1 Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany
2 Abteilung Molekulare Virologie, Hygiene Institut, Universität Heidelberg, Otto-Meyerhof-Zentrum, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany
Correspondence
Michael Frese (at Heidelberg)
michael_frese{at}med.uni-heidelberg.de
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ABSTRACT |
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MAIN TEXT |
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Tumour necrosis factor (TNF)- is a powerful proinflammatory cytokine with pleiotropic properties (Liu & Han, 2001
). In virus infections, TNF-
may contribute to virus clearance and/or to organ damage, depending on the underlying infection and pathogenic agent (Schlüter & Deckert, 2002
). For example, it has been observed that the intrahepatic induction of TNF-
correlates with the inhibition of hepatitis B virus (HBV) replication in HBV-transgenic mice (Gilles et al., 1992
; Guidotti et al., 1996
; McClary et al., 2000
). The hypothesis that TNF-
plays an important role in HBV clearance has been substantiated recently by Pasquetto et al. (2002)
, who demonstrated that TNF-
synergistically enhances the inhibitory effect of IFN-
on HBV gene transcription.
Elevated TNF- serum levels have been found in patients with hepatitis C (Tilg et al., 1992
; Torre et al., 1994
). Furthermore, it has been shown that liver infiltrating cytotoxic T lymphocytes and also, to a lesser extent, hepatocytes produce TNF-
during HCV infections (González-Amaro et al., 1994
; Koziel et al., 1995
; Löhr et al., 1994
). To analyse whether TNF-
has a direct antiviral effect on HCV replication, we employed a panel of Huh-7 cell clones containing various subgenomic and genomic HCV replicons (see Fig. 1
for genomic maps of the replicons used in this study). In a first set of experiments, cells containing the bicistronic, subgenomic HCV replicons I377/NS3-3' or I377/NS2-3', the monocistronic, subgenomic HCV replicon I389/ NS3-3'/Hygubi/5.1 or the bicistronic, full-length HCV genome I389/Core-3'/5.1 (Frese et al., 2002
; Lohmann et al., 1999
, 2001
; Pietschmann et al., 2002
) were seeded onto glass coverslips and incubated with various doses of up to 10 000 IU recombinant human TNF-
ml-1 (Sigma-Aldrich). After 3 days of treatment, the cells were fixed with 3 % paraformaldehyde, permeabilized with 0·5 % Triton X-100 and the non-structural protein NS5A was immunostained using the mouse monoclonal antibody (mAb) 3924-4940-8858 (Biogenesis) and goat antibodies conjugated to the cyanine dye Cy3 (Dianova). In contrast to previous experiments in which the treatment with IFN-
or IFN-
inhibited HCV protein synthesis efficiently (Frese et al., 2001
, 2002
), TNF-
did not affect the expression of NS5A in any of the cell clones tested (Fig. 2
A). To substantiate this finding, we treated additional cell clones containing the subgenomic replicon I389/NS3-3' or the full-length genome I389/Core-3'/5.1 with TNF-
concentrations of up to 100 000 IU ml-1 before analysing the cells for NS5A expression. Again, TNF-
had no effect on the expression of NS5A (data not shown). These results indicate that HCV protein synthesis in Huh-7 cells is not affected by TNF-
.
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Next we wanted to find out whether TNF- inhibits HCV replication in a manner so subtle that it is not readily detectable by Northern blot analysis. A very sensitive and precise quantification of HCV replication can be achieved with replicons encoding the firefly luciferase as a reporter gene (Krieger et al., 2001
). Here, we used cells of the Huh-7 cell clone 9B that contain the replicon I389/NS3-3'/LucUbiNeo-ET. The cells were seeded into multiple cell culture dishes and treated for 48 h with TNF-
or IFN-
or a combination of both cytokines. Finally, cells were lysed and luciferase activities were determined. We observed that TNF-
inhibited slightly the reporter gene activity and enhanced the antiviral activity of IFN-
(Fig. 3
A). However, it should be emphasized that the inhibitory effect of TNF-
was very weak compared to that of IFN-
. For example, treatment with 5000 IU TNF-
ml-1 caused a 2-fold decrease in luciferase activity, whereas as little as 10 IU IFN-
ml-1 reduced reporter activity by more than 875-fold. A more detailed mathematical analysis of the data revealed no statistically significant evidence that TNF-
and IFN-
inhibited luciferase activity in a cooperative manner (data not shown). Taken together, these results corroborate the finding that TNF-
has no or almost no effect on HCV RNA replication in Huh-7 cells.
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One might argue that the Huh-7 cells used in our experiments have an impaired TNF- signalling. To exclude this hypothesis, we measured the activation of NF-
B, a key event in the cellular response to TNF-
(Baeuerle & Baltimore, 1996
). Naive Huh-7 cells and those with the replicons I377/NS3-3' or I389/Core-3'/5.1 were seeded into multiple cell culture dishes, cultivated overnight and co-transfected with p(IL-6-kB)3-50hu.IL6p-luc+ (kindly provided by G. Haegeman, University of Ghent, Belgium) and pRL-SV40 (Promega) using OptiMEM (Life Technologies) and the FuGENE transfection reagent (Roche). Note that p(IL-6-kB)3-50hu.IL6p-luc+ contains the firefly luciferase gene under the control of three NF-
B response elements preceding a 50 bp TATA box containing the IL-6 promoter fragment (Plaisance et al., 1997
), whereas pRL-SV40 encodes the Renilla luciferase gene under the control of the SV40 early enhancer/promoter region. After part of the transfected cells had been treated with 5000 IU TNF-
ml-1, the cells were lysed and the luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega). The firefly luciferase activities were then corrected for transfection efficiency using the corresponding Renilla luciferase readings and the effect of TNF-
on NF-
B activation was calculated. We observed that treatment with TNF-
led to an approximately 10-fold increase in reporter gene expression, irrespective of whether or not the cells contained HCV replicons, demonstrating that our Huh-7 cells did indeed respond to TNF-
(data not shown).
We suspected that non-specific cytotoxic effects of TNF- might account for the observed inhibition of HCV replication after the treatment with high doses of that cytokine, although TNF-
alone usually does not induce apoptosis (Bradham et al., 1998
). Thus, we decided to quantify the cytotoxicity of TNF-
. Naive Huh-7 cells were seeded into 96-well plates and treated for 24 or 48 h with different concentrations of TNF-
(ranging from 5 to 25 000 IU ml-1). The metabolic activity of the cells was then measured using either a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 (Roche) or, in a more sensitive way, the incorporation of [6-3H]thymidine (Amersham). Whereas the WST-1 assay did not reveal any cytotoxicity of TNF-
(data not shown), we did observe a weak decrease in thymidine incorporation with increasing cytokine concentrations. For example, a 48 h treatment with 5000 IU TNF-
ml-1 reduced thymidine incorporation to 69 % of the levels found in untreated control cells (Fig. 3D
). These results demonstrate that TNF-
does not induce massive cell death in Huh-7 cells but still has a subtle inhibitory effect on the rate of cell proliferation. Thus, we think that non-specific cytotoxic effects of TNF-
at least contribute to the observed inhibition of the HCV-driven reporter gene activity.
It would be interesting to know whether HCV replication in general is resistant to TNF- or whether the resistance is host cell-dependent. At the moment, however, this question cannot be answered because Huh-7 cells are the only ones known to support high level HCV replication in cell culture. Nevertheless, there is accumulating evidence that TNF-
does not inhibit HCV replication in vivo. For example, TNF-
alleles with a G
A substitution at position -308 within the promoter region are associated with increased gene transcription and expression (Kroeger et al., 1997
; Louis et al., 1998
; McGuire et al., 1994
; Wilson et al., 1997
), but the genetic capacity to produce higher levels of TNF-
seems not to prevent a persistent HCV infection (Höhler et al., 1998
; Rosen et al., 2002
). On the contrary, enhanced TNF-
expression might even favour virus persistence. In a preliminary analysis of 243 Irish women accidentally exposed to a HCV-contaminated immunoglobulin preparation in 1977, the frequency of TNF-
alleles with an A at position -308 was significantly higher in woman who developed subsequent chronic hepatitis C (43·5 %) than in those who got infected but cleared the virus spontaneously (26·3 %) (McKiernan et al., 1999
).
In summary, our results demonstrate that HCV replication in the human hepatoma cell line Huh-7 is highly resistant to TNF-. This finding suggests that the increased production of TNF-
seen in many hepatitis C patients does not contribute to HCV clearance by inducing antiviral defence mechanisms in infected hepatocytes.
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ACKNOWLEDGEMENTS |
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Received 22 November 2002;
accepted 7 January 2003.