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Journal of Virology, July 2005, p. 8422-8430, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8422-8430.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta{dagger}

Julio C. Vega-Arreguín, Tatiana Timchenko, Bruno Gronenborn,* and Bertha Cecilia Ramírez*

Institut des Sciences du Végétal, CNRS, 91198 Gif sur Yvette Cedex, France

Received 19 November 2004/ Accepted 6 March 2005


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ABSTRACT
 
Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.


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INTRODUCTION
 
Nanoviruses are single-stranded DNA (ssDNA) plant viruses (41). Their multipartite genome consists of at least six to eight circular individually encapsidated ssDNA molecules of ca. 1 kb, with one virion sense open reading frame (ORF) each (5, 7, 23, 35). Nanovirus DNA components and their respective encoded proteins of known function comprise DNA-R encoding the master Rep (M-Rep) protein (39), DNA-S encoding the capsid protein (43), DNA-C encoding the cell cycle link protein Clink (4), DNA-M encoding a movement protein, and DNA-N encoding a nuclear shuttle protein (42). The functions of at least three more proteins, all encoded by additional individual genome components, remain unknown (41).

Nanoviruses multiply in the nucleus of infected cells by rolling-circle replication (RCR). Upon infection of a host cell, short DNA molecules encapsidated together with the viral ssDNA serve as primers for host polymerase(s) to initiate synthesis of the complementary (minus) strand DNA, creating a double strand (16). The double strand serves as transcription template and for RCR, which is initiated and terminated by replication initiator (Rep) proteins. Nanoviruses encode different Rep proteins; however, only the 33-kDa M-Rep protein is required and sufficient to catalyze replication initiation of its coding DNA and of the other virus genome components (22, 39, 40). Faba bean necrotic yellows virus (FBNYV) M-Rep, expressed in Escherichia coli, has origin-specific DNA cleavage and nucleotidyl transfer activities in vitro and is an ATPase, both essential functions for viral DNA replication in vivo (39). During RCR the M-Rep protein cleaves the consensus nonamer sequence TAGTATT{downarrow}AC located at the origin of replication (39), creating a 3'-OH terminus and, by analogy to geminivirus replication, is thought to prime viral (plus) strand DNA synthesis (26).

For geminivirus Rep proteins, interactions with several host proteins have been described (1, 25). All these different proteins interacting with Rep were identified by using the yeast two-hybrid system. However, due to difficulties encountered in purifying the respective protein complexes, very little is known about the in planta interaction of these proteins. For nanoviruses, nothing is known about the host proteins that interact with M-Rep during replication.

We have designed oligohistidine-tagged M-Rep variants of the nanovirus FBNYV that are proficient to catalyze viral DNA replication initiation and termination in Nicotiana benthamiana and report the affinity purification from plant tissue of enzymatically active histidine-tagged M-Rep protein. To the best of our knowledge, this is the first example of a tagged ssDNA virus replication initiator protein that is functional in vivo and that is readily purified from plant tissue. In addition, replicons encoding oligohistidine-tagged M-Rep multiplied and moved systemically along with wild-type FBNYV in its natural host Vicia faba. The replicon encoding the modified M-Rep protein could be transmitted by aphids in a mixed infection with wild-type virus. Finally, we show the in planta interaction of the tagged M-Rep with wild-type M-Rep, suggesting that this protein may be used to identify other protein partners.


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MATERIALS AND METHODS
 
Construction of DNA-R-His replicons of FBNYV. Basic molecular biology protocols were as described previously (34). Cloning of FBNYV DNAs of the Egyptian isolate EVI-93 (FBNYV-EG) has also been described elsewhere (39). A BamHI site was introduced by PCR-based QuikChange mutagenesis (Stratagene) at nucleotide position 54 into the noncoding sequence of FBNYV-EG DNA-R (previously designated C2) (23, 39) by using the primers C2BamHI(+) (5'-GAGTCATCACGTGGATCCCACGTGATATTAG-3') and C2BamHI(–) (5'-CTAATATCACGTGGGATCCACGTGATGACTC-3'). The DNA-R thus modified was subsequently inserted into the BamHI site of pBluescript II KS(+) (Stratagene). This plasmid (pBKSC2-B+) was used to construct DNA-R derivatives (replicons) encoding the oligohistidine-tagged M-Rep proteins. An NheI site was introduced by site-directed mutagenesis at nucleotide position 124 of DNA-R in plasmid pBKSC2-B+ by using the primers C2NheI(+) (5'-GAATAAAATATGGCTAGCCAAGTTATATGC-3') and C2NheI(–) (5'-GCATATAACTTGGCTAGCCATATTTTATTC-3'). Complementary oligonucleotides coding for the His6 tag flanked by an NheI site, 6His-NheI(+) (5'-CTAGCCATCACCATCACCATCACG-3') and 6His-NheI(–) (5'-CTAGCGTGATGGTGATGGTGATGG-3'), were annealed and inserted into the previously generated NheI site of DNA-R, yielding replicon R-6H-S encoding protein 6H-MRep-S (Fig. 1).



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  		FIG. 1. DNA-R-His replicons. The designations of the replicons, their respective encoded proteins, and the N- and C-terminal amino acid sequences of the corresponding proteins (wild-type and oligohistidine-tagged M-Rep) are shown. Amino acid changes other than the oligohistidine tag, indicated inside a rectangle, are in italics and underlined or represented by a dash.

The wild-type sequence at the N terminus of 6H-MRep-S was restored by reintroduction of methionine 1 (wild-type amino acid sequence) and substitution of serine 3 by arginine, leading to replicon R-6H-M encoding protein 6H-MRep-M (Fig. 1). The primers used for site-directed mutagenesis of R-6H-S to create these amino acid changes were 6HisM-Rep2(+) (5'-CATCACCATCACCATCACATGGCTCGGCAAGTTATATGCTGGTGC-3') and 6HisM-Rep2(–) (5'-GCACCAGCATATAACTTGCCGAGCCATGTGATGGTGATGGTGATG-3').

Replicon 6H-MRep-A was generated by substitution of the methionine of 6H-MRep-M by alanine (Fig. 1) by using primers 6HisA-Rep2(+) (5'-CATCACCATCACGCGGCTCGGCAAGTT-3') and 6HisA-Rep2(–) (5'-AACTTGCCGAGCCATGTGATGGTGATG-3'). The replicon containing only four histidines (4H-MRep-A) was obtained fortuitously in the same mutagenesis experiment (Fig. 1).

To generate the replicon R-6H-Ter encoding a M-Rep protein with a His6 tag at its C terminus (MRep-6H-Ter), an NheI site was introduced by site-directed mutagenesis at nucleotide position 970 of DNA-R by using the primers C2Nhe970(+) (5'-GATAGGATTGTCTATGCTAGCGCGTGACGTCATGT-3') and C2Nhe970(–) (5'-ACATGACGTCACGCGCTAGCATAGACAATCCTATC-3'), and the annealed oligonucleotides 6His-NheI(+) and 6His-NheI(–) (see above) were inserted into this NheI site (Fig. 1).

The correctness of all of the DNA-R modifications was verified by sequence analysis. Dimers of the respective molecules, excised by BamHI, were inserted into the BamHI site of plasmid pBin19. Subsequently, the pBin19 derivatives were transferred by electroporation (30) into Agrobacterium tumefaciens strains LBA4404 (31) and COR308 (17), provided by Cornell Research Foundation, Inc.

Agrobacterium-mediated inoculation. Agroinoculation, as well as agroinfection, of plants by DNA virus genomes requires more than one complete unit of viral genetic information to be placed within the T-DNA, so that infectious viral DNA molecules can be produced by recombination or replication in the plant cell (13, 14, 37). N. benthamiana leaf disks were inoculated by A. tumefaciens LBA4404 carrying dimers of the respective FBNYV DNAs in pBin19 and kept on culture media at 24°C with continuous light as described previously (21, 39). Leaves of 4- to 5-week-old N. benthamiana plants were infiltrated at their abaxial face with A. tumefaciens LBA4404 carrying the FBNYV DNAs in pBin19 by using a needleless syringe.

V. faba plants were agroinoculated with A. tumefaciens COR308 grown in YEB medium (0.5% nutrient broth, 0.5% peptone, 0.1% yeast extract, and 5 mM MgSO4, adjusted to pH 7.2) containing 50 µg of kanamycin/ml and 5 µg of tetracycline/ml. An overnight culture was diluted 10-fold in YEB, 50 µg of kanamycin/ml, 5 µg of tetracycline/ml, 10 mM MES (morpholinoethanesulfonic acid; pH 6.0), and 50 µM acetosyringone (Sigma) and then cultured at 30°C until an optical density at 600 nm of ca. 1.5 was reached. The bacteria were pelleted by centrifugation (20 min at 3,500 x g) and resuspended in 1/10 of the original volume of MS medium (Sigma), 10 mM MES, and 150 µM acetosyringone. V. faba plants were injected with ca. 1 ml of the bacterial suspension by using a syringe with a needle.

All agroinoculated or virus-infected N. benthamiana and V. faba plants were cultivated at 25°C, in 50% humidity and with 16 h of light in growth chambers inside a restricted-access S3-confinement facility.

Aphid transmission assays. Virus and DNA-R-His replicon transmission assays were performed by using the insect vector Aphis craccivora (kindly provided by L. Allala, INA, El Harrach, Algeria) and the host V. faba. Viruliferous and nonviruliferous aphids were reared on V. faba plants in cages inside growth chambers in S3 confinement as described above. Healthy plants, 7 to 10 days old, were infected with viruliferous A. craccivora fed on V. faba infected by the FBNYV Algerian isolate (FBNYV-DZ [provided by L. Allala]). The plants were agroinoculated either immediately or 2 days later with A. tumefaciens COR308 carrying dimers of the DNA-R-His replicons in pBin19. The presence of the replicons in the infected symptomatic plants was tested 10 to 15 days postagroinoculation (dpa) by immunocapture-PCR (IC-PCR) with the DNA-R-His-specific oligonucleotides 6His-NheI(+) and C2Nhe970(–). For transmission, nonviruliferous A. craccivora were fed on FBNYV-infected and agroinoculated plants for an acquisition access period of 3 days and subsequently transferred to healthy 7- to 10-day-old V. faba plants for an inoculation access period of 5 days. Insects were killed by treatment with an insecticide (0.2% Dedevap; Bayer). After 2 weeks, samples of newly developed leaves of these plants were tested for viral or replicon DNAs by PCR with purified DNA or by IC-PCR of crude extracts with the primers C2Nhe205(+) (5'-ATGAAGTATCTTGCTAGCCAAACTGAACAA-3') and C2Nhe970(–) for the virus and 6His-NheI(+) and C2Nhe970(–) for the replicons. The primers M13(+) (5'-GTAAAACGACGGCCAGT-3') and M13(–) (5'-GGAAACAGCTATGACCATG-3') were used to confirm the absence of any contaminating pBin19 T-DNA in the samples.

Further transmission of the DNA-R-His replicon by A. craccivora was performed by first agroinoculation with FBNYV-EG cloned DNAs (T. Timchenko et al., unpublished data) and subsequently infection with viruliferous (FBNYV-DZ) A. craccivora. V. faba plants were agroinoculated with A. tumefaciens COR308 cultures, each one carrying a dimer of the eight FBNYV-EG genome components (DNA-R, DNA-C, DNA-M, DNA-N, DNA-S, DNA-U1, DNA-U2, and DNA-U4) (41), along with agrobacteria carrying a dimer of the DNA-R-His replicon. Agroinoculated plants that had developed symptoms at 21 dpa (4 of 34 plants) were superinfected with viruliferous A. craccivora for 3 days, after which the insects were removed. After a further 3 days, nonviruliferous insects were allowed access to these primary plants (agroinoculated with eight FBNYV-EG DNAs plus the DNA-R-His replicon and subsequently exposed to viruliferous insects) for a 6-day acquisition period. The aphids were then transferred to healthy V. faba (secondary plants), and transmission of the DNA-R-His replicon to secondary plants was tested after 8 and 25 days by PCR with the DNA-R-His-specific primers 6His-NheI(+) and C2Nhe970(–).

IC-PCR. Detection of FBNYV DNA-R and DNA-R-His replicons was carried out by IC-PCR as described previously (12) with a monoclonal antibody raised against FBNYV (kindly provided by H.-J. Vetten, BBA, Braunschweig, Germany). Primers C2Nhe205(+) and C2Nhe970(–) were used to detect DNA-R (797-bp fragment), and primers 6His-NheI(+) and C2Nhe970(–) were used to detect the DNA-R-His replicons (891-bp fragment).

Analysis of DNA replication by Southern blot. Replication in N. benthamiana leaf disks of the DNA-R-His replicons was analyzed as described previously (39, 40). DNA was extracted from N. benthamiana leaf disks at 4 to 5 dpa, fractionated by electrophoresis in 1% agarose gels, transferred to nylon membranes, and detected by hybridization with component-specific 32P-labeled probes. The probe used for DNA-R was the entire component (1003 bp) of R-6H-A linearized by BamHI. Probes for DNA-S (a fragment of 608 bp) and DNA-C (a fragment of 543 bp) were generated by PCR with the following sets of primers: C5Nhe328(+) (5'-AAAATGGCTAGCAAATGGAATTGGTCTGGTACGAA-3') and FBIR2B(–) (5'-TCCGCTGAACCTGGGGCGGGGGTAATACTAAGCCC-3'), followed by HincII digestion for DNA-S; C10BamHI(+) (5'-CGTTGTTCTTGGATCCAAGATGGGTCTGAA-3') and C10EcoRI(–) (5'-TTTAATTACGAATTCTCAACTAATTACAATATCC-3') for DNA-C. DNA fragments were fractionated by 1% agarose gel electrophoresis, purified by MiniElute columns (Qiagen), and 32P labeled (Amersham Megaprime Kit).

For replication assays of other FBNYV DNAs, leaf disks were coagroinoculated with equal volumes of two cultures of A. tumefaciens LBA4404: one carrying in pBin19 dimers of one of the DNA-R-His replicons and another carrying in pBin19 dimers of DNA-S or DNA-C, respectively. Quantification of the replicative DNA forms was done by using a Storm 840 PhosphorImager and ImageQuant software (Molecular Dynamics).

Purification of oligohistidine-tagged proteins. Since it had been shown that FBNYV Clink protein enhances replication of FBNYV DNA up to 7-fold (3), agrobacteria containing the Clink-encoding DNA-C was always included, along with the DNA-R or DNA-R-His replicons in transfection experiments for protein expression. For analytical purposes, total protein from N. benthamiana leaf disks harvested 4 or 5 dpa was extracted (at 1:5 fresh wt/vol) in HEPES buffer (50 mM HEPES [pH 8.0], 150 mM NaCl, 50 mM EDTA, 0.05% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.1 KI units of aprotinin/ml [Roche], 10 mM ß-mercaptoethanol). The extract was centrifuged at 15,000 x g for 15 min at 4°C. The protein concentration of the supernatant was determined by the Bradford assay (6). Then, 10 µg of total protein was fractionated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE). Proteins were electrotransferred for 1 h at 200 mA to a polyvinylidene difluoride membrane (Amersham), which then was blocked for 2 h at room temperature with 5% (wt/vol) skimmed milk in TBS buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl) containing 0.1% Tween 20. The membranes were incubated with polyclonal anti-M-Rep antibodies (1:2,000 dilution in TBS buffer of a sixth bleeding serum, preadsorbed by overnight incubation at 4°C with total protein extracts of N. benthamiana, Arabidopsis thaliana, and V. faba). Detection of antigen was performed with anti-rabbit immunoglobulin G coupled to alkaline phosphatase (Sigma-Aldrich). For antibody production, oligohistidine-tagged M-Rep protein purified from Escherichia coli containing plasmid pQE30-rep2 was used (39). Antisera were raised in rabbits and kindly provided by H.-J. Vetten.

Purification of oligohistidine-tagged M-Rep proteins by immobilized metal ion affinity chromatography (IMAC) was essentially as described previously (3) with slight modifications. Agroinfiltrated N. benthamiana leaves or agroinoculated leaf disks were harvested at 3 or 4 days postinfiltration. Total protein from leaves (10 g [fresh weight]) was extracted at a 1:5 (wt/vol) ratio in TN buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1 mM PMSF, 0.1 KI units of aprotinin/ml, 5 mM ß-mercaptoethanol, 5 mM imidazole, and one tablet of Complete Mini, EDTA-free, protease inhibitor cocktail [Roche] per 10 ml of buffer). The extract was centrifuged at 15,000 x g for 15 min. All extraction and purification steps were carried out at 4°C. The protein concentration was determined by the Bradford assay (6). Total protein (30 mg) was incubated with 500 µl of Talon metal (Co2+) affinity resin (Clontech) for 30 min. The resin was washed once with 40 ml of TN buffer and once with 20 ml of TN buffer containing 20 mM imidazole. Oligohistidine-tagged M-Rep proteins were eluted with 500 mM imidazole in TN buffer. Otherwise, the resin was stored at –20°C until proteins were analyzed by SDS-PAGE and Western blotting as described above.

For purification of enzymatically active tagged M-Rep, total protein was extracted from 9 g of leaf disks (ca. 300 disks) at 5 dpa with 45 ml of modified TN buffer (without ß-mercaptoethanol and containing 10% glycerol and 0.5% Tween 20). The extract was incubated for 30 min on ice with DNase and RNase (10 µg/ml each) prior to centrifugation. For protein purification by IMAC, 500 µl of Talon metal (Co2+) affinity resin were used. After protein binding, the column was washed as described above, and the tagged M-Rep protein was eluted with 5 column volumes of modified TN buffer containing 400 mM imidazole. Samples were kept on ice at 4°C for a maximum of 1 week.

Purification of M-Rep from E. coli. The 6H-MRep-M coding sequence was excised from the replicon R-6H-M by digestion with NheI and BamHI. The fragment was purified from an agarose gel, digested with Sau3AI, and cloned into pET21a (Novagen) at the NheI and BamHI sites. The resulting plasmid pET21a-R-6H-M was introduced into E. coli BL21(DE3)-recA (2, 38), harboring plasmid pRep4 (Qiagen) for tightly controlled expression of the recombinant protein. Bacteria were grown at 37°C in LB medium to an optical density at 600 nm of ca. 0.6 and transferred to 18°C for 1 h before induction at 18°C with 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 5 h. The bacteria were centrifuged, and the pellet was resuspended in buffer A (50 mM phosphate buffer [pH 8.0] containing 500 mM NaCl, 10% glycerol, 0.5% Tween 20, and 1 mM PMSF). Bacterial lysis and protein purification from E. coli was by IMAC as described previously (34), except that the washes were performed as follows: two washes with 40 column volumes of buffer A containing 10 mM imidazole and one wash with 20 column volumes of buffer A containing 40 mM imidazole. The 6H-MRep-M protein was eluted with 5 volumes of buffer A containing 400 mM imidazole. Samples for enzymatic activity were kept on ice at 4°C for 1 week.

ATPase assay. The ATPase activity of the tagged M-Rep purified from plant tissue and from E. coli was determined as described previously (39). Totals of 175 ng of tagged M-Rep from N. benthamiana and 3.5 µg from E. coli were incubated either at 37°C for 45 min, at room temperature for 45 min, or at room temperature for 16 h with 5 nM [{gamma}-33P]ATP and 5 µM nonlabeled ATP. The reaction products were separated by thin-layer chromatography on polyethyleneimine cellulose F plastic sheets (PEI; Merck) using 0.5 M LiCl-1 M formic acid as a running buffer. The amount of [33P]orthophosphate liberated by M-Rep was quantified by using a PhosphorImager (Molecular Dynamics).


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RESULTS
 
Oligohistidine-tagged FBNYV M-Rep proteins are functional in planta. To determine whether the addition of a series of histidines to the N or C terminus of FBNYV M-Rep interferes with protein function, the replication initiation activity of several oligohistidine-tagged protein variants was studied in planta. Agrobacteria containing FBNYV DNA-R or one of the replicons encoding an M-Rep protein with an N- or C-terminal oligohistidine tag were used (Fig. 1), and agrobacteria with the T-DNA vector pBin19 alone served as a control. Total DNA was extracted from leaf disks at 4 or 5 dpa and analyzed by Southern blot with DNA-R as the probe. Wild-type M-Rep protein initiated replication of its coding DNA (Fig. 2A, lanes 7 and 9). In contrast, the oligohistidine-tagged M-Rep protein lacking methionine 1 (of the wild-type protein) and whose arginine 3 was replaced by serine (6H-MRep-S, encoded by replicon R-6H-S; Fig. 1) was not able to catalyze replication initiation (Fig. 2A, lane 2). The same result was obtained when six histidines were added to the C terminus of M-Rep (MRep-6H-Ter, encoded by replicon R-6H-Ter; Fig. 2A, lane 1). Therefore, changing arginine 3 and deleting methionine 1 at the N terminus in conjunction with addition of a His6 tag, as well as deleting two amino acids at the C terminus of M-Rep in conjunction with addition of a His6 tag (Fig. 1), abolished the capacity of the respective modified M-Rep proteins to initiate FBNYV DNA replication.



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  		FIG. 2. Replication of FBNYV DNA-R-His replicons in N. benthamiana leaf disks. (A) Total DNA extracted from leaf disks agroinoculated with FBNYV DNA-R-His replicons was analyzed by Southern blotting with a DNA-R-specific probe. DNA from inoculation with pBin19 alone was used as a control (lane 3). Lanes 1 and 2 are from a different gel. Positions of replicative viral DNA forms and input DNA are indicated. ccc, covalently closed circular DNA; oc, open circular DNA. M, molecular marker. (B and C) Quantification of the ds- and ssDNA forms from three independent replication assays. The amount of the respective DNA forms is given as a percentage of wild-type DNA (DNA-R). Replication of R-6H-M and DNA-R in the presence of DNA-S (encoding the capsid protein) and its quantification are shown in panel A, lanes 8 and 9, and panels B and C as indicated.

In contrast, replicon R-6H-M encoding the 6H-MRep-M protein with no changes in the M-Rep sequence other than the N-terminal addition of the tag (Fig. 1) replicated in leaf disks (Fig. 2A, lanes 6 and 8). Protein analysis suggested that only a single M-Rep species was produced from the R-6H-M replicon (see below). To rule out the possibility of any low-level translation restart after the tag at methionine 1 of the wild-type sequence that might have been undetected, the replicon R-6H-A coding for 6H-MRep-A in which the wild-type methionine 1 of 6H-MRep-M was replaced by alanine was generated (Fig. 1). Replicon R-4H-A (coding for 4H-MRep-A) was obtained fortuitously. These two replicons also replicated in leaf disks (Fig. 2A, lanes 4 and 5), albeit less efficiently. This result excluded the possibility that the observed replication of R-6H-M was due to undetected amounts of wild-type Rep expressed by internal initiation. Replication experiments of DNA-R and replicon R-6H-M in the presence of DNA-S expressing the virus capsid protein revealed a higher level of ssDNA, probably due to protection of the ssDNA by capsid protein (Fig. 2A, lanes 8 and 9, and Fig. 2C).

The amount of replicative DNA species in three independent experiments was quantified. Replicons R-6H-M, R-6H-A, and R-4H-A led to less replicative DNA than wild-type DNA-R. In the case of replicon R-6H-M compared to DNA-R the reduction was 2-fold for double-stranded DNA (dsDNA) (Fig. 2B) and 4-fold for ssDNA (Fig. 2C); it was about 5-fold for dsDNA and 14-fold for ssDNA in the case of R-6H-A compared to DNA-R (Fig. 2B and 2C). Since R-6H-M (Fig. 2A, lane 6) replicated more efficiently than R-6H-A and R-4H-A (Fig. 2A, lanes 4 and 5), methionine 1 of the wild-type protein may be important for M-Rep function. In the presence of DNA-S, the increase of ssDNA (Fig. 2C) seems to correlate with a decrease of dsDNA (Fig. 2B). Collectively, the results demonstrate that the addition of an oligohistidine tag to the amino terminus of FBNYV M-Rep (6H-MRep-M, 6H-MRep-A, and 4H-MRep-A; Fig. 1) does not abolish replication in planta.

Initiation of replication of other FBNYV genome components by the oligohistidine-tagged M-Rep proteins. M-Rep is the only viral protein necessary to initiate replication of all the genomic DNAs of a nanovirus (39, 40). To determine whether the tagged M-Rep proteins also catalyzed replication initiation of other FBNYV DNAs, we tested replication of FBNYV DNA-S (capsid protein) and DNA-C (Clink protein) in the presence of the DNA-R-His replicons. N. benthamiana leaf disks were coagroinoculated with different combinations of two cultures of agrobacteria: one carrying in pBin19 dimers of either DNA-R, R-6H-M, R-6H-A, or R-4H-A and the the other carrying dimers of FBNYV DNA-S or DNA-C, respectively (39). Wild-type M-Rep protein initiated replication of FBNYV DNA-S (Fig. 3A, lane 9) and DNA-C (Fig. 3B, lane 8). Oligohistidine-tagged M-Rep proteins 6H-MRep-M, 6H-MRep-A (not shown), and 4H-MRep-A (not shown) proved capable of catalyzing replication of FBNYV DNA-S (Fig. 3A, lane 6) and DNA-C (Fig. 3B, lane 5). Quantification of the transreplication data of DNA-S and DNA-C from three independent experiments showed reduced replication levels with the modified M-Rep protein (Fig. 3C), probably due to the reduced levels of autonomous replication observed in the autoreplication assays (Fig. 2). These results demonstrate that the oligohistidine-tagged M-Rep proteins act as master Rep proteins.



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  		FIG. 3. Trans-replication of FBNYV DNA-S and DNA-C. (A and B) Total DNA extracted from N. benthamiana leaf disks agroinoculated with DNA-R and the R-6H-M replicon, along with DNA-S (A) or DNA-C (B) as indicated, was analyzed by Southern blotting with component-specific probes. Replicative viral DNA forms and input DNA are indicated as in Fig. 2. (C) Quantification of viral DNA forms. The amount of DNA-S and DNA-C (ds- and ssDNA) was quantified in three experiments, and the mean for each DNA-S and DNA-C replicated in the presence of the R-6H-M replicon is given as a percentage of that obtained in the presence of wild-type DNA-R.

Systemic spread of the DNA-R-His replicons in FBNYV-infected V. faba plants. To determine whether the DNA-R-His replicons spread systemically within the host plant in the context of a virus infection, V. faba was infected with wild-type FBNYV by A. craccivora and subsequently agroinoculated with A. tumefaciens strain COR308 carrying in pBin19 dimers of replicons R-6H-M or R-4H-A. Disease symptoms were monitored, and the presence of the DNA-R-His replicons was analyzed by IC-PCR of noninoculated symptomatic V. faba leaves that had developed 2 weeks after agroinoculation. The R-6H-M replicon was detected in 100% (15 of 15) of the symptomatic plants (Fig. 4A shows four representative examples). In two other experiments the R-6H-M replicon was detected in 87% (13 of 15) and 75% (6 of 8) of the symptomatic plants. For R-4H-A the experiment was performed twice, and the replicon was detected in 67% (10 of 15) and 70% (7 of 10) of the symptomatic plants. T-DNA-specific primer sequences (see Materials and Methods) were used to confirm the absence of pBin19 DNA in the leaves tested (not shown). These results indicate that the R-6H-M and R-4H-A replicons are able to spread systemically along with the wild-type FBNYV DNAs. Systemic spread of the R-6H-M and R-4H-A replicons was not detected in control plants, which were agroinoculated with R-6H-M or R-4H-A alone (Fig. 4A).



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  		FIG. 4. Systemic spread and transmission of FBNYV DNA-R-His replicons. (A) Detection in primary plants of the R-6H-M replicon by IC-PCR with DNA-R-His-specific primers in newly developed leaves of V. faba infected with FBNYV by A. craccivora and agroinoculated with the R-6H-M replicon. Lanes 1 to 4, different individual aphid-infected and agroinoculated primary plants; non-inf, agroinoculated but not aphid-infected plant. (B) Detection in secondary plants of DNA-R and R-6H-M replicons by IC-PCR in newly developed leaves of V. faba infected by aphids fed on primary FBNYV-infected and agroinoculated plants. DNA-R (left panel)- and DNA-R-His (right panel)-specific primers were used. Lanes 1 to 4, samples from different individual secondary plants. Numbers in the left and right panels correspond to samples from the same plants. inf, infected but not agroinoculated primary plant; p, sample from an aphid-infected and agroinoculated primary plant. (C) Detection in primary symptomatic plants of the DNA-R (lanes 1 and 2) and R-6H-M (lanes 3 and 4) replicons by PCR in newly developed leaves of V. faba agroinoculated with all eight FBNYV-EG DNA components, along with the R-6H-M replicon. (D) Detection in secondary plants of DNA-R and R-6H-M replicons by PCR at 8 and 25 dpi as indicated. Numbers in the left and right panels correspond to the plant number. R, DNA-R-specific primers; H, DNA-R-His-specific primers; M, molecular marker (in kilobases). Arrows indicate amplified DNA-R- or DNA-R-His-specific fragments (797 and 891 base pairs, respectively).

Transmission of FBNYV DNA-R-His replicons by aphids. To investigate whether the replicons are transmitted by aphids to healthy plants, FBNYV-infected symptomatic plants in which the R-6H-M or R-4H-A replicons had moved systemically were used for transmission assays. For this purpose, nonviruliferous A. craccivora were starved for 5 h prior to an acquisition access period of 3 days on FBNYV-infected and R-6H-M or R-4H-A replicon-positive symptomatic plants (primary plants). The aphids were then transferred to healthy plants (secondary plants) and maintained for an inoculation access period of 5 days. Secondary plants were monitored for symptom development and analyzed by IC-PCR 2 weeks after aphid access. Figure 4B shows a representative experiment in which only wild-type DNA-R was detected in four symptomatic secondary plants tested 2 weeks after inoculation access (Fig. 4B, left panel). This indicates that wild-type FBNYV is readily transmitted by the aphids and that virus transmission is not affected by the multiplication of the R-6H-M or R-4H-A replicons. However, replicon R-6H-M was not detected in the secondary plants that had scored positive for DNA-R, i.e., had received virus from the aphids (Fig. 4B, right panel). The experiments were repeated three times with replicons R-6H-M and R-4H-A, respectively. Using these conditions, replicons expressing a tagged M-Rep were not transmitted by aphids along with the virus. These results show that, despite the systemic movement of the R-6H-M and R-4H-A replicons in a simultaneous infection with FBNYV, no transmission by aphids along with wild-type virus occurred, or that the replicons were rapidly lost after transmission.

In a different experimental setup, we used the established FBNYV infection by agroinoculating V. faba plants with eight cloned viral DNAs (T. Timchenko, et al., unpublished data). In addition to these eight DNAs, the R-GH-7 replicon was agroinoculated into V. faba. Agroinoculated primary plants that developed symptoms at 21 dpa (4 of 34) were superinfected with viruliferous A. craccivora carrying the isolate FBNYV-DZ for a 3-day inoculation access period. Viruliferous insects were removed and after a further 3 days nonviruliferous insects were allowed access to the primary plants for a 6-day acquisition access period. They were then transferred to healthy V. faba (secondary plants) for transmission, and the presence of replicon R-6H-M was assayed by PCR. DNA-R (no distinction is possible in this experimental set up between agroinoculated or aphid-delivered DNA-R) was found in primary plants 1 and 2 (Fig. 4C, lanes 1 and 2). By PCR, replicon R-6H-M could only be detected in primary plant 2 (Fig. 4C, lane 4), whereas it was detected in plant 1 by IC-PCR (data not shown). Transmission to secondary plants was analyzed at 8 and 25 dpi. DNA-R and replicon R-6H-M were detected in secondary plants 1 and 2 at 8 dpi (Fig. 4D). At 25 dpi, the amount of R-6H-M DNA had increased in both plants analyzed (Fig. 4D, right panel). These results show that aphids are able to transmit the modified replicon R-6H-M, provided it is agroinoculated, along with all eight FBNYV-EG DNAs and has spread systemically. Superinfection by viruliferous aphids containing FBNYV-DZ then allows for acquisition and transmission of the R-6H-M replicon.

Affinity purification of oligohistidine-tagged FBNYV M-Rep proteins from plant tissue. The difficulty in purifying Rep proteins of nano- and geminiviruses under conditions of natural infection has been a major problem in studying them in planta (24). Having demonstrated that oligohistidine-tagged FBNYV master Rep proteins were functional in planta, we assayed the expression of the tagged proteins by Western blotting with M-Rep-specific antisera. Figure 5A shows an example of 6H-MRep-M expressed in N. benthamiana leaf disks (lane 2). Similar levels of protein expression were observed for 6H-MRep-A and 4H-MRep-A, and comparable results were obtained with agroinfiltrated N. benthamiana leaves (data not shown). Only one major protein species is produced from the R-6H-M replicon (Fig. 5A, lane 2), and the difference in size between the wild-type and tagged protein indicates the presence of the tag (Fig. 5A, lanes 2 and 3, and Fig. 6). Therefore, we conclude that there is no translation initiation at the internal methionine (methionine 1 of the wild-type M-Rep) following the tag sequence. This was proven by replication of the 6H-MRep-A and 4H-MRep-A replicons (see above). To rule out that the Rep proteins detected were due to expression in A. tumefaciens, Western blots of protein extracts from agrobacteria carrying pBin19 alone (Fig. 5B, lane 3) or dimers of R-6H-M (Fig. 5B, lane 4) or DNA-R (Fig. 5B, lane5) were performed. Only nonspecific cross-reaction with other proteins was observed in A. tumefaciens irrespective of whether pBin19 carried a dimer of DNA-R or R-6H-M (Fig. 5B, lanes 3, 4, and 5, respectively). Similarly, nonspecific cross-reaction was observed in extracts of N. benthamiana inoculated with agrobacteria carrying pBin19 (Fig. 5B, lane 2), whereas in extracts of N. benthamiana inoculated with agrobacteria carrying R-6H-M, 6H-MRep-M protein was detected (Fig. 5B, lane 1). Lane 6 shows 6H-MRep-M protein from E. coli that served as control.



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  		FIG. 5. In planta expression and purification of oligohistidine-tagged M-Rep proteins. (A) Western blot analysis of crude extracts from N. benthamiana leaf disks 4 days after inoculation with agrobacteria containing the FBNYV DNA-R (lane 3) and the R-6H-M replicon (lane 2). The positions of wild-type M-Rep of ca. 33 kDa (lane 3) and 6H-MRep-M (lane 2) proteins are indicated. The difference in migration between these two proteins is due to the additional His tag. An extract from inoculation with agrobacteria containing pBin19 was used as a control (lane 1). (B) Western blot analysis of extracts from N. benthamiana compared to extracts from A. tumefaciens. Extracts from N. benthamiana inoculated with agrobacteria carrying a dimer of R-6H-M in pBin19 (lane 1) or pBin19 alone (lane 2) and extracts from agrobacteria carrying pBin19 alone (lane 3), a dimer of R-6H-M (lane 4), or DNA-R (lane 5) were assayed. Lane 6 contains 6H-MRep-M protein from E. coli. (C) Purification of 6H-MRep-M by affinity chromatography from N. benthamiana leaves agroinfiltrated with the R-6H-M replicon. Western blot analysis of total extracts (lanes 1 and 2), bound proteins (lanes 3 and 4), and eluted proteins (lanes 5 and 6). (D) SDS-PAGE of the purified 6H-MRep-M protein stained with Coomassie brilliant blue (lane 2). The arrow indicates 6H-MRep-M.



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  		FIG. 6. ATPase activity of plant- and E. coli-expressed 6H-MRep-M proteins. (A) 6H-MRep-M purified from N. benthamiana and E. coli was incubated with {gamma}-33P-labeled ATP. Liberation of 33Pi from {gamma}-33P-labeled ATP by 6H-MRep-M was analyzed by thin-layer chromatography on PEI sheets. Lanes 1 and 4, no protein; lanes 2 and 5, 6H-MRep-M from N. benthamiana; lanes 3 and 6, 6H-MRep-M from E. coli. Lanes 1 to 3 indicate reactions performed at room temperature for 16 h; lanes 4 to 6 indicate reactions performed at 37°C for 45 min. (B) Amount of protein (175 ng of 6H-MRep-M from N. benthamiana and 3.5 µg from E. coli) used for the ATPase assay analyzed by Western blotting.

We then progressed to purify oligohistidine-tagged M-Rep protein from N. benthamiana plants by IMAC. The R-6H-M replicon was introduced by infiltration of agrobacteria into the youngest leaves. The leaves were harvested 4 days postinfiltration, and the tagged protein was extracted from plant tissue under nondenaturing conditions and bound to the Co2+-resin. The majority of 6H-MRep-M protein was eluted in the first four fractions containing 500 mM imidazole; fractions 1 and 2 are shown in Fig. 5C, lanes 5 and 6. The eluted fractions were pooled, analyzed by SDS-PAGE, and stained with Coomassie brilliant blue (Fig. 5D, lane 2). The results show that oligohistidine-tagged M-Rep proteins can readily be isolated from N. benthamiana plants by a single purification step.

The 6H-MRep-M purified from plant tissue is enzymatically active. M-Rep possesses an ATPase activity that is essential for viral DNA replication in vivo (39). This ATPase activity and its requirement for DNA replication is also a characteristic feature of geminivirus Rep proteins (11, 18). To prove that 6H-MRep-M isolated from N. benthamiana possesses ATPase activity, the hydrolysis of [{gamma}33P]ATP by the protein purified from plant tissue was assayed. The same 6H-MRep-M expressed in and purified from E. coli served as a control. Figure 6A shows that the oligohistidine-tagged M-Rep has ATPase activity after purification from E. coli (Fig. 6A, lanes 3 and 6) and from N. benthamiana (Fig. 6A, lanes 2 and 5). Lanes 1 to 3 were from an assay at room temperature and 16 h of incubation; lanes 4 to 6 show the reaction products after 45 min at 37°C. A Western blot (Fig. 6B) illustrates the amount of protein used for the ATPase assay (175 ng from N. benthamiana and 3.5 µg from E. coli). These results show that 6H-MRep-M purified from plant tissue possesses comparable ATPase activity.

Similarly, origin DNA cleavage activity of 6H-MRep-M from N. benthamiana was compared to that of the protein purified from E. coli (see Fig. S1 in the supplemental material). Both proteins were proficient in origin cleavage activity.

In planta interaction between FBNYV 6H-MRep-M and wild-type M-Rep. It had been reported that geminivirus Rep proteins form oligomers in solution (29, 33) and that the oligomerization state of Rep in vitro is pH dependent (15, 29). Further results suggested that an oligomeric complex may be essential for the initiation of DNA replication of geminiviruses (32). The nanovirus M-Rep protein also oligomerizes (T. Timchenko et al., unpublished results); however, and yet oligomerization was never shown in planta. Using the 6H-MRep-M protein, we demonstrated that interaction between tagged and wild-type proteins occurs in planta. Figure 7 (lanes 1 to 4) shows total protein extracts from N. benthamiana leaf disks agroinoculated with pBin19 (lane 1), DNA-R (lane 2), R-6H-M (lane 3), and coagroinoculated with R-6H-M and DNA-R (lane 4). The proteins eluted after incubation with the affinity resin are shown in lanes 5 to 8. M-Rep coelutes with 6H-MRep-M (Fig. 7, lane 8). M-Rep by itself was not bound by the resin (Fig. 7, lane 6) and was only detected in conjunction with 6H-MRep-M in extracts of plants expressing both wild-type M-Rep and 6H-MRep-M. This strongly suggests that both proteins interacted in planta, probably by forming mixed oligomers. Thus, the use of functional oligohistidine-tagged FBNYV master Rep variants allows isolation and purification of M-Rep protein complexes ex planta.



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  		FIG. 7. In planta interaction between FBNYV 6H-MRep-M and M-Rep. Total protein extracts (lanes 1 to 4) and eluted proteins after purification by IMAC (lanes 5 to 8) analyzed by Western blotting. Proteins from N. benthamiana leaf disks agroinoculated with pBin19 (lanes 1 and 5), DNA-R (lanes 2 and 6), the R-6H-M replicon (lanes 3 and 7), and the R-6H-M replicon plus DNA-R (lanes 4 and 8) are shown. The positions of the tagged (6H-MRep-M) and wild-type (M-Rep) protein are indicated.


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DISCUSSION
 
Proteins involved in replication of ssDNA plant viruses have mostly been identified by virtue of their interaction with replication initiator proteins by using the yeast two-hybrid system. In vitro pull down or immunoprecipitation of recombinant proteins confirmed complex formation by the respective interacting partners. For example, the interaction of the Wheat dwarf virus Rep protein with the large subunit of the wheat replication factor C has been observed (28). Moreover, it has been established that Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep binds to the tomato proliferating cell nuclear antigen (9), and Tomato golden mosaic virus (TGMV) Rep binds to a retinoblastoma-related protein (1, 25). An N. benthamiana sumoylation enzyme, NbSCE1, was also shown to interact with the Rep proteins from TYLCSV, TGMV, and African cassava mosaic virus-Kenia (10); curiously, the interaction site on Rep was mapped to a protein sequence that extends over two opposite sides of the protein domain as revealed by its tertiary structure (8). This conundrum calls all the more for in planta confirmation of the complexes found by yeast two-hybrid assays and for a demonstration of the physiological relevance of such interactions. For example, in planta complex formation has been demonstrated for the nanovirus F-box protein Clink and Medicago sativa SKP1, a constituent of the ubiquitin-dependent protein turnover pathway (3).

The engineering of a functional Rep protein of an ssDNA plant virus, carrying a tag that allows its purification from the plant in the course of an infection, has been a challenge for the in planta study of gemini- and nanovirus replication. Hong et al. (20) showed that fusing green fluorescent protein (GFP) to the C terminus of ACMV Rep results in a protein with properties similar to those of Rep with respect to viral DNA replication and subcellular localization. The GFP tag permits visual detection but no affinity purification. A functional replication initiator protein with such characteristics has not been reported for any ssDNA virus. Here we present the design of an oligohistidine-tagged FBNYV M-Rep protein that is functional in planta and describe its use for the isolation and identification of proteins that interact in vivo with the tagged M-Rep. The tagged M-Rep was shown to trigger replication initiation in N. benthamiana of its cognate DNA component as well as that of other FBNYV DNAs.

The artificial replicon encoding the tagged M-Rep protein was able to spread throughout a plant when movement, encapsidation, and other essential virus functions were provided by coinfection with a helper virus. Furthermore, when all essential virus FBNYV DNAs were introduced by agroinoculation along with the R-6H-M replicon, it was also transmitted by the aphid vector after superinfection with a helper virus. This proves that the R-6H-M replicon has all of the features of a genuine M-Rep-encoding artificial FBNYV genome component.

In addition, the tagged protein was readily purified from N. benthamiana leaves by native IMAC, and two of its key enzymatic functions, the ATPase activity and the origin-specific ssDNA cleavage were demonstrated. To the best of our knowledge, this represents the first example of affinity purification of an enzymatically active ssDNA virus replication initiator protein from the natural host of the virus. Moreover, we have shown the in planta interaction between an oligohistidine-tagged M-Rep and wild-type M-Rep, suggesting the formation of mixed protein oligomers. This will further allow the copurification of other viral and cellular partner proteins of M-Rep. Oligomerization is a common feature of replication initiator proteins, as has been shown for geminiviruses (32, 33) and replication-associated proteins of animal DNA viruses, such as the simian virus 40 large T antigen (36) and the adeno-associated virus Rep78 protein (19).

The possibility that multifunctional Rep proteins are subject to posttranslational modifications to regulate their different functions is certainly possible. In the case of the SV40 large T antigen, some activities are regulated by phosphorylation (36). Concerning Rep proteins of ssDNA plant viruses, there is very limited information about such modifications. Kong and Hanley-Bowdoin (24) have shown that the TGMV Rep interacts with a protein kinase (GRIK), but no evidence of phosphorylation of Rep in plant or insect cells was reported. Having at hand a tagged and functional M-Rep protein, the biological significance of phosphorylation or other protein modifications can now be readily studied in vivo.

Obtaining functionally intact modified replication initiator proteins of ssDNA viruses is difficult since, in the case of FBNYV, alteration of two amino acids at the N or C terminus of the M-Rep protein already abolishes its activity. The importance of methionine 1 and arginine 3 for M-Rep is reflected by the fact that 6H-MRep-S is not functional, whereas 6H-MRep-M in which, apart from the tag, the wild-type amino acid sequence is conserved catalyzes replication initiation in planta. The lower replication level of the replicon R-6H-M compared to wild-type DNA-R (Fig. 2) indicates that the oligohistidine tag impairs to some extent the function of the protein. The replacement of methionine 1 by alanine in 6H-MRep-A or 4H-MRep-A results in a further reduction of DNA replication. Whether the reduced activity of 6H-MRep-A and 4H-MRep-A is solely due to the methionine 1 to alanine change or whether it is also influenced by the oligohistidine tag immediately preceding methionine 1 remains to be determined. Basic amino acids at the N terminus of the TYLCSV Rep have been suggested to be implicated in DNA recognition by the protein (8), an idea in line with the results presented here. An alternative or additional explanation for the observed reduction of DNA replication efficiency may be that important DNA-R elements required in cis were located in the sequence immediately preceding and/or encoding the amino terminus of M-Rep. The addition of the 27 nucleotides of the tag and base changes within the following sequence may interfere with the correct function of such cis-acting elements. Experiments uncoupling M-Rep expression from cognate DNA (template) replication will provide distinctive information.

The ability of the DNA-R-His replicons to spread systemically in the context of an infection with FBNYV indicates that the replicons move from cell to cell, either as DNA-protein complexes similar to geminivirus cell-to-cell movement (27) or as virions. The fact that we were able to amplify by IC-PCR the oligohistidine-tagged M-Rep encoding DNAs shows that they are at least tightly associated with virus capsid protein. Since the DNA-R-His replicons were transmitted by aphids, it is quite possible that they are also encapsidated into true virions.

A tagged replication initiator protein of an ssDNA virus that is functional in planta represents a very useful tool for studying in planta protein-protein interactions and for identifying its viral and host partner proteins. It will also allow the study of host cell-dependent posttranslational modifications of Rep and its interacting partner proteins in the course of a virus infection.


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ACKNOWLEDGMENTS
 
We thank H.-J. Vetten for providing monoclonal and polyclonal antibodies to FBNYV and polyclonal antibodies to M-Rep. We are indebted to L. Allala for providing the Algerian isolate of FBNYV and its vector A. craccivora and to the Cornell Research Foundation, Inc., for A. tumefaciens strain COR308. We thank A.-L. Haenni, F. Bernardi, and two anonymous reviewers for valuable suggestions.

J.C.V.-A. was supported by a fellowship from CONACYT (Mexico).


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut des Sciences du Végétal, CNRS, Avenue de la Terrasse, Bât. 23, 91198 Gif sur Yvette Cedex, France. Phone: (33) 169823571. Fax: (33) 169823695. E-mail for B. C. Ramírez: ramirez{at}isv.cnrs-gif.fr. E-mail for B. Gronenborn: gronenborn{at}isv.cnrs-gif.fr. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back


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Journal of Virology, July 2005, p. 8422-8430, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8422-8430.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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  • Timchenko, T., Katul, L., Aronson, M., Vega-Arreguin, J. C., Ramirez, B. C., Vetten, H. J., Gronenborn, B. (2006). Infectivity of nanovirus DNAs: induction of disease by cloned genome components of Faba bean necrotic yellows virus. J. Gen. Virol. 87: 1735-1743 [Abstract] [Full Text]  

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