Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany1
Institut für Virologie, Universität Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany2
Abteilung Innere Medizin II, Medizinische Universitätsklinik Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany3
Author for correspondence: Ralf Bartenschlager. Fax +49 6131 393 5604. e-mail bartnsch{at}mail.uni-mainz.de
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Abstract |
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Introduction |
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The antiviral effects of type I (/
) IFNs are mediated by a number of effector proteins including double-stranded RNA-activated protein kinase (PKR), 2'5' oligoadenylate synthetase (OAS) and Mx (reviewed in Stark et al., 1998
). Mx proteins are highly conserved, large GTPases that have been found in many mammalian, avian and fish species (reviewed in Arnheiter et al., 1996
; Leong et al., 1998
). The human MxA protein has antiviral activity against both negative- and positive-strand RNA viruses (for a review see Haller et al., 1998
). Transfected cells expressing MxA under the control of a constitutive promoter are highly resistant to various viruses of the families Orthomyxoviridae (Pavlovic et al., 1990
, 1992
; Marschall et al., 2000
; Frese et al., 1995
), Paramyxoviridae (Schnorr et al., 1993
; Schneider-Schaulies et al., 1994
; Zhao et al., 1996
), Rhabdoviridae (Pavlovic et al., 1990
), Bunyaviridae (Frese et al., 1996
; Kanerva et al., 1996
) and Togaviridae (Landis et al., 1998
). Transgenic mice that constitutively express MxA are highly resistant to Thogoto virus (THOV), a tick-borne orthomyxovirus (Pavlovic et al., 1995
), and they exhibit increased resistance to Influenza A virus, Vesicular stomatitis virus, La Crosse virus and Semliki Forest virus (Pavlovic et al., 1995
; Hefti et al., 1999
), indicating that MxA plays an important role in IFN-induced antiviral defence against RNA viruses.
The mechanism(s) by which IFN- inhibits HCV replication is presently not understood and may involve effects mediated by both the innate and the adaptive immune systems. Investigations have been hampered by the lack of efficient cell culture systems and small animal models permissive for HCV infection and replication. The recent development of selectable subgenomic RNAs (replicons) now allows studies on genuine HCV RNA replication in cell culture (Lohmann et al., 1999
). Here, we show that IFN-
efficiently inhibits the replication of HCV subgenomic RNAs in human hepatoma cells in a dose-dependent manner. Furthermore, we used the HCV replicon system to investigate whether MxA plays a role in the inhibition of HCV. Endogenous MxA that was expressed after stimulation with IFN-
as well as recombinant MxA that was transiently overexpressed in transfected cells did not inhibit HCV replicons. Therefore, we conclude that IFN-induced effector proteins other than MxA are responsible for the inhibition of HCV.
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Methods |
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The Sicilian SiAr126 strain of THOV (Albanese et al., 1972 ) was grown in BHK-21 cells. Stock virus contained 8·3x107 p.f.u./ml.
Plasmids.
The entire open reading frames of MxA (Aebi et al., 1989 ) and MxA(T103A) (Ponten et al., 1997
) were amplified by PCR by using the forward primer 5'Hind-MxA (5' CGACAAAGCTTCACCACCATGGTTGTTTCCGAAGTGGACATCG 3') and the reverse primer 3'Not-MxA (5' CGACAAAAGAGCGGCCGCTTAACCGGGGAACTGGGCAAG 3'). PCR products were digested with HindIII and NotI and inserted into the eukaryotic expression vector pSUPERCATCH (kindly provided by C. M. Hovens, Institut für Medizinische Virologie, Universität Zürich, Switzerland) after restriction with the same enzymes. The resulting plasmids pSC-MxA and pSC-MxA(T103A) allow the expression of wild-type MxA and MxA(T103A) under the transcriptional control of the strong constitutive cytomegalovirus immediate-early promoter.
Transfections.
Cells were transfected by using the Effectene reagent (Qiagen) as specified by the manufacturer. Note that the replication of HCV subgenomic RNAs was extremely sensitive to toxic or cytostatic effects caused by certain other transfection procedures (data not shown).
Interferon treatment.
Recombinant human IFN-2 (kindly provided by K. Weyer and E. K. Weibel, HoffmannLa Roche Ltd, Basel, Switzerland) and IFN-
B/D (a gift from Ciba Geigy Ltd, Basel, Switzerland) were used.
Immunofluorescence analysis.
Cells grown on glass coverslips were fixed with 3% paraformaldehyde and permeabilized with 0·5% Triton X-100. Immunostaining was performed according to standard protocols. The NS5A protein of HCV was labelled by using the mouse monoclonal antibody (MAb) 11H (kindly provided by J. A. Hellings, Organon Teknika, Boxtel, The Netherlands). THOV proteins were labelled with the hyperimmune guinea pig antiserum gp457 (Jones & Nuttall, 1989 ) (kindly provided by P. A. Nuttall, NERC Institute of Virology and Environmental Microbiology, Oxford, UK) or with MAb 3D11, which is directed against the nucleoprotein (kindly provided by A. R. Filipe, Centre for Zoonoses Research, National Institute of Health, Lisbon, Portugal). MxA proteins were labelled by using either a polyclonal rabbit antibody directed against recombinant histidine-tagged MxA or MAb 2C12 (Staeheli & Haller, 1985
). Bound primary antibodies were visualized with goat antibodies conjugated to Alexa Fluor 488 (Molecular Probes) or Cy3 (Dianova).
Western blot analysis.
About 8x105 parental HuH-7 cells and those of clone 9-13 were seeded into 8·5 cm diameter dishes. One day after seeding, the cell culture medium was replaced by medium containing 5000 U/ml IFN-B/D (control cells were not stimulated with IFN but otherwise were treated identically). Three days after seeding, cells were harvested and total cell extracts were prepared in sample buffer (Laemmli, 1970
). Protein samples were separated by SDSPAGE, transferred to microporous PVDF membranes (Immobilon-P, Millipore) and immunostained according to standard protocols. The HCV proteins NS3, NS5A and NS5B were specifically labelled by using MAbs 1B6 (Wölk et al., 2000
), 11H (see above) and 5B-3B1 (D. Moradpour, unpublished results), respectively.
Northern (RNA) blot analysis.
About 3x105 cells of clone 9-13 were seeded into 6 cm diameter dishes and maintained in culture medium supplemented with 500 µg/ml G418. Three days after seeding, cells were washed once with PBS and the medium was replaced by medium without G418 but with 1000 U/ml IFN-2 (control cells were not stimulated with IFN but otherwise were treated identically). Cells were harvested at various time-points. Total RNA was prepared by the guanidinium thiocyanatephenolchloroform procedure (Chomczynski & Sacchi, 1987
), denatured by treatment with 5·9% glyoxal in 50% DMSO and 10 mM sodium phosphate buffer, pH 7·0, separated by denaturing agarose gel electrophoresis and analysed by Northern blot following standard protocols (Ausubel et al., 1997
).
Quantification of HCV replicon RNA.
Northern blot analysis was performed as described above. Prior to hybridization, the membrane was stained with methylene blue and cut roughly 1 cm below the 28S rRNA band. The upper part of the blot containing the HCV replicon RNA was hybridized with a 32P-labelled, negative-sense riboprobe complementary to the internal ribosome entry site (IRES) of HCV and the neo gene. The lower strip was hybridized with a 32P-labelled, antisense riboprobe to detect -actin mRNAs. HCV- and
-actin-specific signals were quantified by phosphorimaging with a BAS 2500 scanner (Fuji). HCV signals were corrected for total amounts of RNA loaded in each lane of the gel and compared to a serially diluted standard of in vitro transcripts of the HCV replicon in order to calculate the number of replicon molecules.
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Results |
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In order to determine whether type I IFNs inhibit the replication of HCV subgenomic RNAs, cells of clone 9-13 were treated with 5000 U/ml IFN-2 or IFN-
B/D, fixed and double-immunostained for NS5A and MxA. Fig. 1(A)
shows that NS5A expression was reduced dramatically in cells treated with IFN-
, whereas more than 95% of untreated control cells produced large amounts of the HCV protein (bottom panels). However, in a small number of IFN-treated cells (estimated to be fewer than 1%), weak NS5A-specific staining was observed (data not shown). In this experiment, MxA was used as a marker to assess the biological activity of the IFN preparations. As expected, treatment with IFN-
induced the expression of MxA in nearly 100% of the cells (upper middle and right panels). In contrast, MxA was not detectable in untreated, control cells (upper left panel). This indicates that the replication of subgenomic HCV RNAs did not induce the synthesis of type I IFN to a level sufficient to induce the expression of MxA. To exclude the possibility that the observed inhibition of HCV replicons was a peculiarity of cell clone 9-13, two additional cell clones were tested: (i) cell clone 11-7, carrying the HCV I377/NS2-3' replicon, which has the NS2NS5B coding sequence with two adaptive mutations in NS3 and NS4B, at positions 1261 and 1846 (V. Lohmann and R. Bartenschlager, unpublished results); (ii) cell clone 5-15, carrying the HCV I389/NS3-3' replicon, which contains a part of the core coding sequence slightly longer than that in HCV I377/NS3-3' (Lohmann et al., 1999
). The replicon in this cell line has one adaptive mutation in NS5A, at position 2197 (N. Krieger, V. Lohmann and R. Bartenschlager, unpublished results). When tested for inhibition by IFN-
, a reduction in HCV protein expression similar to that observed with cell clone 9-13 was found with cell clones 11-7 (Fig. 1B
) and 5-15 (data not shown). Synthesis of HCV proteins was further assessed by the Western blot technique. Cells of clone 9-13 were treated with 5000 U/ml IFN-
B/D and total cell extracts were analysed with antibodies against NS3, NS5A and NS5B. None of the proteins were detectable after the IFN-
treatment, whereas untreated, control cells expressed easily detectable levels of NS3, NS5A and NS5B (Fig. 1C
). These results indicate that HCV protein synthesis in general, and not only that of NS5A, is impaired after IFN-
treatment.
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MxA does not inhibit the replication of HCV subgenomic RNAs
The ability of Mx proteins to inhibit the multiplication of various RNA viruses (Haller et al., 1998 ) prompted us to investigate their role in cellular defence against HCV. Keskinen et al. (1999)
reported that HuH-7 cells express MxA and MxB after stimulation with high doses of IFN-
. To confirm their findings, HuH-7 cells were treated with 1000 U/ml IFN-
B/D, total cell extracts were prepared 12, 24 and 36 h later and the expression of MxA and MxB was analysed by Western blotting. As expected, HuH-7 cells responded quickly to stimulation with IFN-
. MxA and MxB expression had already reached maximum levels 12 h after IFN was added to the cell culture (data not shown). During the next 36 h, only a slight reduction in the amount of both Mx proteins was observed, indicating that Mx proteins are not rapidly degraded in HuH-7 cells (data not shown).
We investigated the role of MxA in the IFN-mediated inhibition of HCV RNA replication by blocking its antiviral activity by expression of a dominant-negative mutant. For that purpose, we used MxA(T103A), which contains a point mutation located between the first and second GTP-binding consensus motifs (Ponten et al., 1997 ). MxA(T103A) produced in E. coli and highly purified does not bind GTP and, as a consequence, has no GTPase activity (Ponten et al., 1997
). When overexpressed transiently in mouse 3T3 cells, the mutant protein forms large aggregates in the cytoplasm and does not show any antiviral activity (Ponten et al., 1997
). Furthermore, co-expression of wild-type MxA and MxA(T103A) in mouse 3T3 cells leads to the formation of antivirally inactive heterooligomers (Ponten et al., 1997
). To demonstrate that recombinant MxA(T103A) also blocks the antiviral activity of endogenous MxA that is expressed in human hepatoma cells after IFN-
treatment, HuH-7 cells were transiently transfected with an expression vector encoding MxA(T103A), treated with 5000 U/ml IFN-
B/D and subsequently infected with THOV. We chose THOV for this experiment because this virus is extremely sensitive to MxA (Frese et al., 1995
). The dominant-negative effect of recombinant MxA(T103A) on endogenous MxA protein was analysed by double immunofluorescence with specific antibodies directed against MxA and virus proteins (Fig. 3A
). Transfected cells expressed large amounts of MxA(T103A) that accumulated in characteristic cytoplasmic aggregates (upper left panel). In contrast, endogenous wild-type MxA protein accumulated in the form of small granules in the cytoplasm of cells that were treated with IFN-
(upper middle panel). As expected, THOV replicated unhindered in transfected cells expressing MxA(T103A) (bottom left panel) but not in cells expressing MxA (bottom middle panel). However, in cells co-expressing both MxA(T103A) and MxA (upper right panel), THOV antigens were easily detectable (bottom right panel), indicating virus replication. This experiment proved that MxA(T103A) is indeed useful for blocking the antiviral activity of the endogenous MxA of HuH-7 cells. Next, we expressed MxA(T103A) in cells of clone 9-13, subsequently stimulated the cells with 5000 U/ml IFN-
B/D and analysed MxA and HCV protein expression by double immunofluorescence (Fig. 3B
). Note that expression of MxA(T103A) alone did not inhibit NS5A synthesis, indicating that neither the transfection procedure itself nor the expression of the inactive mutant had any deleterious effects on HCV replication (Fig. 3B
, left panels). If endogenous MxA was indeed the effector protein that mediated the IFN-induced inhibition of HCV replicons, MxA(T103A) would be expected to interfere in a dominant-negative way and restore HCV protein expression in these cells. This was clearly not the case (Fig. 3B
, right panels), suggesting that IFN-
most likely acts through an MxA-independent mechanism.
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Discussion |
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Most recently, the HCV replicons of the cell clones 9-13, 5-15 and 11-7 have been recloned and sequenced. Cell culture-adaptive mutations that enhance the number of G418-selectable, replicon-harbouring cell colonies as well as RNA replication have been identified at various positions of the HCV polyprotein. These are located in NS5B at position 2884 (ArgGly) in the case of the replicon in cell line 9-13 (Lohmann et al., 2001
), in NS5A at position 2197 (Ser
Pro) with the replicon in cell line 5-15 and in both NS3 and NS4B at positions 1261 (Thr
Ser) and 1846 (Lys
Thr), respectively, in the case of the replicon in cell line 11-7 (N. Krieger, V. Lohmann and R. Bartenschlager, unpublished results). As shown here, these mutations do not counteract the IFN-induced antiviral response of the host cell.
One potential drawback of the HCV replicon system used in this study is the bicistronic design of the constructs. The original IRES of HCV directs the translation of the neo gene, whereas the expression of the HCV non-structural genes is mediated by the IRES of EMCV. Therefore, we cannot exclude the possibility that the observed inhibition of HCV RNA replication after IFN- treatment is due to a block in EMCV IRES activity reducing HCV protein synthesis and, as a consequence, most likely also reducing RNA replication. In order to exclude this possibility, we have recently developed cell lines harbouring monocistronic replicon RNAs in which the HCV IRES directs translation of a hygromycinubiquitinNS3-to-NS5B fusion protein (N. Krieger, V. Lohmann and R. Bartenschlager, unpublished results). In this construct, the heterologous protein sequences are removed from the HCV proteins by host cell enzymes via the ubiquitin-dependent pathway, circumventing the problem of inserting a second, heterologous IRES element. Preliminary data show that translation/replication of this replicon is also inhibited by IFN-
(N. Krieger, V. Lohmann and R. Bartenschlager, unpublished). Thus, the EMCV IRES is not responsible for the IFN-
-mediated inhibition of HCV replicons.
The application of IFN- to patients with chronic hepatitis C boosts both their innate and adaptive immune systems. It was previously not known which part of the antiviral defence is more important in clearing HCV. However, our finding that IFN-
inhibits the replication of HCV replicons in cell culture indicates that IFN-induced effector proteins of the innate immune system are in the front line against HCV. But who is doing the job? It was conceivable that MxA inhibited HCV because (i) this protein is known to inhibit the replication of various other RNA viruses (Haller et al., 1998
) and (ii) a single nucleotide polymorphism in the first IFN-stimulated response element of the MxA gene promoter has been reported to correlate with the response of hepatitis C patients to IFN-
treatment (Hijikata et al., 2000
). Although it has been shown that MxA expression is not induced in PBMCs during the acute phase of HCV infection (Jakschies et al., 1994
), increased MxA levels have been found in PBMCs of patients with chronic hepatitis C (Antonelli et al., 1999
; Fernández et al., 1999
). Furthermore, MxA levels were monitored in PBMCs of hepatitis C patients during IFN-
therapy. Once the treatment had been started, MxA levels increased further and remained high until the end of therapy (Fernández et al., 1999
). Most recently, MxA expression was also analysed in cells of the liver. Biopsies that were taken from hepatitis C patients prior to IFN treatment showed elevated MxA expression levels in hepatocytes and/or macrophages in 82% (n=28) of the samples, indicating that HCV is able, at least in most cases, to persist in the presence of MxA (MacQuillan et al., 2000
). These findings are in line with our observation that MxA fails to inhibit HCV replicons in cell culture. Thus, we conclude that IFN-induced effector proteins other than MxA are responsible for the inhibition of HCV replication. OAS and PKR, two other proteins that contribute to IFN-induced antiviral defence, might interfere with the replication of HCV (Korth & Katze, 2000
; Taylor, 2000
). In addition, other, as yet unknown, IFN-induced proteins with antiviral activity exist (Zhou et al., 1999
). Thus, it will be challenging to identify the IFN-induced effector proteins that inhibit the replication of HCV. In summary, our results demonstrate that replicon-harbouring cell lines are powerful tools in investigating the complex interaction between HCV and the IFN-induced antiviral defence system of the host. Further studies on that subject are needed urgently in order to improve chronic hepatitis C therapy.
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Acknowledgments |
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Received 23 November 2000;
accepted 11 January 2001.