Department of Microbiology, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan1
Department of Bacteriology, Hyogo College of Medicine, Hyogo, Japan2
Author for correspondence: Yasuko Mori. Fax +81 6 6879 3329. e-mail ymori{at}micro.med.osaka-u.ac.jp
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Abstract |
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Introduction |
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Both HHV-6 variants, represented by HHV-6A strain U1102 (Gompels et al., 1995 ) and HHV-6B strains HST (Isegawa et al., 1999
) and Z29 (Dominguez et al., 1999
), contain a linear, double-stranded DNA genome of approximately 161 kbp with 112 potential open reading frames (ORFs). Nucleotide sequencing has shown that HHV-6A and HHV-6B contain an ORF, U94, that encodes a 490-amino-acid protein homologous to Rep 78/68, a non-structural protein from the human parvovirus adeno-associated virus type 2 (AAV-2) (Thomson et al., 1991
; Gompels et al., 1995
; Isegawa et al., 1999
; Dominguez et al., 1999
). Interestingly, the AAV-2 rep gene homologue is unique to HHV-6 and is not present in other human herpesviruses, making the role of HHV-6 REP in the life-cycle of HHV-6 of particular interest. A rep homologue has recently been identified in the genome of rat cytomegalovirus (Vink et al., 2000
).
AAV-2 Rep is known to possess several biological activities, including DNA binding and site- and strand-specific endonuclease, helicase and ATPase activities, all of which are required for AAV-2 DNA replication (Im & Muzyczka, 1990 , 1992
). A distinctive feature of AAV-2 is that the virus DNA integrates preferentially within a defined region of the cellular genome (Linden et al., 1996
). HHV-6 has also been reported to integrate into the human genome (Luppi et al., 1993
, 1994
; Torelli et al., 1995
; Daibata et al., 1998
). Conservation between HHV-6 and AAV-2 may mean, therefore, that HHV-6 REP possesses a similar range of functions advantageous to the survival of HHV-6 within the host. In support of this idea, HHV-6 REP has been shown to complement replication of a rep-deficient AAV-2 genome (Thomson et al., 1994
), suggesting that they might have similar biological functions.
Recently, we reported the results of immunohistochemistry experiments using a polyclonal antibody that showed that HHV-6 REP was localized to the nucleus of HHV-6-infected cells and that the protein was expressed at the late stage of virus infection (Mori et al., 2000 ). However, we could not detect the REP protein in HHV-6-infected T cells by either Western blotting or immunoprecipitation using the polyclonal antibody. This observation prompted us to make monoclonal antibodies (mAbs) against REP. In this study, we have isolated mAbs against REP and detected the protein in HHV-6-infected T cells by Western blotting. Furthermore, we report here that REP possesses single-stranded (ss) DNA-binding activity.
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Methods |
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Expression of the rep gene in E. coli.
A DNA fragment comprising the entire amino acid coding region of HHV-6B rep was amplified by PCR and cloned, in-frame, into the pMALTM-c2 bacterial expression vector (New England Biolabs) at the BamHI and SalI sites. This vector also contained the coding region for maltose-binding protein (MBP) and the resultant fusion protein (MBPREP) was expressed in E. coli DH5.
Preparation of REP by using recombinant baculovirus.
A DNA fragment comprising the full-length rep gene ORF was amplified by PCR and inserted into the BamHI and HindIII sites of the pFastBac donor plasmid (Gibco BRL). The nucleotide sequences of the primers used for the PCR were: sense, 5' ggatccCCACCATGTTTTCCATAATAAATCCAAGT 3'; antisense, 5' aagcttGTTAAAATTTTTGGAACCGTGTAGTC 3' (lower-case letters indicate additional restriction sites). The pFastBac-recombinant virus containing REP was prepared in accordance with the manufacturers protocol (Bac to BAC Baculovirus expression systems; Gibco BRL).
Establishment of mAbs.
BALB/c mice were immunized with AcGSTREP, a purified fusion of glutathione S-transferase (GST)REPmyc (Mori et al., 2000 ), and boosted with MBPREPN1, the purified fusion of MBPREP (Mori et al., 2000
), as described previously (Okuno et al., 1990
). The first immunization was carried out with 100 µg GSTREPmyc in complete Freunds adjuvant and followed by three boosters with 100 µg MBPREPN1, 34 weeks apart, in incomplete Freunds adjuvant.
Hybridomas were established by fusing splenocytes from the hyperimmune mice with the non-producing myeloma cell line Sp2/0-Ag14. After selection in medium containing hypoxanthine/aminopterin/thymidine, cells secreting mAbs were screened by indirect immunofluorescence assays (IFA). Clones secreting antibodies reactive with HHV-6B- (HST strain) infected MT4 cells and baculovirusREP- (BacREP) infected Sf9 cells were expanded and cloned by limiting dilutions. Ascites fluids with high antibody titres were then accumulated by injecting cloned hybrid cells intraperitoneally into mice treated with Pristane (Sigma).
Immunohistochemical analysis of HST-infected MT4 cells.
In order to confirm the REP expression patterns, immunohistochemical analysis was carried out using the anti-REP mAb as described previously (Mori et al., 2000 ). HST-infected MT4 cells were collected at 12, 24, 48 and 72 h post-infection (p.i.). The cells were fixed in cold acetone and incubated at 37 °C for 1 h with primary antibodies: the anti-REP mAb, OHV-2, which recognizes a nuclear protein expressed in the early stage, or OHV-3, which recognizes HHV-6B glycoprotein H (gH) expressed in the late stage. After washing with PBS for 10 min, fluorescein-conjugated goat antibodies against mouse IgG were added with saturated 4',6'-diamidino-2-phenylindole (DAPI) at a 1:100 dilution. The cells were incubated for 20 min. After washing as above, signals were detected by confocal microscopy.
Western blot analysis.
Western blot analysis was carried out as described previously (Mori et al., 2000 ). Cell lysates were prepared from mock- and HST-infected MT4 cells, mock- and BacREP-infected Sf9 cells or mock- and vaccinia virus-infected 293T cells transfected with pcDNArepB (Mori et al., 2000
) at 72 h p.i. Samples were subjected to 8% SDSPAGE, transferred to PVDF membranes (Bio-Rad) and reacted with the primary antibodies. Reactive bands were visualized using horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescence detection reagents (ECL; Amersham Pharmacia).
Preparation of nuclear extracts.
Nuclear extracts were prepared as described previously (Ni et al., 1994 ). After harvesting the cells by centrifugation, 5x106 SupT1 cells were washed once by suspension in buffer A (20 mM HEPESKOH, pH 7·5, 5 mM KCl, 0·5 mM MgCl2, 0·5 mM DTT) containing 10% sucrose. The cells were then suspended in 500 µl buffer A and incubated on ice for 30 min. They were then lysed by sonication. Nuclei were collected by centrifugation at 2000 g for 10 min and resuspended in 100 µl buffer B (50 mM HEPESKOH, pH 7·5, 10% sucrose, 0·4 mM EDTA, 0·3 mM PMSF, 2 µg/ml leupeptin and 1 µg/ml pepstatin). The suspension was adjusted to 1 M NaCl and incubated at 4 °C for 1 h and then spun at 100000 g for 1 h at 4 °C and frozen at -70 °C.
Preparation of cytoplasmic and nuclear extracts from baculovirus-infected Sf9 cells.
Sf9 cells (2x107) were grown in Graces insect cell culture medium (Gibco BRL). The cells were infected with recombinant baculovirus (m.o.i. of 510) and incubated at 27 °C for 3 days. Cytoplasmic and nuclear extracts were prepared as described previously (Ni et al., 1994 ). Briefly, after harvesting by centrifugation, the cells were washed once with buffer A supplemented with 10% sucrose. The cells were then suspended in 1 ml buffer A, incubated on ice for 30 min, lysed by sonication and spun at 2000 g for 10 min. The supernatant was collected as the cytoplasmic fraction and the pellet containing the nuclei was resuspended in 200 µl buffer B. The suspension was adjusted to 1 M NaCl and incubated at 4 °C for 1 h and then spun at 100000 g for 1 h at 4 °C. Five µl of each fraction was subjected to 8% SDSPAGE, immunoblotted onto PVDF membranes and probed with the anti-REP mAb.
REP DNA-binding assay.
The affinity between REP and DNA was examined by monitoring the elution profile of REP by DNA-affinity column chromatography (Tanaka et al., 1999 ). Sixty µg each of the fusion protein MBPREP and MBP in binding buffer (100 mM NaCl, 10 mM TrisHCl, pH 7·4, 25 mM KCl, 0·05% Tween 20, 1 mM PMSF and 0·5 mM EDTA) was applied to 500 µl unmodified cellulose or ssDNAcellulose (Pharmacia Biotech) in polyprep columns (Bio-Rad). Each column was washed with 250 ml binding buffer and the materials were eluted step-wise with 500 µl volumes of 0·1, 0·2, 0·3, 0·4, 0·6 and 1 M NaCl in binding buffer. Twenty µl each of the input, flow-through, first wash and fractions of each salt concentration were subjected to Western blot analysis with the anti-MBP polyclonal antibody.
In order to determine whether a cellular factor is involved in the binding of REP to ssDNA, nuclear extracts from 5x106 SupT cells were mixed with 30 µg of each protein and applied to unmodified cellulose or ssDNAcellulose columns. The binding assay was carried out as described above.
Probes.
The DNA fragments used in the mobility-shift assay were prepared by synthesis of oligonucleotides. The DNA fragments were 5'-end labelled with [-32P]ATP and T4 polynucleotide kinase. The
ITR probes (Chiorini et al., 1994
) for double-stranded (ds) DNA binding were made by 5'-end labelling either oligomer and annealing with the unlabelled complementary oligonucleotide.
ITR oligonucleotides were produced synthetically as described previously (Chiorini et al., 1994
). The probe sequences were: AD' (
ITR sense), 5' GATCAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC 3'; A'D (
ITR antisense), 5' CTAGGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT 3'; and 68-mer oligonucleotide, 5' CTCGGTACCTCGAGTGAAGCTTGA(N25)GGGAATTCGGATCCGCGGTAAC 3'.
Mobility-shift assays.
DNAprotein complexes were detected by monitoring the electrophoretic mobility of 32P-labelled probes on a non-denaturing gel. For ssDNA binding, 32P-end-labelled 68-mer oligonucleotide and ITR AD' were used as probes. The reaction was performed in buffer (30 mM HEPESKOH, pH 7·5, 7 mM MgCl2, 4 mM ATP, 1 mM DTT, 0·1 mg/ml BSA, 5% glycerol) with either MBPREP or MBP and DNA for 30 min at room temperature. Experiments involving competition between the radiolabelled DNA substrates were performed as described in the figure legends. The samples were loaded on composite gels (2·5% acrylamide and 0·6% agarose) and subjected to electrophoresis in TBE buffer.
For dsDNA binding, radiolabelled probes were incubated with protein at 30 °C for 15 min in 25 µl. The reaction mixture contained 10 mM TrisHCl (pH 7·5), 1 mM EDTA, 1 mM DTT, 0·1% Triton X-100, 4% glycerol and 0·5 µg poly(dI.dC). MBPRep68 (Chiorini et al., 1994
), used as a positive control for dsDNA binding, was kindly provided by Robert M. Kotin (Molecular Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD, USA).
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Results |
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We also analysed the proteins from the in vitro translation (Fig. 3c, lane 8) and found that the molecular mass was similar to that seen in HST-infected cells. This suggests that there was no major post-translational modification. In addition, we could not detect HHV-6A REP in HHV-6A-infected CBMCs (data not shown) or in an in vitro translation of HHV-6A REP (Fig. 3b
, lane 2) using the anti-REP mAb. As shown in Fig. 3(a
, b
), the anti-REP mAb reacted with in vitro translation products of pcDNArepB (HHV-6B) (Fig. 3a
, b
; lanes 3) but not pcDNArepA (HHV-6A) (Fig. 3a
, b
; lanes 2). Furthermore, the mAb reacted with the amino terminus of REP expressed from pcDNA3.1 (Fig. 3 a
, b
; lanes 4) but not the carboxy terminus (Fig. 3a
, b
; lanes 5). Therefore, the mAb reacted with HHV-6B REP specifically and recognized the amino terminus of REP. The major products corresponding to the REP amino terminus (Fig. 3a
, b
; lanes 4) and full-length REP (Fig. 3c
, lanes 6 and 8) were seen as two bands; these protein mobilities correspond to those predicted from the use of two different start codons, as reported by Rapp et al. (2000)
.
REP has ssDNA-binding activity
DNAcellulose chromatography was performed in order to determine whether HHV-6 REP could bind ssDNA. The MBPREP fusion protein or MBP protein was applied to ssDNAcellulose, as described in Methods. As shown in Fig. 4(a), the ssDNAcellulose column retained MBPREP, but did not retain MBP. To examine this more precisely, the ssDNA-binding activity of HHV-6 REP was also tested by gel-shift experiments using the 5'-end-labelled ssDNA oligonucleotides 68-mer nucleotide and
ITR AD'.
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The dsDNA-binding activity of HHV-6 REP was also tested by using DNAcellulose chromatography with the same methods as the ssDNA column. The dsDNAcellulose column did not retain MBPREP, indicating that HHV-6 REP could not bind dsDNA non-specifically (data not shown). As AAV-2 Rep has been reported to bind specifically to dsDNA, we investigated whether HHV-6 REP would also bind to specific dsDNA sequences. To examine this, a mobility-shift assay was performed by using 32P-labelled ITR, a specific sequence for AAV-2 Rep binding. MBPRep68
, the positive control, formed a stable complex with ds
ITR (Fig. 5c
, lane 2), but MBPREP did not form a complex (Fig. 5c
, lane 3). Even when the amount of MBPREP protein was increased (maximum 5 µg), MBPREP
ITR complexes were not found (data not shown). Although we also examined whether MBPREP could bind HHV-6 DNA fragments by using random pSTY clones (Isegawa et al., 1999
), MBPREP did not bind the HHV-6 DNA fragments that we tested (data not shown), indicating that HHV-6 REP does not have dsDNA-binding characteristics similar to those of its counterpart from AAV-2.
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Discussion |
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In our previous study, we initially detected the HHV-6B REP protein in the nucleus at 24 h p.i. and found that it accumulated to high levels, mainly in the nucleus and, to a lesser extent, in the cytoplasm, within 72 h. However, an anti-REP polyclonal antibody did not detect the REP protein in the presence of phosphonoformic acid or cycloheximide and actinomycin D at any time (Mori et al., 2000 ). To allow further investigation, for this study, we produced an anti-REP mAb. Using this mAb, we detected the REP protein in the nucleus and cytoplasm of HST-infected MT4 cells 24 h p.i. by IFA (data not shown). However, REP could be detected in few cells, even when gH, which is expressed at the late stage of virus infection, was detected in almost all cells, indicating that REP might be expressed only at very low levels in HST-infected cells. Rapp et al. (2000)
also reported that the U94 transcript is expressed at low levels and that its expression is tightly regulated in HHV-6-infected cells. They also suggested that the U94 protein might be required only in small amounts during infection.
We also investigated the pattern of REP expression in recombinant baculovirus-infected cells using our anti-REP mAb. By Western blotting and IFA, we detected REP expression in the nucleus and cytoplasm at high levels. These results may indicate that the titre of the anti-REP mAb is high. In contrast, we did not detect HHV-6A REP as an in vitro translation product or in HHV-6A (U1102)-infected CBMCs with the anti-REP mAb. Therefore, the anti-REP mAb seemed to recognize only HHV-6B REP. In addition, the mAb reacted with the amino terminus of the REP protein and there are six amino acid differences between HHV-6A and HHV-6B in the amino terminus of REP; one of them could therefore be an epitope of the mAb.
In an in vitro translation and in pcDNA-transfected cells, the major products corresponding to the amino terminus of REP and full-length REP were detected as two bands. These protein mobilities correspond to those predicted from the use of two different start codons, as reported by Rapp et al. (2000) . However, only one band, of 56 kDa, was found in HHV-6B-infected MT4 cells and baculovirus-infected Sf9 cells, suggesting that the first start codon might be used for translation mainly in virus-infected cells.
In this study, by using two different methods, we report that a bacterially expressed MBPREP fusion protein possesses ssDNA-binding activity and that, when MBPREP fusion protein was mixed with nuclear extracts of SupT1 cells, the ssDNA-binding capacity increased. Although our results from the MBPREP fusion protein-binding assay indicate that REP can bind ssDNA weakly in the absence of other proteins, the increased binding in the presence of the SupT1 nuclear factors supports the idea that REP may interact with other cellular proteins that themselves bind DNA, and that these interactions result in the strong and tight binding of ssDNA.
It has been reported that AAV Rep 68 and Rep 78 directly bind the transcriptional coactivator PC4, an ssDNA-binding protein (Weger et al., 1999 ). PC4 also interacts with components of the general transcription machinery, namely TFIIA or the TATA box-binding protein (TBP), in a manner that is dependent upon the presence of TFIIA (Ge & Roeder, 1994
). It is believed that PC4 functions early, during formation of the TFIIATFIIDpromoter (DA) complex (Kaiser et al., 1995
), and that its function is dependent both on TBP-associated factors and on TFIIH. Previously, we reported that HHV-6 REP could bind TBP in vitro and in vivo (Mori et al., 2000
). Taken together, the published results and those concerning ssDNA binding from this study indicate that HHV-6 REP may play a role in DA complex formation and that its function is in virus gene regulation.
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Acknowledgments |
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References |
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Received 25 September 2001;
accepted 7 December 2001.