Laboratory of Virology and Immunology, Royal Veterinary and Agricultural University, Bülowsvej 17, 1870 Frederiksberg C, Denmark1
Department of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark2
Author for correspondence: Gert Bolt. Fax +45 35282742. e-mail gb{at}kvl.dk
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Abstract |
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Introduction |
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MV can cause apoptosis (Esolen et al., 1995 ; Fugier-Vivier et al., 1997
; Grosjean et al., 1997
; Okada et al., 2000
), inhibit the lymphoproliferative response (reviewed by Borrow & Oldstone, 1995
; Schneider-Schaulies & ter Meulen, 1999
), and induce interferons and other cytokines (see, for instance, Schneider-Schaulies et al., 1993
; Volckaert-Vervliet et al., 1978
). However, our understanding of the molecular pathways triggered in the cell by MV remains fragmentary.
With the DNA microarray technology, the expression of several thousand individual genes can be monitored (reviewed by Eisen & Brown, 1999 ). This technology has been used to identify cellular genes that are differentially expressed in response to infection with different viruses (see, for instance, Khodarev et al., 1999
; Nees et al., 2001
; Taylor et al., 2000
; Zhu et al., 1998
).
In the present study, we examined the influence of MV infection on cellular gene expression using cDNA microarrays.
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Methods |
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cDNA microarray analysis.
RNA was extracted from MV- or mock-infected primary PBMCs with the NucleoSpin RNA II kit (Clontech). The mRNA populations were used as templates for radiolabelled cDNA probes, which were produced with the Atlas Pure Total RNA Labelling System (Clontech), with primer mixes specific for each microarray (Clontech) and [-32P]dATP (Amersham Pharmacia Biotech). The probes were then hybridized overnight to cDNA microarrays on nylon membranes as recommended by the manufacturer (Clontech). After hybridization, the arrays were washed and incubated at -80 °C with BioMax MS film in BioMax Transcreen LE Intensifying Screens (Kodak) for intervals of 6 h to 14 days. The autoradiographs were developed, scanned and analysed with the AtlasImage 1.5 software (Clontech). Probes produced from each RNA preparation were hybridized to the Atlas human 1.2 I, 1.2 II and 1.2 III arrays each containing cDNA from 1176 human genes. Thus, the individual expression levels of more than 3000 genes of uninfected PBMCs and of PBMCs infected with vaccine strain MV or wild-type MV were compared.
RTPCR.
Results obtained in the microarray assays were confirmed by RTPCR on independent RNA extractions. The expression levels of genes found to be upregulated by MV infection using microarrays were compared for mock- and MV-infected cells. RNA extraction and RTPCR were carried out with the SV Total RNA Isolation System (Promega) and the Superscript One-Step RTPCR System (Life Technologies), respectively. Primer sequences for the individual genes were obtained from Clontech. For genes that were not included on the microarrays, primers were constructed as previously described (Bolt, 2001 ). The intensities of RTPCR bands in agarose gels after ethidium bromide staining were compared by densitometric scanning using the Quantiscan 2.1 software (Biosoft).
RNA dot blotting.
RNA was dot blotted to Hybond-N nylon membranes (Amersham Pharmacia Biotech) with a Bio-Dot Microfiltration Apparatus (Bio-Rad) as recommended by the manufacturer of the membranes. cDNA inserts of the MV haemagglutinin (H) protein gene and the human GAPDH gene were excised from the peH1SK construct (Schmid et al., 1987 ) and from a previously described construct (Bolt, 2001
), respectively. The inserts were purified, radiolabelled with [
-32P]dCTP, and hybridized to the dot blots, all as previously described (Bolt, 2001
). The hybridizations were analysed by autoradiography and densitometric scanning, as described above.
Western blotting.
PBMCs were boiled in SDSPAGE loading buffer. The lysates were electrophoresed and electroblotted to Hybond-C membranes, which were assayed with primary antibodies, sheep anti-mouse or donkey anti-rabbit horseradish peroxidase (HRP) conjugates (Amersham Pharmacia Biotech) diluted 1:300, and the HRP Conjugate Substrate kit (Bio-Rad). The following primary antibodies were employed: mouse anti-actin monoclonal antibody (mAb) (Neomarkers), rabbit anti-Bcl-3 (Upstate Biotechnology), rabbit anti-NF-B p50 (Zymed Laboratories), rabbit anti-NF-
B p52 (Upstate Biotechnology) and mouse anti-MV nucleocapsid (N) protein mAb clone 16AC5 (Sheshberadaran et al., 1983
).
Interferon ELISA.
Dilutions of supernatants from PBMC cultures and standard samples were tested for interferon (IFN)- and IFN-
by standard ELISA procedures using sheep anti-IFN-
(Endogen) or mouse anti-IFN-
(R&D Systems) mAb for coating and biotinylated mouse anti-IFN-
(Endogen) mAb or goat anti-IFN-
(R&D Systems) for detection.
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Results |
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When comparing the signals from infected cells with those of uninfected cells, we considered genes on the microarrays to be upregulated if their signal was increased by more than a factor of two for both Edmonston- and DK96A1-infected cells or by more than a factor of three for only one of the MV strains. The expression of genes that were upregulated according to these criteria was also examined by RTPCR on independent RNA extractions from mock, Edmonston- or DK96A1-infected primary PBMCs of the same donor. We identified nine genes that were upregulated by MV infection according to both the microarray and the RTPCR analyses (Fig. 2, Table 1
).
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The present finding that MV upregulated the activating transcription factor 4 (ATF-4) and the pro-apoptotic and growth arrest-inducing CHOP/GADD153 genes indicated that MV infection induces the endoplasmic reticulum (ER) stress response in PBMCs. We therefore examined the expression of other genes connected to this response. These genes were not included on the arrays, and their expression was only analysed by RTPCR. The mRNA levels of calreticulin, GRP78 (BiP), GRP94 (endoplasmin), calnexin, TFII-I, Herp and ERp57 were increased in MV-infected PBMCs (Fig. 2, Table 1
). We have previously demonstrated upregulation of the former four genes in MV-infected HEp-2 cells (Bolt, 2001
). Thus, MV appears to induce the ER stress response.
Taken together, we identified a total of 17 genes upregulated by MV infection of human PBMCs. These genes are presented in Table 1. To further validate the above findings, we examined the expression of the 17 genes by RTPCR on RNA extractions from primary PBMCs derived from another human donor and found that all 17 genes were still upregulated by MV Edmonston infection (not shown).
The Edmonston strain was more efficient than the wild-type in upregulating the 17 host cell genes (Table 1, Fig. 2
). We therefore compared the multiplication of the two viruses. RNA from the extractions used for array analysis and RTPCR was examined by dot blot hybridization. One µg of the RNA from Edmonston-infected PBMCs used for array analysis gave a stronger signal after hybridization with a radiolabelled probe of the MV H gene than 1 µg RNA from the similar wild-type-infected cells (Fig. 3A
). According to densitometric scanning, the ratio of the signal obtained with the MV H gene probe to the signal obtained with the GAPDH housekeeping gene probe was 3·5 times higher for the Edmonston strain than for the wild-type. The MV titre of the cell culture supernatant of the cells used for these two RNA extractions 48 h p.i. was 1·1±0·5x105 infectious doses (ID)/ml for the Edmonston-infected cells and 1·3±0·6x104 ID/ml for the wild-type-infected cells (means and standard deviations of triplicate assays). However, these two cultures were not grown in parallel. We therefore also examined the RNA analysed by RTPCR, as the Edmonston- and wild-type-infected cells used for those RNA extractions were grown in parallel. Again a stronger signal was obtained with the MV H gene probe from RNA of Edmonston-infected PBMCs than from RNA of PBMCs infected with the wild-type virus (Fig. 3B
). According to densitometric scanning, the above ratio was 6·2 times higher for the Edmonston strain than for the wild-type. The MV titre of the cell culture supernatant was 8·0±4·0x104 ID/ml and 1·7±0·6x104 ID/ml for the Edmonston- and wild-type-infected PBMCs, respectively.
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MV infection upregulates the IFN-, Bcl-3, and NF-
B p52 proteins in PBMCs
We then examined whether upregulation of proteins encoded by MV-induced genes could be detected. Supernatants from cultures of PBMCs from the same donor that was used for the expression array analyses were examined for type 1 IFN. Using ELISA, we measured < 0·8, 18·9±0·4, and 70·2±1·6 pg/ml interferon- (means and standard deviations of triplicate assays) in the medium of mock-infected, wild-type-infected, and Edmonston-infected PBMCs, respectively. Thus, for IFN-
, mRNA and protein levels appeared to be well correlated, and the Edmonston virus was a stronger IFN-
inducer than the DK96A1 wild-type (Fig. 2
, Table 1
). According to both expression array analysis and RTPCR, the Edmonston strain was also a strong inducer of IFN-
. Nevertheless, we were unable to detect IFN-
in any of the tested supernatants. Nees et al. (2001)
recently reported a similar finding. In that study, papillomavirus-induced IFN-
mRNA was also detected with an expression microarray, but IFN-
could neither be detected in cell culture supernatants nor in cell lysates. Discrepancies between the mRNA and protein levels of IFN-
may thus be a general phenomenon.
Other proteins of MV-induced genes were assayed by Western blot analysis of lysates from cultures of PBMCs from the same donor that was used for the expression array analyses. Initially, we tried to compare the intensities of bands from undiluted lysates, but that proved to be unreliable (not shown). Instead, we compared parallel dilutions of each lysate (Fig. 4). Bcl-3 was detected in MV-infected cells but not in uninfected PBMCs. In agreement with the mRNA levels, the amount of Bcl-3 seemed to be higher in Edmonston-infected cells than in DK96A1-infected cells (Fig. 4
). The NF-
B p52 subunit was detected in Edmonston-infected but not in DK96A1- or mock-infected cells (Fig. 4
). NF-
B are transcription factors consisting of dimers of the Rel protein family. The prototype NF-
B is a heterodimer of the p50 and p65 subunits (reviewed by Ghosh et al., 1998
). We therefore also compared the levels of p50 in MV-infected and uninfected cells. The p50 subunit was present in equal levels in mock-, DK96A1- and Edmonston-infected PBMCs (Fig. 4
). Thus, the MV-induced upregulation of the p52 NF-
B subunit was selective for that particular subunit. The MV N protein was detected in both DK96A1- and Edmonston-infected cells (Fig. 4
). In accordance with the above analyses on virus multiplication, PBMCs infected with the Edmonston virus appeared to contain more MV N protein than cells infected with the wild-type virus.
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Discussion |
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The ER stress response can play a key role in induction of apoptosis, and increasing evidence also points to a role in the IFN response (reviewed by Kaufman, 1999 ). We believe that MV infection induces the ER stress response due to the flux of viral glycoproteins through the ER. The migration of MV glycoproteins towards the cell surface is slow and incomplete (Cattaneo & Rose, 1993
; Hu et al., 1994
; Ogura et al., 1991
). In pulsechase experiments, MV glycoproteins were associated with ER chaperones for prolonged intervals (Bolt, 2001
), indicating that folding of the MV glycoproteins into their correct conformation is an extensive task for the ER folding machinery. ERp57 is a protein disulphide isomerase, which forms complexes with the ER chaperones calreticulin and calnexin. These complexes are believed to mediate folding of nascent glycoproteins in the ER (Oliver et al., 1999
; Molinari & Helenius, 1999
). Induction of these three genes as well as that of GRP78, which is also an ER chaperone, improves the capacity of the ER to handle incoming viral glycoproteins, thus protecting the cell against protein aggregation. Herp is a recently discovered stress-induced integral protein of the ER membrane. Herp has a ubiquitin-like domain, but the function of Herp and its role in the ER stress response remains to be determined (Kokame et al., 2000
). Activating transcription factor 4 (ATF-4), also called cAMP response element binding protein 2 (CREB-2), and transcription factor II-I (TFII-I) are both involved in the activation of promotors of genes upregulated by ER stress (Fawcett et al., 1999
; Parker et al., 2001
). ATF-4 was also found to be upregulated by other viruses in microarray studies (Khodarev et al., 1999
; Taylor et al., 2000
). The CHOP/GADD153 protein has been reported to block transition from the G1 to the S phase of the cell cycle (Barone et al., 1994
). Arrest of lymphocytes in the G0 or G1 phase of the cell cycle is believed to be an important mechanism behind the reduced lymphoproliferative response and the immunosuppression in patients with measles (McChesney et al., 1987
; Naniche et al., 1999
; Schnorr et al., 1997
). Thus, although purely speculative, it is possible that the ER stress response, through the induction of CHOP/GADD153, is involved in the MV-induced reduction of lymphocyte proliferation.
MV-induced upregulation of 2',5'-oligoadenylate synthetase (2-5A) and intercellular adhesion molecule-1 (ICAM-1) has previously been described (Soilu-Hänninen et al., 1996 ; Tilles et al., 1987
). Membrane protein E16 (CD98 light chain) is an amino acid transporter, which is disulphide-linked to the 4F2 (CD98 heavy chain) glycoprotein (Mastroberadino et al., 1998
). Interestingly, the CD98 heavy chain appears to modulate cell fusion by Newcastle disease virus and human parainfluenza virus type 2 (Okamoto et al., 1997
). E16 was also upregulated by cytomegalovirus (Zhu et al., 1998
).
A strong and broad IFN response comprising several IFN subtypes requires the positive feedback regulation of interferon regulatory factor (IRF) 3 and 7 in virus-infected cells. IRF-3 is constitutively expressed and activated post-translationally, whereas IRF-7 is regulated at the transcriptional level (reviewed by Nakaya et al., 2001 ). Servant et al. (2001)
recently demonstrated MV-induced IRF-3 activation, and in the present study we demonstrate MV-induced upregulation of the IRF-7 gene.
Several studies have shown that MV strains differ in their IFN-inducing properties, and it has been suggested that virulent MVs can inhibit the induction of type 1 IFN (see, for instance, Mirchamsy & Rapp, 1969 ; Naniche et al., 2000
; Volckaert-Vervliet et al., 1978
). Naniche et al. (2000)
recently reported that wild-type isolates, which had been isolated and passaged on lymphoid cells, were much weaker inducers of type 1 IFN than the Edmonston strain and the Edmonston-derived Moraten vaccine strain. The present study is in agreement with this finding, since the wild-type MV isolate was a weaker inducer of the IFN-
and -
genes than the Edmonston strain. However, this difference in induction of host cell genes was not confined to the type 1 IFN genes. The Edmonston strain and the DK96A1 wild-type appeared to upregulate the same cellular genes, but the wild-type isolate was a weaker inducer of almost all the studied MV-induced genes than the Edmonston strain. In the present study, this difference could be explained by a better in vitro growth of the Edmonston than the wild-type isolate.
This is the first large-scale study of the role of MV infection in host cell gene expression. As a starting point, we chose to examine non-selected primary PBMCs and to minimize the influence of non-viral factors by using viruses (and mock material) passed on PBMC cultures. Since MV does not grow to high titres in PBMC cultures, only a low m.o.i. could be obtained. The PBMCs were analysed when most cells were infected, but at a time where cells at all stages of infection were present in the culture. Future studies may preferably be carried out with high-titre virus stocks in order to establish more synchronized infections, which can be assayed at different time points. More pronounced modulation of the expression levels than seen in the present study are also likely to be obtained with homogeneous cell populations such as cell lines or selected PBMC subpopulations. Microarray-based analyses of virus-induced modulation of host cell gene expression have been reported for several other viruses (see, for instance, Khodarev et al., 1999 ; Nees et al., 2001
; Taylor et al., 2000
; Zhu et al., 1998
). The number of genes found to be differentially expressed in virus-infected cells was highly variable, but most of these studies identified a higher number of genes than the present study, and both up- and downregulated genes were found. Several factors including characteristics of the individual viruses and the experimental strategies are likely to influence the number of differentially regulated genes that can be identified. In the present study, the use of mitogen-stimulated PBMCs may be an important factor, since PHA-L treatment is likely to induce a wide range of cellular genes. We speculate that virus-induced downregulating signals may be overridden and virus-induced upregulations may to a large extent drown in PHA-L-stimulated PBMCs.
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Acknowledgments |
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References |
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Received 25 October 2001;
accepted 7 January 2002.