Department of Microbiology and Immunology1 and Department of Obstetrics and Gynecology2, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
CREST Program of the Japan Science and Technology Corporation, Sendai 980-8575, Japan3
Author for correspondence: Kazuo Sugamura. Fax +81 22 717 8097. e-mail sugamura{at}mail.cc.tohoku.ac.jp
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Abstract |
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Introduction |
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B19 carries three major viral proteins: VP1 and VP2, virus capsid proteins, and NS1, a non-structural protein (Ozawa et al., 1987 ). NS1 is known to be implicated in virus replication (Ozawa et al., 1986
), activation of virus and cellular gene transcription (Doerig et al., 1990
; Moffatt et al., 1996
; Sol et al., 1993
) and target-cell cytotoxicity (Moffatt et al., 1998
; Momoeda et al., 1994
; Ozawa et al., 1988
). We showed previously that NS1-induced cytotoxicity was mediated by caspase 3 and inhibited by the overexpression of Bcl-2, suggesting that NS1 is an apoptotic protein of B19 (Moffatt et al., 1998
). In addition, the commitment of B19-infected cells to undergo apoptosis is combined with their accumulation in the G2/M phase of the cell cycle (Sol et al., 1999
). Based on these observations, we speculated that the severe anaemia of the B19-infected foetus might result from the expression of cytotoxic NS1 in erythroid precursor cells. However, there has been no direct evidence that expression of NS1 induces NIHF in vivo. Hence, to establish a mouse model of human NIHF, we generated NS1-transgenic mice in which NS1 expression was strictly confined to erythroid-lineage cells.
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Methods |
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Cell culture.
K562 is an erythroleukaemia cell line, UT-7/Epo is a megakaryocytic cell line adapted for growth dependent on erythropoietin (Shimomura et al., 1992 ), COS7 is a simian virus 40-transformed fibroblast cell line and MT-1 is a human adult T-cell leukaemia cell line. The cell lines were maintained in RPMI 1640 medium containing 10% FCS and 2 U erythropoietin (Kirin Brewery Pharm. Res. Lab.) was added to the medium for the UT-7/Epo cells.
Immunoprecipitation and immunoblotting.
Immunoprecipitation and immunoblotting were performed as described previously (Moffatt et al., 1996 , 1998
). In brief, cells were lysed in RIPA buffer (pH 7·5) containing 10 mM TrisHCl (pH 7·5), 1% NP-40, 0·1% sodium deoxycholate, 0·1% SDS, 150 mM NaCl, 1 mM EDTA and 10 µg/ml aprotinin. The lysates were then immunoprecipitated with a mAb specific for NS1 coupled to protein ASepharose beads and anti-mouse IgG (Zymed). The immunoprecipitates were separated on a 10% polyacrylamide gel and transferred to PVDF filters (Millipore). After being incubated in PBS containing 3% BSA, the blots were probed with a mAb against NS1 protein and immune complexes were visualized by enhanced chemiluminescence according to the manufacturers instructions (Amersham).
Southern blot analysis.
Tissue DNA was isolated from tails of mice using the QIAamp Tissue kit (Qiagen). An aliquot (10 µg) of the DNA was digested with XbaI, separated on a 1·0% agarose gel and transferred onto Gene Screen Plus (NEN Research Products) and then hybridized overnight at 42 °C in 6xSSC/0·5% SDS using 32P-labelled probes corresponding to the NS1 coding region (398 bp) or the Cre coding region (328 bp). The membrane was washed in 2xSSC/0·1% SDS and exposed to X-ray film. The signal was quantified with an image analyser (BAS2000, Fuji) and the copy number was estimated by using the control plasmid to generate the transgene.
PCR analysis to genotype the transgenic mice.
Mouse tails and embryos were lysed with 0·5% NP-40, 1 µg/µl proteinase K and Taq TM PCR buffer (Takara Shuzo) for 2 h and incubated at 95 °C. Approximately 1 µg of DNA extracted from the lysates was subjected to 35 cycles of amplification on a thermal cycler. The primers used for amplification of sequences within the NS1 gene were N1 (5' CACAGACACCAGTATCAGCAGCAGT 3') and N2 (5' CACACATAATCAACCCCAACTAACG 3'), which produced 261-bp DNA fragments. The primers used for amplification of sequences within the cre gene were C1 (5' AAAAACTATCCAGCAACATT 3') and C2 (5' TAACATTCTCCCACCGTCAG 3'), which produced 328-bp DNA fragments. The primers used for amplification of sequences within the lacZ gene were L1 (5' GCGTTACCCAACTTAATCG 3') and L2 (5' TGTGAGCGAGTAACAACC 3'), which produced 320-bp DNA fragments (Sakai & Miyazaki, 1997 ). PCR products were analysed by electrophoresis in 2% agarose gels.
Detection of
-galactosidase in whole embryos.
For whole-mount X-Gal staining, embryos were removed at embryonic day 10·5 (E10·5). They were fixed at 4 °C for 2 h in 1% formaldehyde, 0·2% glutaraldehyde and 0·02% NP-40 in PBS. After washing with PBS, they were incubated at 37 °C for 5 h in 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 and 1 mg/ml X-Gal in PBS.
Blood analysis.
Embryonic blood was obtained from umbilical cord vessels using microhematocrit tubes (Microcaps, Drummond Scientific). Haematocrit measurements of embryonic blood were taken after centrifugation for 3 min. Giemsa staining of blood smears was performed as described previously (Socolovsky et al., 1999 ).
RNA isolation and RTPCR analysis.
To confirm expression of the NS1 gene mRNA, we performed RTPCR. Total RNA was isolated from liver and spleen with Trizol reagent (Technologies TM) according to the manufacturers instructions. The RNA concentration was determined from the absorbance. The isolated RNA was reverse-transcribed to cDNA using random hexamers as primers and Superscript II reverse transcriptase (Life Technologies) according to the manufacturers instructions and the cDNA was then used as a template for PCR. PCR was performed under the same conditions as the genotype analysis.
Real-time RTPCR.
Real-time RTPCR was performed as described previously (Barbany et al., 2000 ; Bièche et al., 1999
). cDNA was synthesized from 3 µg total RNA extracted from each embryonic liver and then subjected to PCR amplification. The PCR was performed in a 50 µl volume. The PCR mixture contained 2 µl of each appropriate RT reaction cDNA sample, 5 µl 10xSYBR Green PCR buffer, 5 µl 25 mM MgCl2, 4 µl dNTP mix, 0·5 µl AmpErase UNG, 15 pmol forward and reverse primer and 0·25 µl AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems). All PCRs and detection of real-time fluorescent energy were performed using an ABI Prism 7700 Sequence Detection system (Perkin-Elmer Applied Biosystems). The thermal cycling conditions consisted of an initial denaturation step at 95 °C for 10 min and 50 cycles at 95 °C for 15 s and 60 °C for 1 min. Each PCR result was calculated from a standard curve, constructed by using various concentrations of NS1 cDNA (10-fold serially diluted cDNA ranging between 10-10 and 10-14 g). A strong linear relationship between the threshold cycle (Ct) and the log of the starting cDNA copy number was continually demonstrated (r2>0·98; data not shown). The
-actin signal was used to normalize the amount of input cDNA. Experiments were performed with duplicates for each data point.
Whole-mount in situ hybridization.
Embryos were fixed in 4% paraformaldehyde on ice for several hours and processed for whole-mount in situ hybridization. After dehydration and rehydration, the embryos were incubated with digoxigenin-labelled probe and stained with the alkaline phosphate substrate, NBT/BCIP, under the conditions recommended by the manufacturer (Boehringer Mannheim). Antisense and sense RNAs of NS1 were labelled with digoxigeninUTP and used as probes.
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Results |
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Establishment of transgenic mouse lines
We first established a GATA1-Cre transgenic mouse line (line 453) expressing IE3.9int-cre. In order to confirm that the Cre recombinase in GATA1-Cre mice was functional, we used transgenic mice bearing a reporter gene construct, CAG-CAT-Z, which directs the expression of the E. coli lacZ gene upon the Cre-mediated excision of the CAT gene located between the CAG promoter and the lacZ gene (Sakai & Miyazaki, 1997 ). GATA1-Cre mice were crossed with heterozygous CAG-CAT-Z mice. The yolk sacs of the F1 progeny mice were examined at E10 for LacZ expression by X-Gal staining, because the yolk sac is an active haematopoietic centre at E10. In the F1 progeny, positive X-Gal staining was observed in the yolk sacs derived from embryos that carried both the CAG-CAT-Z transgene and the IE3.9int-cre transgene, indicating that the Cre recombinase of the GATA1-Cre mice was functional in vivo (data not shown).
We next established three independent GATA1-Neo-NS1-transgenic mouse lines (lines 464, 468 and 175) containing IE3.9int-loxP-neo-loxP-NS1 transgenes. In all three lines, the transgene was unable to drive expression of the NS1 gene because the neo gene was inserted between the IE3.9int promoter and the NS1 gene (Fig. 1A). The F1 progeny of the GATA1-Neo-NS1 lines 464, 468 and 175 respectively had 5, 30 and 70 copies of the NS1 gene, as determined by Southern blot analysis (data not shown). We also confirmed that the founder mice carried the transgenes in a heterozygous manner and that the transgenes were transmitted to their progeny according to Mendelian expectations.
In order to obtain transgenic mice expressing NS1 in erythroid-lineage cells, we crossed heterozygous GATA1-Neo-NS1 mice with heterozygous GATA1-Cre mice; a quarter of their progeny were expected to express the NS1 gene due to direct recombination between the IE3.9int promoter and the NS1 gene. The F1 progeny were genotyped and shown to include loxP-NS+/cre+ mice carrying both the IE3.9int-cre and IE3.9int-loxP-neo-loxP-NS1 transgenes, loxP-NS+/cre- mice carrying only IE3.9int-loxP-neo-loxP-NS1, loxP-NS-/cre+ mice carrying only IE3.9int-cre and loxP-NS-/cre- wild-type mice.
NS1 expression in the haematopoietic organs of F1 progeny obtained from crossing GATA1-Neo-NS1 mice with GATA1-Cre mice
We first analysed 195 F1 progeny obtained from crossing line 468 of the heterozygous GATA1-Neo-NS1 mice with heterozygous GATA1-Cre mice at weaning. The number of loxP-NS+/cre+ mice was approximately half that of the loxP-NS+/cre-, loxP-NS-/cre+ or wild-type mice at weaning (Table 1). These weanlings were examined by RTPCR for expression of the NS1 gene in the spleen, which is a haematopoietic organ. NS1 gene expression was detected in loxP-NS+/cre+ mice but not in the other genotypes (Fig. 2A
). Hence, we genotyped the F1 progeny further at various days of gestation. The number of loxP-NS+/cre+ embryos was not significantly less before E13·5 compared with the other F1 littermates, but decreased appreciably after E13·5 (Table 1
). We also examined the embryos by RTPCR at E12·5 for expression of the NS1 gene in the liver, which is an embryonic haematopoietic organ. Of more than ten embryos in the same uterus, NS1 gene expression was detected in only three loxP-NS+/cre+ embryos (Fig. 2B
) and not in the other genotypes. Furthermore, we examined the quantitative expression of the NS1 gene in these three loxP-NS+/cre+ embryos by real-time RTPCR. The cDNA copy number of the lane 3 embryo was approximately twofold higher compared with the lane 2 and lane 4 embryos (Fig. 2B
). These data may suggest that the embryos lived to different stages because NS1 was expressed at different levels.
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Discussion |
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In the F1 progeny from crosses between line 468 GATA1-Neo-NS1 mice and GATA1-Cre mice, approximately half the loxP-NS+/cre+ genotype mice, which expressed NS1, were born alive and the other half died in utero, presenting with hydropic features and severe anaemia, probably due to the disruption of erythropoiesis. These phenotypes of the NS1-transgenic mice are similar to those of human NIHF, which occurs when there is an intrauterine B19 infection during pregnancy, and indicate that the NS1-transgenic mice can be used as an animal model of this disease. In contrast, GATA-1-deficient mice, despite showing completely defective erythropoiesis in the primitive haematopoietic centre (Fujiwara et al., 1996 ; Pevny et al., 1991
; Shivdasani & Orkin, 1996
), do not show hydropic characteristics. The NS1 protein is known to have a variety of functions in addition to inducing apoptosis, as outlined above. Recently, we demonstrated B19-induced G2 cell-cycle arrest with suppression of nuclear import of cyclin B1, which was thought to be mediated by NS1 expression (Morita et al., 2001
). Thus, it is possible that these various functions associated with the NS1 protein influence the occurrence of hydropic changes in the foetus.
The severity of embryonic damage in the loxP-NS+/cre+ F1 progeny varied with the transgenic line: loxP-NS+/cre+ F1 progeny resulting from crosses with line 175 mice were recovered at weaning, but in a significantly lower proportion than those resulting from line 468, and none were recovered after E7·5 from line 464, indicating that the NS1 gene induced the most severe embryonic damage in line 464. We believe that the severity of embryonic damage is primarily dependent on the level of NS1 expression in the F1 progeny, but also on the leakiness of NS1 expression during embryogenesis. Since line 464 showed the lowest NS1 copy number of the three transgenic lines, the level of NS1 gene expression did not correlate with its copy number and it is possible that the level of expression was dependent on either its integration site or modifications such as acetylation and methylation. Thus, our NS1-transgenic mice include early foetal loss prior to liver haematopoiesis. In humans, similar early foetal loss associated with B19 infection has also been reported (Miller et al., 1998 ). Furthermore, we showed that F1 progeny derived from the same line lived to different stages and that the levels of NS1 expression were different among the littermates. We think that the differences in NS1 expression among members of the same F1 generation may be due to differences in individual mice. Further research will be necessary to clarify the mechanisms involved.
We previously detected B19-infected erythroid-lineage cells in tissues derived from foetal patients with NIHF (Yaegashi et al., 1999 ). These cells exhibited apoptotic features similar to UT7/Epo cells infected with B19 in vitro (Yaegashi et al., 1999
), suggesting that anaemia observed in patients with NIHF results from B19-induced cytotoxicity of erythroid-lineage cells. Since NS1 is known to possess cytotoxic activity through apoptosis, the anaemia is thought to be caused by NS1 expression in erythroid-lineage cells. Our NS1-transgenic mice provide direct evidence that NS1 expression in vivo in erythroid-lineage cells induces anaemia.
Previously, it has been shown that B19 infects human erythroid-lineage cells in vivo as well as in vitro preferentially through its receptor, P antigen (Brown et al., 1993 , 1994
). However, the P antigen is reportedly expressed on foetal cardiac myocytes (Rouger et al., 1987
). Interestingly, B19 was found to infect myocardial cells of patients with NIHF, resulting in myocarditis (Lambot et al., 1999
; Porter et al., 1988
). Hence, not only anaemia but also myocarditis is thought to be implicated in the mechanism of B19-induced human NIHF (Morey et al., 1992
; Naides & Weiner, 1989
). Although the GATA1 promoter is known to be activated specifically in erythroid-lineage cells, we investigated whether our transgenic mice showed NS1-induced damage to the myocardium by leakiness of transgene expression. We observed that our NS1-transgenic mice showed no damage to the myocardium, which is compatible with our previous study that B19 infection was not detected in cardiac myocytes derived from patients with NIHF (Yaegashi et al., 1999
). These observations suggest that NS1 expression in erythroid-lineage cells is sufficient to cause an adverse murine foetal outcome like human NIHF, which may be mediated primarily by anaemia.
In the present study, we have shown hydropic change and embryonic death induced by NS1 expression in erythroid-lineage cells in mice, suggesting that the main cause of adverse outcome by intrauterine B19 infection in human is NS1-mediated cytotoxicity of erythroid-lineage cells. Thus, our NS1-transgenic mice are expected to be a useful model for analysing the stages of the disease in utero.
Recently, some cases of B19-associated non-hydropic foetal loss in the late-second and third trimesters have been reported (Tolfvenstam et al., 2001 ). Similarly, we have often encountered intrauterine foetal death with pale appearance and no hydropic change when B19 infection was detected in the foetus. These observations suggest that B19 infection may induce foetal adverse outcome without hydropic changes. Hence, further study is required to clarify whether other B19 components in addition to NS1 are involved in the foetal adverse outcome in B19 infection.
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Acknowledgments |
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References |
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Received 26 July 2001;
accepted 25 October 2001.