Human parvovirus B19 non-structural (NS) protein is supposed to play a major role in B19 replication and transcription, and therefore in B19 patho- genicity.
Journal of General Virology (1997), 78, 215–219. Printed in Great Britain ...............................................................................................................................................................................................................
SHORT COMMUNICATION
Establishment of a cell line expressing human parvovirus B19 non-structural protein from an inducible promoter Marianne Leruez-Ville,1,2 Isabelle Vassias,1 Coralie Pallier,1 Agnes Cecille,1 Uriel Hazan3 and Frederic Morinet1 Service de Microbiologie, Unite! de Virologie, Ho# pital Saint-Louis, and CNRS UPR9051, 1 avenue Claude Vellefaux, 75010 Paris, France Laboratoire de Bacte! riologie–Virologie, Ho# pital Avicenne, 125 avenue de Stalingrad, 93009 Bobigny, France 3 INSERM U380, Institut Cochin de Ge! ne! tique Mole! culaire, 22 rue Me! chain, 75014 Paris, France 1 2
Human parvovirus B19 non-structural (NS) protein is supposed to play a major role in B19 replication and transcription, and therefore in B19 pathogenicity. Constitutive expression of NS protein in stable cell lines has failed so far, presumably because of its cytotoxicity. To avoid this cytotoxic effect, we have cloned the NS gene in an Epstein– Barr virus episomal vector under the control of a steroid inducible promoter (5xGRE) and transfected this construction into HeLa cells. We obtained stable cell lines inducibly expressing high level of NS protein, with 50 % of the cells demonstrating specific nucleo-cytoplasmic staining. In Western blot analysis, three B19 NS proteins (72, 68 and 60 kDa) were found but a unique NS transcript was detected by Northern blotting. The NS protein expressed in HeLa cell lines was demonstrated to be functional as it trans-activates the B19 P6 promoter. These cell lines might be major tools for further study and characterization of B19 NS protein.
Human parvovirus B19 is the causative agent of erythema infectiosum, of transient aplastic crisis in patients with underlying anaemia and is associated with intra-uterine foetal, acute and chronic arthritis and chronic anaemia in immunocompromised patients. Parvovirus B19 contains a 5±4 kb, linear, single-stranded DNA genome and initiates all transcripts at a strong left side promoter at map unit 6 (P6) (Doerig et al., 1987). The right side of the B19 genome encodes two capsid proteins (VP1, VP2), and the left side one non-structural protein (NS ; 77 kDa) (Ozawa et al., 1987). Human erythroid progenitors are the target cells of parvovirus B19 and in vitro replication occurs only in these cells (Ozawa et al., 1986). The Author for correspondence : Frederic Morinet (mail to be sent to address 1). Fax 33 1 42 49 92 00.
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tropism of the virus for erythroid cells is due to the presence of its receptor, the P antigen (Brown et al., 1993), and probably to undetermined intracellular factors present only in these permissive cells. Megakaryocytic progenitor cells and some cell lines with erythroid characteristics are semi-permissive to B19 infection (Srivastava et al., 1990 ; Leruez et al., 1994). In these cells there is no B19 replication and mRNA encoding NS protein is preferentially synthesized because of a functional block in full-length transcription (Liu et al., 1992) ; therefore, NS protein is the only viral protein expressed. NS protein is presumably cytotoxic because after transient transfection it is detected in HeLa cells but stable transfected clones cannot be obtained (Momoeda et al., 1994 ; Ozawa et al., 1988). NS protein cytotoxicity might explain the lytic effect of B19 virus in semi-permissive cells such as megakaryocytes in vitro (Srivastava et al., 1990) and the fall of platelet counts described in experimental and natural infection. The B19 NS protein has significant homology with the T antigens of polyomaviruses and with the E1 proteins of papillomavirus (Astell et al., 1987). The homologous sequence is a nucleoside triphosphate (NTP)binding site, and NS protein may be an ATP-dependent helicase essential for B19 replication. Furthermore, NS protein increases transcription from the P6 promoter (Doerig et al., 1990) and transactivates the human immunodeficiency virus type 1 LTR in the presence of Tat protein (Sol et al., 1993). NS protein and}or the immune response against it might therefore play a major role in the pathogenicity of B19 infection. B19 NS protein has not been studied extensively. It has recently been produced in a prokaryotic system (Von Poblotzki et al., 1995), but no stable transfected eukaryotic clones have been obtained to date. We have established the first cell lines which express this protein from an inducible promoter. We have used an Epstein–Barr virus episomal vector (pGRE52}EBV) containing the EBNA-1 gene, the hygromycin B resistance gene and the glucocorticoid response elements (5xGRE) (Mader & White, 1993). This promoter is highly inducible by dexamethasone and displays very low basal activity in HeLa cells and very high activity after induction because these cells express high levels of glucocorticoid receptor.
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Fig. 1. Indirect immunofluorescence assay with a specific NS antibody. In the HeLa-NS-21 cell line, 50 % of the cells demonstrate nuclear or nucleo-cytoplasmic staining after 48 h dexamethasone induction (b, ¬400). No HeLa-NS-21 cells were positive in the absence of induction (a, ¬200).
We first cloned the entire NS gene in plasmid pcDNAI}neo (Invitrogen). The NS gene was obtained after PCR amplification of plasmid pEMBL-B19 (Doerig et al., 1987). The NS gene from nucleotides 436 to 2442 was amplified using primers containing NotI restriction sites (underlined) : 5« GGG GCG GCC GCA TGG AGC TAT TTA GAG CG 3« and 5« GGG GCG GCC GCA ATC TAC AAA GCT TTG CA 3«. The pcDNAI}neo-NS plasmid was sequenced (T7 Sequencing Kit, Pharmacia) to confirm the correct sequence of the entire cloned NS gene. In vitro transcription and translation of the NS gene (T7 Coupled Reticulocyte Lysate System, Promega) allowed us to check for the presence of NS protein of the expected size. The NS gene obtained by NotI restriction of pcDNAI}neo-NS was subcloned in the baculovirus transfer vector pVL1392 (Invitrogen). A BglII–BamHI NS gene fragment was obtained by restriction of pVL1392-NS and cloned in pGRE5-2}EBV downstream of the 5xGRE promoter. pGRE5-2}EBV-NS and pGRE5-2}EBV were transfected into HeLa cells with lipofectin. One day before transfection, 2¬10& cells were plated on to 3±5-cm-diameter well dishes. Briefly, 200 µl of serum-free medium (Opti-MEM I, Gibco BRL) containing 3 µg DNA were incubated for 30 min with 5 µg lipofectin (Gibco BRL) diluted in 200 µl of Opti-MEM I ; 600 µl of Opti-MEM I was then added to the lipofectin reagent–DNA complexes and used to transfect HeLa cells overnight at 37 °C in 5 % CO . After removal of the lipofectin # reagent–DNA complexes, cells were incubated in 3 ml of growth medium with or without 10−' M dexamethasone (Sigma ; tissue culture grade, tested for long-term stability and lack of toxicity by the supplier) for 48 h. Cells were then harvested and analysed for NS production by indirect immunofluorescence. Briefly, cytocentrifuged cells were fixed for 10 min with 3±7 % paraformaldehyde, and permeabilized with 100 % methanol for 10 min at 4 °C. A rabbit polyclonal antibody (diluted 1 : 250) specific for an NS C-terminal peptide
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was used (a generous gift from C. Astell, U.B.C., Vancouver, Canada). Transient transfections of HeLa cells with pGRE52}EBV-NS showed 5 to 10 % positive NS expressing cells. Stable cell lines expressing NS protein were obtained by hygromycin B (Boehringer Mannheim) selection at 300 µg}ml started 24 h after transfection. After 10 to 15 days, the surviving clones were isolated, amplified and assayed for NS protein expression. A total of 91 hygromycin B resistant clones was analysed ; among them 10 were found to express NS protein. In these 10 clones, 5 to 50 % of the cell population were positive for NS protein expression ; the intensity of immunofluorescence (indicating the amount of NS protein) was heterogeneous among positive cells and the staining was nuclear or nucleo-cytoplasmic. Two clones, HeLa-NS-21 and HeLa-NS-46, showed a high level of NS protein expression with respectively 50 % and 40 % of positive cells after induction (Fig. 1 b). No NS positive cells were observed in these clones without dexamethasone (Fig. 1 a) and in HeLa-pGRE5-2}EBV or basic HeLa cells with or without dexamethasone. In order to obtain a higher percentage of NS positive cells, HeLa-NS-21 cells were subcloned by limiting dilution, generating 53 subclones. Upon induction, all of those subclones displayed 30 to 50 % of NS expressing cells ; none of them displayed more than 50 %. We think that NS negative cells obtained after induction of HeLa-NS-21 and HeLa-NS-46 clones are more likely to be cells unable to express NS rather than cells lacking the NS gene. We noticed that the presence of the NS gene results in a slower growth rate of our NS expressing clones when compared to normal cells. If NS gene negative cells were present in HeLa-NS-21 and HeLa-NS-46 cell populations, they should have grown faster than NS gene containing cells and become the predominant cell type within the months that these cell lines were maintained in culture. Furthermore, if HeLa-NS-21 or HeLa-NS-46 NS negative cells were lacking the NS gene, we would have expected to obtain
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Fig. 2. (a) Western blot analysis of proteins extracted from HeLa, HeLapGRE5-2/EBV and HeLa-NS-21 cell lines induced or not induced by dexamethasone, revealed by a specific NS antibody. Three NS specific bands of 72, 68 and 60 kDa were found in HeLa-NS-21 cells only after induction (arrows). (b) Northern blot analysis of mRNAs extracted from HeLa, HeLa-pGRE5-2/EBV and HeLa-NS-21 cells induced or not induced by dexamethasone. Hybridization was performed using labelled NS gene as a probe. NS mRNA of 2±3 kb was present only in induced HeLa-NS-21 cells (arrow). A GAPDH probe was used as an internal control (arrow).
at least one completely negative clone among the 53 subclones studied. This stable percentage of NS expressing cells could be explained by a cycle-dependent regulation of NS expression. Western blot analysis was performed on cells extracts. Proteins were run on SDS–PAGE and electro-transferred for 90 min at 12 V onto PVDF membranes (Immobilon-P, Millipore) in a 20 % methanol, 25 mM Tris–HCl (pH 8±0), 192 mM glycine transfer buffer. Membranes were reacted with a 1 : 500 diluted rabbit polyclonal anti-NS antibody, a generous gift from S. Modrow (IMMHIM, Regensburg, Germany). When induced by dexamethasone HeLa-NS-21 and HeLa-NS-46 clones expressed NS protein, which was seen on Western blots as three bands of 72, 68 and 60 kDa ; the 72 kDa band was the more intense (Fig. 2 a). The three bands were not present in
HeLa cells and in non-induced HeLa-NS-21 extracts (Fig. 2 a). Three NS bands have already been described when NS was obtained by in vitro translation or transient transfection (Doerig et al., 1990 ; Ozawa et al., 1987, 1988). In order to find out if those three NS proteins could be derived from different transcriptional products, we did a Northern blot. Total cellular RNAs were extracted (RNAzol B, Bioprobe Systems) ; 10 µg was then loaded onto a formaldehyde–agarose gel, blotted to a nitrocellulose filter (Optitran BA-S 85, Schleicher and Schuell) and hybridized using the entire NS gene as a probe. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used as an internal control. The two probes were labelled with [$#P]dCTP (Amersham) using a nick-translation kit (Boehringer Mannheim). We found a unique NS RNA transcript of 2±3 kb in induced HeLa-NS-21 cells (Fig. 2 b) as described in B19 infected bone marrow cells (Ozawa et al., 1987). In non-induced HeLa-NS-21 and in induced and noninduced HeLa-pGRE5-2}EBV and HeLa cells no NS transcripts were detected (Fig. 2 b). This suggests that post-transcriptional modification or protein processing are responsible for the presence of the three NS proteins. Either the 68 and 60 kDa proteins may correspond to premature termination of translation or they are secondary to phosphorylation of the B19 NS protein, as has been described for the mouse minute virus (MMV) NS1 protein (Cotmore & Tattersall, 1986) and suggested for B19 NS protein itself (Kawase et al., 1995). To confirm the cytotoxic effects of B19 NS protein, 200 HeLa or HeLa derived cell clones were plated on 10-cmdiameter dishes with 9 ml of growth medium. Twenty-four hours later 10−' M dexamethasone was added or not. After 14 days the plates were fixed for 10 min in methanol and colonies were stained overnight with crystal violet and counted. Expression of NS protein in induced HeLa-NS-21 cells led to a drastic diminution (" 90 %) of plating efficiency. Dexamethasone alone also had a cytotoxic effect : we observed a 30 to 40 % decrease of plating efficiency when control cell line HeLapGRE5-2}EBV and basic HeLa were plated in the presence of dexamethasone. However, the NS protein related cytotoxicity was significantly higher (P ! 0±001) as indicated by χ#-analysis. The number of NS gene copies present in HeLa-NS-21 cells was estimated by Southern blot analysis. Briefly low-molecular-mass DNA was extracted from 10( HeLa-NS-21 cells (Hirt, 1967). Extracted DNA (10, 5, 3 and 1 µg samples) was run on a 0±6 % agarose gel after BglII restriction. BglII linearized pGRE5-2}EBV-NS plasmid (1 µg, 100, 10 and 1 ng, 100, 10 and 1 pg samples) was run on the same gel to be used as a comparative scale. Plasmid pGRE5-2}EBV-NS was used as a probe and was labelled as described above. After hybridization the 12±3 kb band (corresponding to the size of plasmid pGRE52}EBV-NS) observed in Hirt extracts of 10' HeLa-NS-21 cells was estimated at 10 pg. We considered that 1 pg of our plasmid contained 10' DNA molecules (as 10& EBV molecules ¯ 1 pg) and that HeLa-NS-21 contains a high NS gene episomal copy number (about 10 per cell) (data not shown).
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M. Leruez-Ville and others Canada), P. Tattersall (Yale University School of Medicine, New Haven, Connecticut, USA) and S. Modrow (Institut fu$ r medizinische Mikrobiologie und Hygiene and Innere Medizin, Universita$ t Regensburg, Germany) for the generous gifts of NS polyclonal antibodies without which this study would not have been possible. We thank P. Beard (ISREC, Epalinges, Switzerland) for the kind gift of BP-06 plasmid DNA.
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Fig. 3. P6 transactivation when 1±5 µg of pGL2-B-P6 was transfected in HeLa, HeLa-pGRE5-2/EBV and HeLa-NS-21 cell lines. Twelve hours after transfection cells were induced where indicated by dexamethasone. Luc activities were determined after 72 h induction. *, ®dexamethasone ; +, dexamethasone.
In order to determine whether NS expressed in the HeLaNS-21 clone was functional we demonstrated that it was able to enhance transcription from the P6 promoter. We used plasmid pSV0CAT-P6 given to us by P. Beard (Doerig et al., 1990) and we constructed plasmid pGL2-B-P6 by subcloning the HindIII fragment obtained from pSV0CAT-P6 in pGL2Basic vector (Pharmacia). Cell lines were transfected in four different assays with 0±5, 1, 1±5, 2 and 3 µg of pSV0CAT-P6 or pGL2-B-P6, maintained for 12 h, further incubated with or without 10−' M dexamethasone for 72 h and harvested. CAT activities were determined using thin-layer chromatography and Luc activity was measured as previously described (Sol et al., 1993). We found an average of 21-fold transactivation of the P6 promoter when NS protein was expressed (Fig. 3). When pGL2-B-P6 was transfected in basic HeLa cells or the control HeLa-pGRE5-2}EBV cell line, we obtained a 2- to 4fold transactivation after induction, demonstrating that dexamethasone by itself was responsible for a low level of P6 activation. But the transactivation of P6 promoter was significantly higher (P ! 0±001) when NS protein was present. We have confirmed those results when using pSV0CAT-P6 for transfection (data not shown). The aim of this study was to establish cell lines expressing B19 NS protein. Since NS protein may be cytotoxic in transfected cells (Momoeda et al., 1994 ; Ozawa et al., 1988), we used the 5xGRE promoter, a steroid inducible promoter. As reported previously for adeno-associated virus (Ho$ lscher et al., 1994 ; Yang et al., 1994) and MMV (Caillet-Fauquet et al., 1990), it seems that the use of an inducible promoter is necessary for the establishment of transfected cell lines expressing parvoviral NS proteins. The 5xGRE promoter proved to be essential for successful establishment of the first stable cell lines producing B19 NS protein. These cell lines, which proved to express functional NS protein, will be of major interest to study the interaction between this protein and the cell cycle or different cellular promoters. We thank C. Astell (University of British Columbia, Vancouver,
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Received 10 July 1996 ; Accepted 3 September 1996
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