘Jubilee’ seedlings (Starke Ayres) and quantification of disease symptoms by image analysis were carried out as described previously
(Martin et al., 1999). In each of three to six repeated agroinoculation
experiments, the ten chimaeras, plus wt MSV-Kom and MSV-Set,
were each used to infect 14 plants. In each experiment, the number of
plants showing symptomatic infections with the various chimaeric
and wt viruses was recorded after 29 days. Viral genomic DNA was
isolated from the agroinfected plants, and Southern blot analysis or
degenerate-primer PCR amplification and restriction fragment-length
polymorphism (RFLP) analysis with CfoI were used to confirm the
identity of agroinfectious clones, as described previously (Willment
et al., 2001).
RESULTS AND DISCUSSION
Upper bounds of the minimal LIR sequence
required for MSV-Kom replication
BMS cells were bombarded either with rep-containing
LIR-1 deletion mutants (pKori0-1-based constructs; Supplementary Fig. S1) on their own, or with rep-replacement
LIR-2 and LIR-1+LIR-2 deletion mutants (pKori0-2-based
constructs; Supplementary Fig. S1) together with pMSVKom or pKomRep (an MSV-Kom Rep expression cassette).
Each bombarded LIR construct had two LIR copies (one wt
and one truncated), which are necessary for efficient replicational release of the genome from the plasmid background
(Supplementary Fig. S1). pKori0-1 and pKori0-2 had only
one LIR copy and were therefore not replication-proficient
(Fig. 2).
Three days after bombardment, RF-DNA was detectable by
both RF-specific PCR and Southern analyses in BMS cells
bombarded with pLIR1[2163,+148] (two wt MSV-Kom LIRs)
and pLIR1[2163,+54] (39-truncated LIR-1 and wt LIR-2;
Fig. 2), but not in those bombarded with pLIR1[2163,+27]
(39-truncated LIR-1 and wt LIR-2; Fig. 2). This indicates that
the 39 boundary of the minimal LIR sequence required for
replication lies between +27 and +54 nt 39 of the V-ori
(defined as the last A of the conserved TAATATTAC
sequence in the loop of the potential stem–loop structure,
163 nt 39 of the first MSV-Kom LIR nucleotide; Fig. 1).
Both Southern and RF-specific PCR analyses of the LIR-2
mutants co-bombarded with pMSV-Kom indicated that
only the construct with two wt LIR sequences,
pLIR2[2163,+148], was trans-replicated efficiently (Fig. 2).
However, when the LIR-2 mutants were co-bombarded
with pKomRep, both Southern and PCR analyses indicated
the presence of small amounts of pLIR2[290,+148] and
pLIR2[2106,+148] (both containing a wt LIR-1 and
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59-truncated LIR-2) RF-DNA. No RF-DNA was detectable
in BMS cells co-bombarded with pKori0-2 (containing
only a wt LIR-1 and no LIR-2) and either pMSV-Kom
or pKomRep, demonstrating that the absence of LIR-2
prevents replication completely.
We suspected that some replication may have occurred
directly from the input DNA when pLIR2[290,+148] and
pLIR2[2106,+148] were co-bombarded with the Rep expression cassette, but that the released RF-DNAs were
probably replication-incompetent. This hypothesis was
supported by the fact that no RF-DNA was detected
by either Southern or PCR analysis following cobombardment of pLIR1[290,+148],LIR2[290,+148] and
pLIR1[2106,+148],LIR2[2106,+148] with either pMSV-Kom
and
or
pKomRep.
pLIR1[290,+148],LIR2[290,+148]
[2106,+148]
,LIR2[2106,+148] respectively contain the
pLIR1
same LIR-2 deletions as pLIR2[290,+148] and
pLIR2[2106,+148], but also contain these same deletions in
their LIR-1. Therefore, whilst the region of nt 2163 to
290 59 of the V-ori is probably not absolutely required for
the termination of replicational release from the LIR-2
deletion mutants, the 59 boundary for the minimal LIR
sequence required for efficient replication resides between
nt 2163 and 2106 59 of the V-ori (Fig. 3).
The extent of the minimal MSV LIR sequences required for
replication is therefore very similar to those determined for
WDV. Whilst the region ranges from between 2163 and
2106 nt 59 of the V-ori to between +27 and +54 nt 39 of
the V-ori in MSV (Fig. 3), in WDV, it ranges between 2188
and 2178 nt 59, and +25 and +28 nt 39, of the V-ori
(Sanz-Burgos & Gutierrez, 1998). This indicates that,
despite the viruses sharing only approximately 48 % LIR
sequence identity and the WDV LIR being 101 nt larger
than that of MSV, the 59 and 39 boundaries of the core LIR
sequences required for replication have been conserved
between the viruses. It also suggests that the spatial
arrangements of other features determined within the
WDV LIR may have similarly conserved analogues within
the MSV LIR.
Identification of replication specificity between
MSV-Kom and other viruses
The ability of different MSV and PanSV isolates to complement the replication functions of MSV-Kom was tested
by co-bombarding BMS cells with the MSV-Kom repreplacement construct pLIR2[2163,+148] (containing two wt
LIRs, with rep replaced by the CaMV 35S promoter and cat
gene; Supplementary Fig. S1) and pMSV-VM, pMSV-VW,
pMSV-Tas, pMSV-Set or pPanSV-Kar. Respectively, the
Reps of these wt cloned viruses share 98, 87, 88, 82 and
65 % amino acid sequence identity with that of MSV-Kom.
Southern and RF-specific PCR analyses determined that
pLIR2[2163,+148] was trans-replicated by all of the wt MSV
isolates and PanSV-Kar (Fig. 4; Supplementary Fig. S2,
available in JGV Online). This result is somewhat
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J. A. Willment and others
Fig. 3. Comparison of the MSV-Kom LIR
sequence with those of MSV-Set and
PanSV-Kar. (a) Features within the LIRs of
these viruses. Probable virion-sense ori (V-ori)
TAATATTAC and inverted-repeat, RPI, TATA
box and rightward promoter element (rpe)
sequences are drawn to scale with the plot in
(b). The 59 and 39 boundaries of the minimal
MSV-Kom LIR sequence required for replication are within the indicated regions. (b)
Relatedness of MSV-Set and PanSV-Kar LIR
sequences to that of MSV-Kom. The plot was
constructed with RDP2 (Martin et al., 2005),
moving a 15 nt window along an alignment
of each of the sequences with MSV-Kom
(aligned by using CLUSTAL_X; Thompson et al.,
1997) 1 nt at a time, counting the number of
matches between the sequences within each
window and plotting these at the central
window position. The LIR region around the
RPI where MSV-Kom is on average more
similar to PanSV-Kar than it is to MSV-Set is
indicated by a thick black line.
surprising, as the Rep proteins of PanSV-Kar and MSVKom share only 65 % amino acid sequence identity.
Although such leniency in trans-replication specificity has
been observed between begomovirus Rep proteins and
both defective satellite (Dry et al., 1997; Lin et al., 2003)
and DNA-b (Briddon & Stanley, 2006) molecules, in
begomo- and curtoviruses, trans-replication has only been
demonstrated between isolates sharing .80 % Rep amino
acid sequence identity (Gilbertson et al., 1993; Frischmuth
et al., 1997; Hill et al., 1998; Unseld et al., 2000; Brown
et al., 2002; Saunders et al., 2002; Ramos et al., 2003). In
Fig. 4. trans-Replication of a Rep-deficient MSV-Kom construct
(pLIR2[”163,+148]) by various wt viruses. (a) Detection of replicating wt viruses by RF-specific PCR using the degenerate primers
215–234 and 1770–1792 (Willment et al., 2001), showing that
the wt viruses are replication-proficient. (b) Detection of
pLIR2[”163,+148] RFs by RF-specific PCR using primers 215–
234 and CaMV 35S. As controls, DNA from unbombarded cells
(BMS) and cells bombarded with pLIR2[–163,+148] only were also
analysed for the presence of RF-DNA. The approximate sizes
(in nt) of PCR products compared with PstI-digested l DNA
(lPstI) are indicated on the right.
1836
some cases, begomovirus strains with Reps sharing .90 %
amino acid sequence identity exhibit specificity for their
cognate origins (Chatterji et al., 2000). Intriguingly, in
triplicate experiments, we also observed consistently that
the PanSV-Kar Rep provided better complementation of
MSV-Kom replication functions than did the much more
closely related MSV-Set Rep, which shares 82 % amino acid
sequence identity with that of MSV-Kom. Despite its
inability to trans-replicate MSV-Kom efficiently, the MSVSet Rep is perfectly functional, as evidenced by MSV-Set
being both agroinfectious (Schnippenkoetter et al., 2001a)
and replication-proficient in BMS cells (Fig. 4a). It is also
very unlikely that pLIR2[2163,+148] RF-DNAs detected in
these trans-replication experiments arose via a recombinational mechanism (rather than through trans-replication),
as BMS cells bombarded with the MSV-Kom rep-replacement construct alone yielded no evidence of RF-DNAs in
either Southern or PCR analysis.
Efficient trans-replication presumably requires the sharing
of common RSDs within the LIR. Although mastrevirus
intergenic regions are highly variable (Hughes et al., 1992;
Rybicki, 1994; Padidam et al., 1995; Martin et al., 2001),
the iterative sequence elements identified by ArgüelloAstorga et al. (1994a, b) as potential RSDs are found within
the LIRs of all currently sequenced mastreviruses. The
sequences of these elements are, however, not conserved
between MSV isolates such as MSV-Kom and MSV-Set
(Schnippenkoetter et al., 2001a). In the PanSV and MSV
isolates that we have analysed, there are also considerable
differences in the spacing of the RPI relative to the rep
TATA box and the V-ori (Fig. 3). The RPI is 103, 87
and 65 nt 59 of the V-ori and 39, 22 and 11 nt 59 of the
rep TATA box in MSV-Set, MSV-Kom and PanSV-Kar,
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Journal of General Virology 88
MSV sequences involved in replication
respectively. Only in the case of MSV-Set do these differences (potentially acting as secondary RSDs; Fontes et al.,
1994a) potentially impact on the ability of its Rep to
recognize and trans-replicate MSV-Kom.
previously (Schnippenkoetter et al., 2001b). The infectivity
of all of these LIR chimaeras was tested by agroinoculation
of maize.
Identification of MSV-Kom RSDs
In all the plants agroinfected with the two predominantly
MSV-Kom chimaeras, pSekA.2 and pKRAS, pKRAS consistently produced the most severe symptoms (Fig. 5). This
chimaera, which has the 48 nt MSV-Kom inverted-repeat
sequence replaced by that of MSV-Set (which differs at
eight sites from the MSV-Kom sequence), infected a
similar number of plants to, and produced only slightly
milder symptoms in maize than, MSV-Kom (Fig. 5).
However, the predominantly MSV-Set chimaera containing the MSV-Kom inverted-repeat sequence, pSRAK, was
substantially less infectious and produced much milder
symptoms in maize than did MSV-Set. pSRAK only
induced symptoms on the second and third leaves of
infected plants, with all symptomatic plants apparently
recovering by the fourth-leaf stage. These results indicated
that, whilst the inverted repeat does not contain a primary
RSD that would prevent MSV-Kom from trans-replicating
MSV-Set efficiently, the region might contain the primary
RSD that prevented MSV-Set from trans-replicating MSVKom efficiently.
Based on the replication-specificity results above, we constructed a series of MSV-Kom–MSV-Set chimaeras to
identify the region(s) within the MSV-Kom LIR sequence
responsible for its presumably inefficient interaction with
the MSV-Set Rep. The infectivity of another MSV-Kom–
MSV-Set LIR chimera, pSekA.2, has been described
However, when 67 nt of MSV-Kom between 2120 and
253 nt 59 of the V-ori were used to replace the corresponding pSRAK sequence (which differs from that of MSV
Kom at 51 of 87 sites; Fig. 3), the resulting chimaera,
pSSAK, was as infectious as, and produced only slightly
milder symptoms in maize than, MSV-Set. This indicates
An additional, if not more obvious, factor that may have
contributed to PanSV-Kar trans-replicating MSV-Kom
better than MSV-Set is that the latter virus has tracts of
highly divergent sequence within its LIR. For example, the
MSV-Kom LIR sequence around the RPI from 2102 to
278 nt 59 of the V-ori resembles that of PanSV-Kar significantly more than that of MSV-Set (Fig. 3). Conversely,
the inverted-repeat sequences of the MSV-Kom and MSVSet potential stem–loops are much more similar to one
another than they are to that of PanSV-Kar. This similarity
extends to the MSV-Kom iteron sequences in the inverted
repeat, which have a 4 nt overlap with those of MSV-Set,
but none with those of PanSV-Kar. This suggested to us
that the RSD of MSV-Kom, which is more compatible with
that of PanSV-Kar than that of MSV-Set, probably resides
within sequences at, or around, the RPI and not in the
iteron sequences in the stem–loop.
Fig. 5. Relative infectivities and symptom severities of MSV-Kom, MSV-Set and various viruses with chimaeric MSV-Kom/
MSV-Set LIR sequences. In the cartoon of chimaeric LIR sequences given to the right of the chimaera names, regions in black
and white are MSV-Kom- and MSV-Set-derived sequences, respectively. The inverted repeat (stem–loop) is represented by a
triangle. Nucleotide co-ordinates are relative to the V-ori (+1) and refer to the co-ordinates of the enzyme sites used to make
the chimaeric exchanges between the two genomes. For the histograms, the left panel indicates the mean percentage of maize
plants that developed symptoms in between three and six agroinoculation experiments involving different chimaeras and wt
viruses. The right panel represents the mean chlorotic leaf areas observed on leaves 3–6 of plants that developed symptoms.
Error bars represent SD.
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1837
J. A. Willment and others
that the attenuation in symptoms observed in pSSRK,
rather than being due to replacement of a primary RSD, is
probably caused by disruption of a sequence-specific interaction between an element in the MSV-Set inverted repeat
and this 67 nt 59 region. The possibility of an interaction
between the region 2120 to 253 nt 59 of the V-ori and the
inverted repeat is supported by the observation that
pSSRK, the predominantly MSV-Set chimaera containing
the MSV-Kom 67 nt 59 region and the MSV-Set inverted
repeat, produces symptoms almost as mild as those
produced by pSRAK (Fig. 5).
fragments, whereas the wt genomes remain uncut (1306 nt
for MSV-Kom and 1321 nt for MSV-Set; expected PCR–
RFLP fragments for all wt and chimaeric virus genomes are
shown in Table 1). With CfoI, the PCR product of wt MSVSet is digested into 11, 25, 601 and 685 nt fragments and wt
MSV-Kom into 2, 16, 37, 80, 319 and 853 nt fragments (in
both cases, only the larger two fragments are resolvable on
a 1.5 % agarose gel). Chimaeric genomes contain mixtures
of MSV-Kom and MSV-Set and all have different restriction patterns from those of the wt viruses. For example, the
predominantly MSV-Set chimaera SSAK, having gained
four extra CfoI sites due to the portion of MSV-Kom that it
contains, is digested into fragments of 2, 11, 16, 25, 80, 216,
357 and 589 nt (only the 216, 357 and 589 nt bands are
visible in Fig. 6). Therefore, fragments that are seen in
addition to the wt fragments are indicative of transreplication of the self-replication-incompetent chimaeric
genomes.
Whilst the agroinfection experiments demonstrated the
viability of various LIR chimaeras, they did not reliably
indicate the location of the RSD that prevented the MSVSet Rep from trans-replicating MSV-Kom efficiently. To
assay more directly the capacity of Reps from MSV-Set and
MSV-Kom to initiate and sustain replication from chimaeric LIR sequences, we developed a competitive transient
trans-replication assay. Self-replication-incompetent PstI
and NsiI mutants of the various chimaeric LIR sequences
were co-bombarded with pMSV-Kom or pMSV-Set and
evidence of their trans-replication was assayed by RFLP
coupled with Southern and RF-specific PCR analyses. The
rationale behind this approach was that, unless the LIR
sequences of the replication-incompetent viruses contain
the appropriate cis-acting replication-specificity elements,
they should be out-competed for Rep binding by the wt
LIR sequences of the replication-competent helper virus.
Within the 4 day time period of the experiment, one would
expect that only chimaeras with appropriate RSDs should
have been trans-replicated to levels approaching that of the
wt helper virus.
Neither pKomPstI nor pKRASPstI (the predominantly
MSV-Kom chimaera containing the MSV-Set invertedrepeat sequence) competed successfully with pMSV-Set for
the MSV-Set Rep (Fig. 6). pKNRSNsiI and pKNASNsiI,
which are also predominantly MSV-Kom chimaeras containing the MSV-Set 77 nt surrounding the RPI between
2130 and 253 nt 59 of the V-ori, did compete successfully
with pMSV-Set for the MSV-Set Rep. Conversely, whilst
pSetNsiI and pSRAKNsiI were unable to compete with
MSV-Kom for the MSV-Kom Rep, all predominantly
MSV-Set chimaeras containing the MSV-Kom 67 nt
surrounding the RPI between 2120 and 253 nt 59 of the
V-ori (pSSBKNsiI, pSSRKNsiI and pSSAKNsiI) did compete successfully with MSV-Kom for its Rep (Fig. 6).
With PstI, the PCR products of the self-replicationincompetent genomes are digested into 712 and 596 nt
These results show clearly that the incompatible RSD
impeding MSV-Set trans-replication of MSV-Kom resides
Table 1. trans-Replication of self-replication-incompetent chimaeric virus genomes by wt
MSV-Kom and MSV-Set: expected PCR–RFLP fragment sizes of replicated genomes
See Fig. 6 for trans-replication results.
NA,
Construct
Kom
KomPstI
Set
KRASPstI
KNRSNsiI
KNASNsiI
SetNsiI
SRAKNsiI
SSAKNsiI
SSRKNsiI
SSBKNsiI
Not applicable.
Expected fragment sizes if replicated (bp)
Full-length PCR
products
PstI digestion
1306
1308
1321
1308
1307
1307
1309
1309
1296
1296
1294
1306
712, 596
1321
712, 596
NA
NA
NA
NA
NA
NA
NA
CfoI digestion*
2, 16, 37, 80, 319, 853
NA
11, 25, 601, 685
NA
2, 11, 16, 37, 309, 318, 614
11, 318, 364, 614
11, 25, 589, 684
2, 11, 16, 25, 309, 357, 589
2, 11, 16, 25, 80, 216, 357, 589
11, 25, 216, 455, 589
2, 11, 16, 25, 37, 80, 216, 318, 589
*Fragment sizes in bold are those that should be resolvable after electrophoresis through a 1.5 % agarose gel.
1838
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Journal of General Virology 88
MSV sequences involved in replication
Fig. 6. trans-Replication of self-replication-incompetent chimaeric virus genomes by wt MSV-Kom and MSV-Set. Chimaeric
LIR sequences are as described in Fig. 5; for chimaeric constructs not shown in Fig. 5, co-ordinates of the enzyme sites used to
make the chimaeric exchanges are shown. All chimaeras were co-bombarded with either pMSV-Kom or pMSV-Set and transreplication was identified by RF-specific PCR–RFLP analysis. +, RF-DNA detected for both the wt and self-replicationincompetent genome; ”, only wt RF-DNA detected; *, wt RF-DNA indistinguishable from that of the co-bombarded selfreplication-incompetent genome; l, molecular mass marker. (a) PstI (lanes 2–9)- and CfoI (lanes 10–17)-digested PCR
products amplified following co-bombardment of cells with wt viruses and predominantly MSV-Kom self-replicationincompetent genomes. (b) CfoI-digested PCR products amplified following co-bombardment of cells with wt viruses and
predominantly MSV-Set self-replication-incompetent genomes. Refer to Table 1 for expected fragment sizes of undigested and
digested PCR products, illustrating how replication of the co-bombarded wt and self-replication-incompetent chimaeric virus
genomes is distinguished after PstI or CfoI digestion. Mixtures of the expected banding patterns indicate the presence of both
wt and self-replication-incompetent RF-DNAs and hence evidence of trans-replication. Some fragments listed in Table 1 are too
small to be detected or cannot be distinguished, if similar in size to fragments of the co-bombarded virus genome.
within this 67 nt region of MSV-Kom. They also indicate
that an analogous primary RSD resides within a 77 nt
region containing the RPI, between 2130 and 253 nt 59 of
the MSV-Set V-ori. Whilst it is possible that the RPI is
the RSD, it is also possible that any combination of the
RPI, sequences surrounding the RPI, and the spatial
arrangement of the RPI or other sequences in this region
in relation to the inverted repeat, constitute the RSD.
Conclusions
In this study, we have identified two major differences
between MSV and begomovirus virion-strand replication
origins. First, like WDV (Sanz-Burgos & Gutierrez, 1998)
but unlike begomoviruses (Orozco et al., 1998), MSV
replication involves sequences 39 of the inverted repeat
(Fig. 3). Despite WDV and MSV sharing lower nucleotide
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sequence identity than do begomoviruses, topocuviruses
and curtoviruses to one another, the extent of the LIR
sequence required for replication in these species is
approximately the same and is possibly conserved in all
mastreviruses.
The second and most striking difference between the MSV
and begomovirus V-oris that we have identified in this
study is that RSDs in MSV are far more flexible than they
are in begomoviruses. Whilst complementation of replication functions is generally only possible between begomoviruses sharing ¢80 % Rep amino acid sequence identity
(Lazarowitz et al., 1992; Gilbertson et al., 1993; Frischmuth
et al., 1997; Hill et al., 1998; Saunders et al., 2002; Ramos
et al., 2003), we have demonstrated reasonably efficient
trans-replication of mastreviruses with Reps sharing only
65 % amino acid sequence identity.
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1839
J. A. Willment and others
Despite these differences, we have established clearly that
the cis-elements required for MSV replication show greater
architectural similarity to those of the other geminivirus
genera than was expected previously (Argüello-Astorga
et al., 1994a, b). Whilst our discovery of a sequence-specific
interaction between the RPI and inverted-repeat sequences
of MSV is not entirely surprising, contrary to expectations,
we have also shown that sequences surrounding the RPI,
rather than the V-ori inverted repeat, constitute the
primary RSD of MSV – a situation that is possibly the
same for other mastreviruses.
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ACKNOWLEDGEMENTS
This work was supported by the South African National Research
Foundation. D. N. S. is supported by the Claude Leon Foundation.
D. P. M. is supported by the Sydney Brenner Fellowship and the
Harry Oppenheimer Trust.
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