National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE12618 Tallinn, Estonia1
Institute of Biotechnology, Viikki Biocenter, University of Helsinki, PO Box 56, FIN-00014 Helsinki, Finland2
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia3
Author for correspondence: Andres Merits (at the Viikki Biocenter). Fax +358 9 191959366. e-mail Merits{at}operoni.helsinki.fi
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Abstract |
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Introduction |
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Rep of CFDV and nanoviruses contains several sequence motifs similar to those in Reps of plant ssDNA viruses belonging to the family Geminiviridae (Boevink et al., 1995 ), which are essential for DNA replication (Laufs et al., 1995
; Palmer & Rybicki, 1998
). Rep catalyses multiple reactions during the replicative cycle of the virus. So far, for nanoviruses only in vitro DNA nicking and joining activity of the BBTV Rep have been described (Hafner et al., 1997
). In contrast, much more is known about Reps of geminiviruses [for recent review, see Palmer & Rybicki (1998)
]. Geminivirus Rep is a sequence-specific DNA-binding protein (Fontes et al., 1992
; Behjatnia et al., 1998
; Castellano et al., 1999
) and has an ATPase activity which is required for its helicase function and for viral DNA replication (Desbiez et al., 1995
). Geminivirus Rep catalyses DNA cleavage and ligation of ssDNA during rolling circle replication. In solution, Rep forms oligomeric complexes and the self-association capability of geminivirus Reps is regulated by ATP (Jupin et al., 1995
; Settlage et al., 1996
; Orozco et al., 1997
, 2000
; Orozco & Hanley-Bowdoin, 1998
; Horvath et al., 1998
). In addition, it has been demonstrated that Reps of geminiviruses form complexes with other geminivirus-encoded proteins and proteins from host plants (Xie et al., 1995
, 1999
; Settlage et al., 1996
; Ach et al., 1997
; Horvath et al., 1998
; Liu et al., 1999
).
In this paper we report that the CFDV-encoded putative Rep has nucleic acid-binding and ATPase/GTPase activities in vitro and forms di- or oligomers in yeast cells. It was also found that CFDV Rep associated predominantly with nuclei and membrane fractions of plant and insect cells. These findings indicate that the putative Rep of CFDV shares several characteristic features with Reps of geminiviruses.
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Methods |
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Expression of 6x HisRep in recombinant baculovirus.
To obtain recombinant baculovirus expressing the 6x His fusion protein of CFDV Rep, the EcoRISal fragment from pQE.Rep was cloned into EcoRI/SalI-digested vector pFastBac1 (Gibco BRL) to obtain clone pFastBac.6xHisRep. This clone was used for obtaining recombinant baculovirus, named 6x HisRep-Bac, using the Bac-to-Bac system (Gibco BRL) according to the manufacturers protocols. Recombinant baculovirus was amplified and titrated in Sf9 cells. For recombinant 6x HisRep expression 1x108 High Five cells (Invitrogen) were infected with 6xHisRep-Bac at an m.o.i. of 5 for 48 h at 27 °C. Cells were collected by centrifugation and recombinant protein purification was carried out as described for E. coli-expressed recombinant protein. To obtain the nuclear fraction (N), harvested cells were treated as described by Patterson et al. (1996) . Post-mitochondrial pellet (P15, membrane fraction) and supernatant (S15, soluble fraction) were obtained as described by Laakkonen et al. (1994)
. All protein fractions from E. coli and recombinant baculovirus-infected insect cells were analysed by SDSPAGE (12% gels) and Western blotting, using monoclonal antibody against the RGS(H)3 epitope (Qiagen).
ATPase/GTPase assay.
This was done as described previously (Kalinina et al., 1996 ). Reactions were incubated for 1 h at 37 °C and stopped by addition of EDTA to a final concentration of 20 mM. Unreacted ATP was precipitated by addition of activated charcoal and radioactivity in the supernatant was determined by liquid scintillation counting of Cerenkov radiation.
Synthesis of radiolabelled RNA and DNA probes.
Labelled RNA transcript was transcribed with T7 RNA polymerase from a cDNA clone of Potato virus A as described by Merits et al. (1998) . To obtain labelled dsDNA probes, two fragments of the CFDV genome were used: fragment ApaIXhoI from the cloned full-length CFDV DNA (570 bp, containing the conserved stemloop region of CFDV) and the XhoISalI fragment from pSK.Rep (580 bp). These fragments were purified from agarose gels, treated with calf intestinal phosphatase and purified using the QIAquick PCR purification kit (Qiagen). One µg of each purified fragment was treated with 10 units of T4 polynucleotide kinase in the presence of 25 µCi [
-32P]ATP for 1 h at 37 °C. Labelled DNA was purified using the QIAquick nucleotide removal kit (Qiagen).
ssDNA probes of the indicated polarity were obtained using the primer extension reaction on linearized templates. To obtain three different ssDNAs, named ssDNA(+), ssDNA(-) and ssDNA(z), three different templates with corresponding primers were used. For ssDNA(+), representing positive-polarity ssDNA containing the stemloop region of the CFDV genome, XhoI-linearized plasmid, containing full-length CFDV DNA (Chernov et al., 1992 ), and primer 5' CAATATGAATCGAGTTATGGGCGGGCCC 3' were used (the ApaI site from the CFDV genome is underlined). To obtain ssDNA(-), complementary to the same sequence, CFDV was linearized by ApaI cleavage and primer 5' CAGGACGAGTCGGGACTCCGTGCTCGAG 3' was used (the XhoI site from the CFDV genome is underlined). To obtain the control, ssDNA(z), plasmid pGEM-3Z (Promega) was linearized by AatII cleavage and primer 5' ATTTAGGTGACACTATAGAATAC 3' (corresponding to the SP6 promoter sequence in the plasmid) was used. ssDNA was synthesized in a reaction mixture consisting of 1 µg of linearized plasmid and 50 pmol of the corresponding primer, 0·25 mM of each dNTP and 5 units of DyNAzyme DNA polymerase (Finnzymes) in DyNAzyme reaction buffer. PCR reactions (25 cycles: 95 °C 30 s, 51 °C 30 s, 72 °C 60 s) were carried out in a thermal cycler, reaction products purified using the QIAquick PCR purification kit (Qiagen), and labelled and purified as described above.
Nucleic acid binding assay.
The purified recombinant CFDV 6x HisRep and control proteins [BSA and maltose-binding protein fusion with the -fragment of
-galactosidase (MBP)] were electroblotted onto an Immobilon-P (Millipore) membrane after SDSPAGE in 12% gels. Membranes were blocked and membrane-bound proteins denatured and renatured as described previously (Merits et al., 1998
). Membranes were incubated for 2 h at room temperature with 32P-labelled DNA or RNA (1x105 c.p.m./ml) in 10 ml of nucleic acid-binding buffer (20 mM HEPES, 6 mM TrisHCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, pH 7·0) with or without addition of NaCl. NaCl concentrations of 25, 100, 300 and 500 mM were used to estimate the strength of proteinnucleic acid interactions. Membranes were washed with nucleic acid-binding buffer (with or without addition of NaCl) four times for 30 min, dried, and autoradiographed.
Yeast two-hybrid system (YTHS).
This was used essentially as described by Vojtek et al. (1993) and Hollenberg et al. (1995)
with modifications described by Guo et al. (1999)
. To obtain the yeast expression constructs pLexA.Rep and pVP16.Rep, the BamHISalI fragment from pSK.Rep was cloned into vectors pLexA and pVP16 (Hollenberg et al., 1995
). pLexA encodes the DNA-binding protein LexA and contains a selection marker for tryptophan auxotrophy, whereas pVP16 encodes transcription activation domains and contains a selection marker for leucine auxotrophy. Yeast strain L40 [MATa his3
200 trp1-901 leu2-3,112 ade2 lys2-801am URA3::(lexAop)8-lacZ] was used for hybrid protein expression. Yeast transformation was performed by the lithium acetate method (Schiestl & Gietz, 1989
). Transformants were selected by plating on minimal media lacking leucine and tryptophan. Expression of the
-galactosidase reporter gene was evaluated by freezing colony filter lifts in liquid nitrogen and subsequently staining the filter with X-Gal in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, pH 7·0, 10 mM KCl, 1 mM MgSO4, 50 mM
-mercaptoethanol and 0·3 mg/ml X-Gal). Quantitative
-galactosidase activity assays were performed as described by Trawick et al. (1989)
.
Transient expression of CFDV Rep in Nicotiana benthamiana cells.
For transient expression in N. benthamiana cells the BamHISalI fragment from pSK.Rep was cloned as an N-terminal fusion with the green fluorescent protein gene GFP5 (Haseloff et al., 1997 ) placed under control of the CaMV 35S RNA promoter. The resulting construct, named p35S.GFP5.Rep, was used in particle bombardment experiments. Particle bombardment was performed using the flying disk method with a high-pressure helium-based apparatus PDS-1000 (Bio-Rad) as described in Morozov et al. (1997)
. GFP fluorescence was detected by a Zeiss-Axiovert Bio-Rad MRC 1024 confocal laser scanning microscope using a 15 mW kryptonargon excitation laser with excitation light of 488 nm.
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Results and Discussion |
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Localization of CFDV Rep in insect cells and in N. benthamiana cells
The processing and subcellular localization of recombinant eukaryotic proteins in baculovirus-infected insect cells can be very similar to their localization in the original (or host) cells (Pascal et al., 1994 ; Patterson et al., 1996
). The use of different monoclonal antibodies against N-terminal epitopes of recombinant protein [(H)4, (H)5, (H)6 and RGS(H)3] to immunostain 6x HisRep expressed in infected Sf9 or High Five cells did not produce consistent results. Typically, fluorescence was close to background levels and showed no specific distribution in the cells (not shown). It is possible that the N-terminal 6x His tag epitope is hidden inside the protein and/or between different subunits of oligomerized protein. In attempts to overcome these difficulties, the infected High Five cells (48 h post-infection) were fractionated and analysed by SDSPAGE and Western blotting. In these experiments most of the expressed recombinant protein (about 70%) was found in the nuclear fraction of infected cells (Fig. 2
). Considerable amounts of 6x HisRep protein were also found in the post-mitochondrial pellet fraction (membrane fraction, about 25% of the recombinant protein) and only small quantities (less than 5% of the recombinant protein) were found in the soluble protein fraction (Fig. 2
). An alternative method, based on particle bombardment of plant leaves, was also used to study the subcellular distribution of CFDV Rep. When GFPRep fusion protein was transiently expressed in the epidermal cells of N. benthamiana leaves, fluorescence was detected predominantly in nuclei and at the cell periphery (Fig. 3A
D
). Free GFP is known to be associated partially with the plant cell nucleus (Reichel et al., 1996
). In control experiments with plants expressing non-fused GFP5 gene from the 35S promoter, the distribution of the fluorescence differed from that of GFPRep fluorescence (Fig. 3E
). No specific targeting of fluorescence was observed and the distribution of fluorescence was typical for free GFP in plant cells (Reichel et al., 1996
; Morozov et al., 1999
). This allowed us to conclude that the distribution of GFP fluorescence, presented in Fig. 3
(A
D
), reflected the distribution of Rep in the plant cell. Thus, the putative Rep of CFDV is largely associated with the nucleus, the cell compartment where Reps normally exert their functions (Palmer & Rybicki, 1998
).
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Acknowledgments |
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References |
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Received 17 May 2000;
accepted 11 August 2000.
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