1 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa
2 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
Correspondence
Edward P. Rybicki
ed{at}science.uct.ac.za
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ABSTRACT |
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A figure and table showing interactions between MSV Rep and RepA protein variants and the maize RBR protein (ZmRb1) are available as supplementary material in JGV Online.
Present address: Division of Pathology and Neuroscience, University of Dundee Medical School, Dundee DD1 9SY, UK.
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INTRODUCTION |
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There is substantial evidence, mainly from work with WDV, that RepA is a multifunctional protein with unique features that are important at different stages during the replicative cycle (Gutierrez, 1999). These potentially include downregulation of replication (Collin et al., 1996
) and interactions with cellular retinoblastoma-related protein (pRBR) (Horvath et al., 1998
; Xie et al., 1995
) and a group of host NAC domain-containing GRAB (geminivirus RepA-binding) proteins (Xie et al., 1999
), which may influence host developmental pathways for the benefit of viral processes (Gutierrez, 2000
; Gutierrez et al., 2004
). Properties common to Rep and RepA include sequence-specific DNA binding (Castellano et al., 1999
; Missich et al., 2000
), hetero- and homo-oligomerization (Horvath et al., 1998
) and transactivation of virion (V)-sense gene promoters (Collin et al., 1996
; Hofer et al., 1992
; McGivern, 2002
; Zhan et al., 1993
).
Rep and RepA proteins contain a conserved amino acid sequence (LxCxE) that is common among oncoproteins of tumour-inducing viruses (Ludlow, 1993; Moran, 1994
; Vousden, 1993
). The LxCxE motif is also present in mammalian (Dowdy et al., 1993
; Ewen et al., 1993
) and plant (Dahl et al., 1995
; Nakagami et al., 1999
; Soni et al., 1995
) D-type cyclins, in the nanovirus protein Clink (Aronson et al., 2000
) and in some RNA viruses (Forng & Atreya, 1999
; Oruetxebarria et al., 2002
), and mediates binding to both the mammalian retinoblastoma protein (pRB) and plant pRBR (reviewed by de Jager & Murray, 1999
). However, in the mastreviruses MSV, BeYDV and WDV, only RepA, and not Rep, interacts with pRBR (Gutierrez et al., 2004
; Horvath et al., 1998
; Liu et al., 1999
).
Mastrevirus replication is dependent on cellular replication factors that are generally absent or not functional in non-dividing cells. However, MSV replication has been detected in terminally differentiated cells (Lucy et al., 1996). The purpose of the LxCxE motif in the mastrevirus RepA may therefore be to induce a cellular S phase-like state that is permissive for viral DNA replication, by interaction with pRBR and subsequent activation of S phase-specific gene transcription.
Mutational analysis of the RepA LxCxE motif in WDV and BeYDV has shown the importance of the three conserved residues in mediating binding to pRBR. A mutation of E to K abolished the ability of WDV RepA to bind to pRB (Xie et al., 1995), whilst Liu et al. (1999)
confirmed the importance of all three conserved residues in BeYDV RepA and showed that an E to Q mutation reduced binding efficiency by 95 %. In the present study, the effects of a two amino acid substitution (LLCNE to LLCLK) in the MSV RepA pRBR-interaction motif on both virus replication in maize culture cells and infectivity in maize were determined.
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METHODS |
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PCR site-directed mutagenesis.
An MSV-Kom Rep gene containing mutations altering the pRBR-interaction domain (described below) was provided by Dr T. Mangwende (University of Cape Town, South Africa). A 3 nt mutation resulting in amino acid changes of LLCNE to LLCLK was introduced into pSKRep by PCR site-directed mutagenesis, using the mutagenic primer set C1N201LE202K and C1RbRev (Table 1), to produce pSKRepRb.
Construction of plant expression cassettes.
A 1·2 kb BamHI/BglII fragment (containing the full-length Rep gene) from pSKRep, pSKRepRb and pSKRepRbC(601)A (see below) was inserted into the BamHI site of pAHC17 (Christensen & Quail, 1996) downstream from the maize ubiquitin promoter. The resulting plasmids were designated pRep, pRepRb and pRepRbC(601)A, respectively.
Construction of RepA genes.
An unspliceable Rep gene of MSV-NG1 (formerly MSV-N; Fauquet & Stanley, 2003) cloned in pMB1657, containing a 3' splice site mutation of A733G734 to T733C734 (Wright et al., 1997
), was used to construct MSV-Kom RepA genes. A fragment containing the splice-site mutation was excised from pMB1657 with XhoI and BglII and used to replace the XhoI/BglII fragment of pKom602, to produce pKomMB1657. The MSV-NG1 sequence remaining in pKomMB1657 is identical to that of MSV-Kom. The RepA gene was amplified from pKomMB1657 using the primer set C1 (F) and RepABglII (R) (see Table 1
) and inserted into the BamHI site of pSK to give pSKRepA. RepA versions of Rb mutants were made similarly, using pKomRb and pKomRbC(601)A (described below) as starting templates. The RepA plasmids, each containing the 3' splice-site mutation, were called pSKRepARb and pSKRepARbC(601)A, respectively.
Construction of an intronless Rep gene.
pSKRep was used as a template to create an intronless Rep gene by inverse PCR, with the forward primer RepI (F) and the reverse primer Rep
I (R), which bind adjacent to the 3' and 5' ends of the intron, respectively. The whole template plasmid was amplified minus the intron, in such a way as to allow in-frame ligation. Primer sequences are given in Table 1
. The PCR product was self-ligated to create the plasmid pSKRep
I.
Plasmid construction for yeast two-hybrid analysis.
The RepA genes from pSKRepA, pSKRepARb and pSKRepARbC(601)A and the intronless Rep gene from pSKRepI were PCR-amplified by using primer sets ADSalIC1 (F) (forward primer for all Rep genes), ADBglIIRepA (R) (reverse primer for RepA genes) and ADBglIIC2 (R) (reverse primer for the Rep
I gene; Table 1
) and inserted into pGAD424 (Clontech) in-frame with the GAL4 activation domain (AD), to create the RepA or Rep
IGAL4 AD fusion plasmids pADRepA, pADRepARb, pADRepARbC(601)A and pADRep
I, respectively. Plasmid GBT9ZmRb1 encodes the GAL4 binding domain (BD) fused to the maize RBR protein ZmRb1 and was provided by Dr G. Horvath (Horvath et al., 1998
).
Yeast two-hybrid analysis.
Interaction of MSV RepA and Rep proteins with ZmRb1 was assessed by using the Matchmaker yeast two-hybrid system (Clontech). Plasmids expressing GAL4 BD fusions (pGBT9; trp1 transformation marker) and GAL4 AD fusions (pGAD424; leu2 transformation marker) were used to transform Saccharomyces cerevisiae strains CG1945 and Y187, respectively, as described by Gietz & Woods (1994). The transformation mixture was plated onto yeast drop-out selection media lacking the appropriate amino acid to select for transformants. Yeast strains CG1945 (MATa; containing pGBT9-based plasmids) and Y187 (MAT
; containing pGAD424-based plasmids) were mated according to a protocol modified from the Clontech Yeast Protocols handbook. The diploid yeasts were grown on synthetic drop-out medium lacking tryptophan (Trp) and leucine (Leu), to select for both plasmids, and also on drop-out medium lacking Trp, Leu and histidine (His) and containing 5 mM 3-amino-1,2,4-triazole (3-AT). Only cells containing interacting fusion proteins can grow on the latter medium.
Transient-replication assays.
Replication of MSV replicons in Black Mexican sweet (BMS) suspension culture cells was assayed as described previously (Palmer et al., 1999). In each experiment (with a minimum of two replicates), up to nine plates of BMS were bombarded with gold microprojectiles by using a Bio-Rad PDS-1000/He particle gun. Particles carried the following plasmid combinations: pKom602+pRep or pRepRb; pMSV-PstI (replication-incompetent MSV)+pRep, pRepRb or pRepRbC(601)A; and pKom602, pKomRb or pKomRbC(601)A without any Rep gene provided in trans. For the co-bombardments, up to nine plates of BMS were also co-bombarded with pKom602+pAHC17 or pMSV-PstI+pAHC17, with the latter as a non-Rep co-bombardment control. Four days after bombardment, total DNA was extracted from BMS cells by using the method of Dellaporta et al. (1983)
, including a step where DNA resuspended in 50 mM Tris/HCl, 10 mM EDTA (pH 8) had 600 µg RNaseA ml1 added and the mixture was incubated for 1 h at 37 °C. After a second precipitation with 2-propanol, DNA was resuspended in water and quantified on a 0·8 % agarose gel by measuring band intensities densitometrically from digital images (GelTrak; Dennis Maeder, Center of Marine Biotechnology, MD, USA). Each sample was diluted to 50 ng µl1 and again quantified on a 0·8 % agarose gel to ensure equal loading. Viral DNA replication was assayed by using a quantitative PCR (QPCR)-based technique (Fig. 1
), using 100 ng total DNA as input. The amplification reaction used the primer set 215234 and 17701792 (Willment et al., 2001
; Table 1
), which amplifies only replicationally released circular genomic DNA, but not linear viral DNA, from pKom602-derived input plasmids. To confirm this, DNA extracted immediately after bombardment (as a control for input DNA amplification) was subjected to the same PCR. Each PCR was spiked with pMSV-PstI as an internal control: this competes in amplification reactions with replicated (circular) viral DNA for primers and other PCR components. Twenty-five amplification cycles were used, as amplification was still exponential at cycle 25 in preliminary assays. PstI digestion of amplified pMSV-PstI yields products of 558 and 747 bp, allowing the competitor to be distinguished from amplified replicated viral DNA (Fig. 2a
). The relative concentration of replicated viral DNA, expressed as pg viral DNA per 100 ng total DNA, was calculated by determining the ratio of the replicated virus band intensity to those of the two pMSV-PstI competitor bands by using GelTrak. From individual replicate data obtained by using QPCR, a mean amount (in pg) of replicated virus in the presence and absence of each Rep gene derivative was calculated.
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Agroinoculation and symptom analysis.
Maize seedlings (Zea mays L. cv. Jubilee) were inoculated with agroinfectious constructs as described previously (Martin et al., 1999). In each of three replicates, groups of 14 3-day-old maize seedlings were agroinoculated with pBIKom602, pBIKomRb or pBIKomRbC(601)A, and 14 control plants were injected with sterile distilled water, for a total of 42 plants agroinoculated with each MSV construct. Chlorotic leaf areas (%) were measured for leaves 2, 3, 5 and 6 of each symptomatic plant by using a microcomputer-based image-analysis technique (Martin & Rybicki, 1998
). Leaves 2 and 3 were assessed 15 days post-agroinoculation (p.i.); leaves 5 and 6 were assessed 29 and 35 days p.i., respectively. For each MSV construct, the mean percentage chlorotic area on leaves 2, 3, 5 and 6 was used as a representative measure of chlorosis.
Cloning and sequencing of MSV DNA from infected plants.
Total DNA was extracted from infected maize plants; Rep genes were PCR-amplified from each sample by using the primer set C1 (F) and C2 (R) (Table 1), inserted into the pGEM-T Easy vector (Promega) and both strands were sequenced.
Analysis of the C(601)A reversion in pKomRb.
For maize seedlings agroinoculated with pBIKomRb, a sample was taken from the leaf tip as it emerged from the whorl, then the leaf was sampled progressively downwards towards the base on or around every 10 days and total DNA was extracted. Sequence-specific forward primers were designed [RbA (F) and RbC (F); see Table 1] to differentially amplify Rep genes containing the A(601) or C(601) nucleotide. The reverse primer was the C2 (R) primer (Table 1
). The plasmids pSKRepRb and pSKRepRbC(601)A were included in each PCR as controls: the former was amplified by the RbC (F) primer, but not by the RepRbA (F) primer; the latter was amplified by the RepRbA (F) primer only.
Analysis of the transition/transversion ratio in MSV.
Twenty-two full-length genomic MSV sequences, including maize-type and grass-type isolates from strains MSV-A, B, C, D and E (Martin et al., 2001), were aligned by using DNAMAN. The rate of each type of substitution relative to G to T was estimated by using PAUP* 4.0 (Swofford, 2001
).
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RESULTS |
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The Rb mutation decreases symptom severity
The relative severities of MSV-Kom and MSV-KomRb infections were established by symptom assessment after agroinoculation of the MSV-sensitive maize cv. Jubilee. MSV-KomRb systemically infected maize, although symptom severity on each of leaves 26 was clearly lower (Fig. 3) and plants were less stunted than those infected with wt virus. However, the timing of symptom development and the percentages of agroinoculated plants that became infected were the same in both mutant and wt infections. The mutant virus was also transmissible to maize by Cicadulina mbila leafhoppers (data not shown).
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The Rep C(601)A reversion provides a selective advantage other than improved pRBR-binding affinity
Whilst it is possible that position 601 of the MSV-Kom Rep (or RepA) gene is a mutational hotspot, it is unlikely that the C(601)A reversion could be maintained without strong selective pressure. It has been noted that transitions (AG and C
T) occur at higher frequencies than transversions (A
T, A
C, G
T and C
G) in virtually all DNA sequences examined from any genome (Yang & Yoder, 1999
). MSV is no exception: our analysis of the relative transition/transversion ratios in MSV (the genome of which has equal base frequencies) revealed a mutational bias in favour of transitions (A
G=1·35; C
T=2·09; A
T=0·99; A
C=0·74; C
G=0·69; relative to G
T=1·0). As a C to A transversion is the second least likely substitution to occur in MSV, it must provide a strong selective advantage to be fixed in preference to the more frequently occurring substitutions.
The first option investigated for possible selection pressure was whether the C(601)A reversion increased the binding affinity of the mutant RepA to pRBR. This was determined by using a yeast two-hybrid assay. Whilst wt RepA interacted with ZmRb1, RepARbL201I,E202K did not (Table 2). Therefore, it appears that the loss of pRBR-interaction ability in RepRbN201L,E202K was not detectably restored by the C(601)A reversion.
The effect of the C(601)A reversion on virus replication was analysed by bombardment of BMS with pKomRbC(601)A and co-bombardment with pMSV-PstI+pRepRbC(601)A. As can be seen in Fig. 2(b), replication levels of MSV-Kom, MSV-KomRb and MSV-KomRbC(601)A were indistinguishable from one another. Similarly, although RepRbL201I,E202K apparently replicated MSV-PstI to slightly higher levels than did either Rep or RepRbN201L,E202K, these differences were not significant (P>0·05).
Finally, MSV-Kom, MSV-KomRb and MSV-KomRbC(601)A infectivities were compared by agroinoculation of maize and analysis of symptom severities. The symptoms induced by MSV-KomRb and MSV-KomRbC(601)A were not significantly different at any point in the infection (Student's t-test; Fig. 3). As A(601) in the Rep gene of MSV-KomRbC(601)A did not obviously increase its virulence relative to that of MSV-KomRb, the selective pressure for the reversion did not appear to be increased pathogenicity. However, as the C(601)A reversion in MSV-KomRb occurs so rapidly (the earliest that infected leaf samples were analysed by PCR was on day 10 p.i., therefore the reversion probably appears earlier), it is impossible to separate the symptom-severity profiles of the mutant and C(601)A revertant viruses. It is possible that, by the time symptoms were analysed, the disadvantage of the C(601) mutation had been overcome and the mutant had effectively become the C(601)A revertant. This is further corroborated by the fact that, in some plants (see Fig. 4
), the reversion was detected before symptoms were even detectable. It can, therefore, only be concluded that the C(601)A reversion did not increase symptom severity to wt levels, which is not surprising, as MSV-KomRbC(601)A and MSV-KomRb shared an inability to interact with maize pRBR.
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DISCUSSION |
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The difference in apparent fitness of BeYDV and MSV mutants relative to wt viruses may be attributable to differential requirements for pRBR interaction in viruses infecting dicots (BeYDV) and monocots (MSV). It has been suggested that BeYDV, when adapting to replicate in dicots, may have gained a begomovirus-type interaction between Rep and pRBR that does not require an LxCxE motif (Gutierrez, 2000). However, our results indicate that a monocot-infecting mastrevirus can also replicate and establish a systemic infection (albeit attenuated) in the absence of RepApRBR interaction. Alternatively, the difference would be explained if BeYDV, unlike MSV, is phloem-limited, as suggested by Hanley-Bowdoin et al. (2004)
and McGivern et al. (2005)
.
Assuming that MSV infectivity depends on V-sense promoter activation by Rep/RepA, it appears that MSV RepApRBR interaction is not required for activation of the MSV V-sense promoter. Although Muñoz-Martín et al. (2003) found that WDV RepA-mediated activation of the MSV V-sense promoter depended on an intact pRBR-interaction domain, they did propose that MSV RepA could potentially activate the MSV V-sense promoter by a pRBR-independent route, as the present study suggests. However, whilst the lack of yeast growth in yeast two-hybrid studies has been interpreted as a lack of interaction between Rb mutant RepAs and pRBR, the possibility exists that an alternative binding domain in the mutant RepAs allows the binding of pRBR with a very low affinity in plants, which was undetectable in our assay. TGMV Rep (AL1) does not have an LxCxE motif and interacts with pRBR through another sequence, designated helix 4. A similar structure is also predicted in MSV and BeYDV RepA (Arguello-Astorga et al., 2004
; Kong et al., 2000
).
It is possible that the attenuation of infection symptoms induced by MSV-KomRb relative to wt virus was due to an abolition or dramatic reduction of pRBR binding by the mutant RepA. The pattern of chlorotic streaks on maize leaves is correlated directly with the pattern of virus accumulation (Lucy et al., 1996). As MSV-KomRb-infected plants developed slightly narrower streaks than those infected by wt virus, the mutation may have lowered virus replication efficiency in terminally differentiated cells or altered the tissue specificity of the virus. For example, it might only replicate in vascular tissue, using pre-existing host replication factors that are present in some phloem-associated cells, as suggested for TGMV by Hanley-Bowdoin et al. (2000)
. This suggestion is supported by the data of McGivern et al. (2005)
, using an MSV RepA mutant that is also unable to bind pRBR. These factors limiting virus replication would not be expected to have an effect in the replication assays used in this study [and in those of Xie et al. (1995)
and Liu et al. (1999)
], which employed the use of actively dividing cell cultures that were already competent for host DNA replication.
Taking into account the high-frequency occurrence of a reversion of C(601)A in the mutated RepA gene, which had no detectable effect on pRBR-binding efficiency, the 3 nt Rb mutation may have had effects separate from pRBR binding. Considering the small size of the MSV genome, it would not be surprising for any particular stretch of sequence to have more than one function in the viral life cycle. The fact that the Rb mutation and the C(601)A reversion had no effect on virus replication in BMS may indicate that it did not affect the functions of Rep, but rather those of RepA. A single nucleotide change could affect splicing of the complementary-sense transcripts, thereby altering the ratio of Rep to RepA. Exonic sequences upstream of the Rep intron could affect splicing efficiency, either by influencing RNA secondary structure or by comprising splicing-enhancer sequences (Manley & Tacke, 1996). In turn, RepA expression is determined by how efficiently the transcript is spliced. Increased splicing efficiency of the Rep transcript could result in lower expression of coat protein (CP), due to both lower RepA-mediated activation and increased Rep-mediated repression (Muñoz-Martín et al., 2003
) of the CP promoter. Thus, decreasing splicing efficiency to wt levels could be the selective pressure for the C(601)A reversion. Alternatively, the MSV genome secondary structure may have inherent biological properties, perhaps influencing virus location or the mechanisms by which the genome is replicated, transported or encapsidated. For example, single-stranded DNA (ssDNA)capsid interactions may be dependent on ssDNA secondary structure. Preliminary experimental results indicate that the predicted structures of wt and C(601)A revertant Rep transcripts (which may be similar to Rep DNA structure) are more stable and thermodynamically favourable over the C(601) Rb mutant structure, providing a possible reason for the C(601)A reversion (data not shown).
Interestingly, a single nucleotide mutation in MSV-NG1, equivalent to one [G(604)A] of the three mutations in MSV-KomRb, which alters the LLCNE motif to LLCNK, does not revert (McGivern et al., 2005). The fact that reversion to a wt motif does not occur, despite the favoured status of A to G transitions in MSV, reinforces the proposal that the selective pressure for the C(601)A reversion in MSV-KomRb may be operating at the nucleic acid (structure) level, rather than at the level of Rep and RepA expression or function.
In conclusion, we have demonstrated that an intact MSV Rep pRBR-interaction motif is not required for virus replication in culture cells or infectivity in maize, although it is possibly required for wt symptom development. As Liu et al. (1999) reported similar results for BeYDV, it appears that WDV is exceptional amongst mastreviruses in that it requires a consensus pRBR-interaction motif, even in wheat suspension cells, where host replication factors should be abundant. The high-frequency single-nucleotide reversion in the DNA encoding the MSV pRBR-interaction motif may shed some light on the reason that the apparently non-essential LxCxE motif has been retained in mastreviruses, whilst it has been lost in begomoviruses. The consistent emergence and eventual dominance and easy detection of the C(601)A revertant viral population can be used as a system to investigate aspects of MSV biology, such as transcript or ssDNA secondary-structure constraints, movement models, replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover.
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ACKNOWLEDGEMENTS |
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Received 14 October 2004;
accepted 6 December 2004.