1 Klinische Kooperationsgruppe, Pädiatrische Tumorimmunologie, Kinderklinik, Universitätsklinikum der Technischen Universität München, Marchioninistr. 25, D-81377 München, Germany
2 Institut für Klinische Molekularbiologie und Tumorgenetik, GSF-Forschungszentrum für Umwelt und Gesundheit2 , Marchioninistr. 25, D-81377 München, Germany
3 Abteilung Genvektoren, GSF-Forschungszentrum für Umwelt und Gesundheit, Marchioninistraße 25, D-81377 München, Germany
4 Methesys GmbH, Gottfried-Hagen-Straße 60, D-51105 Köln, Germany
5 Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
6 Pathologisches Institut, Universität Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany
Correspondence
Falk Nimmerjahn
nimmerjahn{at}gsf.de
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ABSTRACT |
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INTRODUCTION |
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Whereas signalling events triggered after influenza virus infection are beginning to be defined (Ludwig et al., 2003; Root et al., 2000
; Garcia-Sastre, 2001
; Pleschka et al., 2001
; Geiss et al., 2002
; Wurzer et al., 2003
), virtually nothing is known about cellular factors that predetermine the efficiency of virus infection. It has been suggested that certain protein kinase C isoforms are important for virus release from endosomes (Sieczkarski et al., 2003
); however, it is not known whether this pathway is of general importance for influenza virus infection of human cells.
In this report, we have shown that influenza virus infection of human cells is essentially dependent on an active NF-B signalling pathway. Cells with low NF-
B activity were virtually resistant to infection with influenza virus, indicating that the reported activation of the NF-
B signalling pathway by influenza virus infection is not sufficient to allow infection (Flory et al., 2000
). This block of influenza virus infection, however, could be overcome by activation of the NF-
B signalling pathway. We furthermore showed that influenza virus infection of highly susceptible cells, e.g. lung epithelial cells, is severely impaired by inhibition of NF-
B signalling.
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METHODS |
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Antibodies and reagents.
The inhibitors BAY11-7085, BAY11-7082 and Ly294002 were purchased from Calbiochem/Merck. The monoclonal antibody specific for influenza virus haemagglutinin 7 (H7) was kindly provided by Dr H. D. Klenk (Marburg). Maackia amurensis lectin II (MAL; recognizing 2,3-branched sialic acid residues) and Sambucus nigra lectin (recognizing 2,6-sialic acid residues) were from Alexis Biochemicals. The phycoerythrin (PE)-conjugated anti-human nerve growth factor receptor (NGFR) antibody was from Becton Dickinson and the cross-linking anti-mouse F(ab)2 fragment from Dianova. For cross-linking experiments, the supernatant of the mouse hybridoma HB8737 (kindly provided by Dr E. Kremmer) was used at a dilution of 1 : 20. For Western blot analysis, an LMP1-specific antibody (Dako) was used at a dilution of 1 : 200 followed by detection with a horseradish peroxidase-coupled secondary antibody (Dianova).
Plasmid constructs and selection of stable cell lines.
The NGFRLMP1 construct and LMP1 mutants 2078, 2131 and 2134 have been described previously (Gires et al., 1997; Dudziak et al., 2003
). The NGFR2131, NGFR2078 and NGFR2134 constructs were generated by replacing the C terminus of wild-type LMP1 by the respective mutant LMP1 C terminus. To allow selection of stable cell lines, the coding sequences of the NGFR fusion proteins were cloned into the episomally replicating pINCO vector downstream from a cytomegalovirus promoter. The CD40L cDNA was cloned from a human T helper cell cDNA library and inserted into the pINCO plasmid. All constructs were verified by sequencing. Stable cell lines were generated either by electroporation (BL cell lines) or lipofection (293T cells) of the respective plasmids into target cells and subsequent culture in the presence of puromycin (1 µg ml1) for 4 days. To increase the number of positive cells before selection, transfected BL cells were enriched by magnetic cell sorting with antibodies against NGFR. Expression of the transgene was verified by FACS and Western blot analysis.
Induction of LMP1 signalling.
Cells (2x106) were incubated with culture supernatant from the mouse hybridoma HB8737 at a dilution of 1 : 20 for 10 min at room temperature. After washing, cells were incubated with a goat anti-mouse F(ab)2 fragment (Dianova) at 2 µg ml1 for 810 h. Following two more cross-links at 3 h intervals, cells were washed and infected with influenza virus containing GFP (FPVGFP) or VacGFP at the indicated m.o.i. values. In control experiments, cells were either left untreated or only incubated with the anti-NGFR antibody without consecutive cross-linking.
Inhibition of NF-B activation.
LCLs were pre-treated with the inhibitor BAY11-7085, BAY11-7082 or Ly294002 at 2·5 µM for 810 h. BL33 and BL41 cells stably transfected with the NGFR2134 LMP1 mutant were incubated with the inhibitors (2·5 µM) during the last two cross-links. Subconfluent A549 cells, U1752 cells and primary human fibroblasts were pre-treated for 46 h with 10 µM of the inhibitors. After washing, cells were infected with recombinant influenza or vaccinia virus and analysed for GFP expression 810 h later by FACS analysis.
Microarray analysis.
The DNA microarray methods have been described in detail elsewhere (Alizadeh et al., 2000). Fluorescently labelled cDNA probes were generated from mRNA (Fast Track kit; Invitrogen), using the Cy5 dye to label cDNA from the indicated cell lines and the Cy3 dye to label cDNA from a reference pool of mRNA prepared from nine lymphoma cell lines. Lymphochip DNA microarrays containing 18 500 human cDNAs were prepared and used as described previously (Alizadeh et al., 2000
). Initial microarray data selection was based on fluorescence signal intensity, with the requirement of 50 relative fluorescent units (r.f.u.) above background in both the Cy3 and Cy5 channels or 500 r.f.u. above background in either channel alone. DNA microarray analysis of gene expression was done essentially as described previously (Alizadeh et al., 2000
). For the red/green diagram, datasets of different cell lines for a specific target gene were averaged. This mean value is shown in black, whereas deviations from this value are shown as different intensities of green (negative deviation) or red (positive deviation).
Virus attachment studies.
Influenza virus attachment to target cells was analysed by incubating target cells with influenza virus (m.o.i. of 1) on ice for 1 h. After removal of excess influenza virus by washing the cells with ice-cold PBS three times, attached influenza virus was detected by staining with an anti-haemagglutinin antibody (anti-H7) followed by a PE-labelled secondary antibody and subsequent FACS analysis.
Titration of influenza virus.
Influenza virus titres were determined by the classical MDCK plaque assay as described previously (Wagner et al., 2000). Briefly, confluent MDCK cell monolayers were infected with 10-fold dilutions of influenza virus in a total volume of 1 ml PBS/0·2 % BSA for 1 h in 6 cm dishes. After washing, cells were covered with an overlay of DMEM cell culture medium containing 0·5 % purified agar. Cells were incubated at 37 °C under 5 % CO2 and plaque formation was analysed 3 days post-infection.
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RESULTS AND DISCUSSION |
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LMP1 or CD40 signalling renders B cells highly susceptible to infection with influenza virus
LMP1 signalling is mediated mainly by two signalling domains in the C terminus of LMP1, called the C-terminal transactivation regions (CTAR) 1 and 2 (Fig. 3a). To test the role of LMP1 signalling on influenza virus infection, we created several inducible forms of LMP1 and LMP1 mutants defective in the signalling domains by fusing the C termini of these LMP1 variants to the extracellular and transmembrane domains of the NGFR (Fig. 3a
). It has been shown that an NGFRLMP1 fusion protein can functionally replace wild-type LMP1 (Gires et al., 1997
). BL33 and BL41 cells expressing the NGFRLMP1 fusion protein became susceptible to influenza virus infection after induction of LMP1 signalling by cross-linking the NGFR (Fig. 3b
, and data not shown). To test whether EBNA2 itself, which is also known to interfere with cellular signalling pathways, could further increase influenza virus infection efficiency, we made use of a cell line that carries an oestrogen-inducible EBNA2 on an EBV-negative background. This, however, did not lead to an additional increase in susceptibility to influenza virus infection, confirming that LMP1 signalling was mediating this effect (Fig. 3b
, and data not shown). As LMP1 mimics CD40 signalling in B cells (Gires et al., 1997
; Eliopoulos & Rickinson, 1998
), CD40 signalling would be expected to have a similar effect on influenza virus infection. Indeed, after culturing different EBV-negative cell lines and primary B cells on 239T cells stably transfected with CD40L, these cells became highly susceptible to subsequent influenza virus infection (Fig. 4a
). Again, CD40 signalling did not affect the infection efficiency of vaccinia virus as shown for the p493/6 cell line (Fig. 4b
), demonstrating the importance of this signalling pathway, especially for influenza virus.
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As influenza virus can attach to BL cells (Fig. 2a), we reasoned that LMP1-mediated signalling might rescue influenza virus infection if triggered after virus attachment to target cells. As shown in Fig. 3(d)
, this is indeed the case, but the infection rate rapidly declined over time indicating that, in the absence of LMP1 or CD40 signalling, influenza virus bound to target cells is rapidly inactivated, probably by endocytosis and subsequent degradation in the endosomal/lysosomal compartment.
Activation of NF-B is responsible for the level of susceptibility to influenza virus infection
To assess differences in NF-B expression between cells in the highly susceptible and poorly susceptible groups, microarray analysis was performed. As shown in Fig. 5
(a), molecules of the NF-
B family and NF-
B target genes were expressed at significantly higher levels in cells of the highly susceptible group. This correlated well with the observed increase in influenza virus infection of EREB2/5 cells after activation of EBNA2 (Fig. 5b
). To investigate whether blocking NF-
B activation would interfere with influenza virus infection, we tested chemical inhibitors of NF-
B activation under several experimental conditions (Figs 5 and 6
). Taken together, these results indicated that inhibition of NF-
B activation blocked influenza virus infection of otherwise susceptible cell lines. The observed increase in susceptibility to influenza virus infection in BL cells carrying the inducible NGFR2134 mutant was abrogated if the cross-link was performed in the presence of the inhibitors BAY11-7085 or BAY11-7082 (Fig. 5c
, and data not shown). Infection of EBV-immortalized cell lines was severely impaired if cells were pre-treated with these inhibitors (Fig. 5d
), but not with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). The observed level of inhibition was comparable with blocking influenza virus binding to its cellular receptor by pre-incubating the cells with MAL (not shown). Inhibition of NF-
B activation did not interfere with vaccinia virus infection, ruling out non-specific cytotoxic effects of the inhibitors (Fig. 5e
). Moreover, influenza virus infection of primary human fibroblasts and the human lung carcinoma cells U1752 and A549, which are considered as the gold standard for influenza virus infection, were also dramatically reduced by inhibition of NF-
B activation (Fig. 6a
, and data not shown). Importantly, receptor expression and virus attachment to cells was not impaired by inhibition of NF-
B activation (Fig. 6b
, and data not shown). To exclude the possibility that the observed effects were due to the use of recombinant influenza viruses, we repeated the experiment with wild-type influenza virus and quantified the release of progeny virus particles after pre-treating the cells with NF-
B inhibitors. As before, influenza virus propagation was strongly impaired in the presence of NF-
B inhibitors (Fig. 6c
).
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Nevertheless, our data indicate that, besides activation of haemagglutinin by cellular or bacterial proteases (Tashiro et al., 1987), the level of NF-
B activity might be a second determinant of influenza virus tropism in vivo. At first sight our results seem to contradict earlier findings that NF-
B activation is a hallmark of inflammatory and antiviral responses mediated especially by type I interferons that block virus replication. However, many other transcription factors and signalling pathways besides NF-
B (e.g. the JakStat and Toll-like receptor signalling pathways) are specifically activated during these antiviral and inflammatory responses and are important to obtain an efficient cellular response (Hertzog et al., 2003
). In contrast to this, our study most likely deals with qualitatively and quantitatively different levels of NF-
B activation. In the case of the B cell lines, this is due to LMP1 expression or the isolated triggering of CD40 signalling, whereas many factors can lead to a basal level of NF-
B activation in lung epithelial cells, e.g. dust particles, cell density and temperature shifts (Liden et al., 2003
; Inoue et al., 2003
). Therefore, inhibition of NF-
B activation may be effective in preventing or treating influenza virus infection. Interestingly, it has recently been shown that many epithelial cells including lung epithelial cells express CD40, especially under inflammatory conditions (Young et al., 1998
; Kaufman et al., 2001
), implicating that this pathway is of great importance for making these cells highly susceptible to influenza virus infection. Although still speculative, this finding might highlight the role of bacterial co-infections, which sustain inflammatory processes in the lung. Furthermore, our results suggest that NF-
B signalling might modulate specific steps in the endocytosis pathway, necessary for efficient influenza virus infection. We are currently trying to identify NF-
B target genes that might be involved in rendering the cells susceptible to infection with influenza virus.
In addition, one might think of using inhibitors of NF-B activation as a therapeutic agent to block the spread of influenza virus after infection of epithelial tissues in the respiratory tract. Whereas these inhibitors have been used systemically for the treatment of pathogenic inflammatory processes in vivo (Pierce et al., 1997
), a more local application of these inhibitors to the respiratory epithelia might be indicated in the case of influenza virus infection. Indeed, besides the identification of specific NF-
B target genes, one of the next steps will be animal studies to define the potential of these inhibitors to block influenza virus infection in vivo.
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ACKNOWLEDGEMENTS |
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Received 13 January 2004;
accepted 16 April 2004.