Fundación para el Estudio de las Hepatitis Virales, C/Guzmán el Bueno 72, 28015 Madrid, Spain
Correspondence
Vicente Carreño
fehvhpa{at}fehv.org
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ABSTRACT |
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Present address: Cancer Research UK Molecular Oncology Unit, Hammersmith Hospital, Du Cane Road, London W12 0HS, UK.
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INTRODUCTION |
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The IFN signal transduction pathway is well known (Stark et al., 1998). The IFN-
receptor is a heterodimer composed of two subunits, IFNAR-1 and IFNAR-2. Appropriate ligand binding results in the phosphorylation and activation of two tyrosine kinases associated with the subunits of the receptors, Jak1 and Tyk2. Then, these proteins tyrosine-phosphorylate the signal transducer and activator of transcription (Stat) 1 and Stat2. Phosphorylated Stat1 and Stat2 form a complex known as ISGF3 with p48, a DNA-binding protein. The complex translocates to the cell nucleus where it binds to the ISRE sequences in the promoter region of IFN-stimulated genes, thus activating the transcriptional machinery. This pathway appears to be a target site for some viruses to escape host immune survey (Heim et al., 1999
; Miller et al., 1999
).
When patients suffering from chronic hepatitis B are treated with IFN-, up to 40 % of them show clearance of hepatitis B virus (HBV) serum markers and normalization of liver functions (Torresi & Locarnini, 2000
; Lin & Keeffe, 2001
; Zuckerman & Zuckerman, 2000
). In this regard, it is not known what mechanisms are involved in HBV resistance to IFN treatment in the remaining patients. We have shown previously (Fernandez et al., 1997
) that the ability of peripheral blood mononuclear cells (PBMCs) from HBV chronically infected patients to express the MxA protein upon IFN stimulation in vitro is impaired. Recently, Rosmorduc et al. (1999)
have shown that HBV defective particles are implicated in a deficient response to IFN-
in Huh7 cells and that this behaviour appears to be mediated by the accumulation of the HBV capsid protein. Indeed, these authors have proposed the transcriptional inhibition of MxA promoter activity by the core protein. Therefore, in this study, we have analysed what mechanism could be used by HBV to escape from IFN-
-induced antiviral activities. We have examined the IFN signal transduction pathway in cell lines transfected with the precore/core (preC/C) proteins. In addition, we have tested for a direct interaction of these proteins, at a pre-transcriptional level, with the cloned MxA promoter, which is considered a good model for IFN-induced genes.
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METHODS |
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HBV DNA.
The HBV genome used in some experiments was obtained from the pGHBV2 plasmid, which contains two tandem copies of the complete HBV genome cloned in the EcoRI site of the pGEM3Z plasmid (Promega). To recover a recircularized HBV DNA genome, pGHBV2 was digested with EcoRI and the 3·2 kb DNA fragment, corresponding to one copy of the complete HBV genome, was purified and joined using T4 DNA ligase (Promega), according to the manufacturer's instructions. Expression of HBV proteins in transfected cells was checked by testing culture supernatants for the presence of HBsAg and HBeAg by means of enzyme immunoassays available commercially (Roche). To control for transfection efficiency, cells were co-transfected with a green fluorescent protein (GFP) expression plasmid and the amount of GFP was measured by flow cytometry; results were corrected accordingly (average of 10 % transfection efficiency).
HBc and HBe expression plasmids.
Coding regions of the HBV core (HBc) and precore (HBe) proteins were generated by PCR amplification of pGHBV2. The primers used are shown in Table 1. PCR products were digested with NheI/NotI and cloned directly in the pCi mammalian expression vector (Promega). Production of HBeAg by the corresponding expression plasmid was verified by transfecting all cell lines and detection of HBeAg in culture supernatants by an enzyme immunoassay (Roche); intracellular preC/C proteins were assayed by sensitive Western blotting (Campillo et al., 1992
) using specific HBV preC/C mouse monoclonal antibodies (mAbs) (Chemicon International). To verify that cells transfected with the HBcAg expression vector synthesized the protein, HBcAg was tested in culture supernatants by the method described by Quiroga et al. (1985)
. Full-length recombinant (Escherichia coli-derived) HBc and HBe antigen preparations were purchased from Virostat (Portland) and run as standards; purity was checked by Western blotting, indicating 95 % purity for HBc and a pattern compatible with incomplete maturation of HBe expression in E. coli.
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Deletion mutants of the MxA promoter were generated by PCR using pMx4CAT. The PCR conditions used to obtain pMx3CAT, which contains all ISRE sequences and a few bases after the putative start codon, were as follows: 25 cycles of 94 °C for 1 min, 55 °C for 1 min and 68 °C for 2 min, and a prolonged incubation at 68 °C for 15 min. The pMx2CAT and pMx1CAT plasmids, which contain ISRE1/2 and ISRE1, respectively, were obtained as follows: 25 cycles of 94 °C for 15 s, 60 °C for 30 s and 68 °C for 1 min, and a final extension step at 68 °C for 15 min. All PCR products were cloned into the pCR 2.1 vector (Invitrogen) and, after sequencing, they were subcloned into the KpnI/XhoI sites of the pCAT-3 basic plasmid. A schematic representation of all of these constructions is shown in Fig. 1. Primers used in the amplification of the MxA promoter and the deletion mutants are shown in Table 1
.
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Protein extraction and Western blotting.
To assay for the presence of the human MxA protein, PKR and actin (as a housekeeping control) or the signal transducers Jak1, Stat1 and Stat2, cells were rinsed in PBS and lysed in RIPA buffer containing 1 % Nonidet P-40 (ICN Biomedicals), 0·5 % sodium deoxycholate, 0·1 % SDS, 10 mg PMSF ml-1, 10 µmol leupeptin l-1, 1 mmol benzamidine l-1 and 100 µM Na3VO4. Normalized volumes of whole cell extracts according to the protein concentration were boiled for 5 min with SDS-PAGE sample buffer and electrophoresed in gradient (420 %) Tris/glycine gels (Novex). At the end of the run, the gel was blotted onto a Western PVDF membrane (Schleicher&Schuell). After an overnight blocking step at 4 °C with 10 % non-fat dry milk, the membrane was incubated at room temperature for 1 h with the appropriate primary antibody at a 1/1000 dilution. Human MxA protein was detected with a mouse-specific mAb. Stat1, Stat2, PKR and actin were detected with rabbit or goat polyclonal antibodies from Santa Cruz. Jak1 was detected with a mouse mAb from Transduction Laboratories. After extensive washing, the membrane was incubated with a secondary rabbit anti-mouse, rabbit anti-goat or goat anti-rabbit peroxidase-conjugated antibody (Dakopatts) at a 1/10 000 dilution for 30 min at room temperature. Finally, the blot was revealed by autoradiography using the enhanced chemiluminescent substrate Supersignal West Pico Chemiluminescent substrate (Pierce). For immunoprecipitation experiments, 300 µl whole-cell extracts from 20x106 cells were pre-cleared with 30 µl protein A/G PLUS-agarose (Santa Cruz) for 1 h at 4 °C. The lysate was pelleted by centrifugation and the supernatant was incubated overnight at 4 °C with 5 µg Stat1 or Stat2 antibodies. After precipitation with 30 µl A/G PLUS-agarose, samples were washed twice with RIPA buffer and three times with 50 mM Tris/HCl pH 7·5. Pellets were then boiled with SDS-PAGE sample buffer and analysed by Western blot using the PY20 anti-phosphotyrosine antibody (Transduction Laboratories). The bands in the autoradiograms were scanned for identification and semiquantified by densitometry using Scion Imaging software (Scion).
Electrophoretic mobility shift assay (EMSA).
For each EMSA, 1 µg HBc or HBe (Virostat) were incubated with 3 µl 87 % glycerol, 1 µl poly(dI : dC) and 2 µl of binding buffer containing 20 mM Tris pH 7·5, 75 mM KCl, 1 mM DTT and 5 µg BSA ml-1 on ice for 15 min. In the case of the supershift assay, samples were incubated for 30 min with appropriate HBV-specific (or species-matched irrelevant) antibodies. Then, 50 000 c.p.m. 32P-labelled DNA probes were added and incubated at room temperature for 30 min. Samples were separated on polyacrylamide gels made with MDE gel solution (FMC) in 0·5x TBE as running buffer. The gel was then dried and exposed to PDS film (Kodak). Two fragments were obtained by PCR from pMx4CAT as probes for EMSA. The MxISRE3 probe contains the first fully functional ISRE3 near the putative start codon of the human MxA promoter and MxISRE2 contains the second functional ISRE2 of the MxA promoter. Two additional probes, termed MxISRE2 and
MxISRE3, which do not contain ISRE2 and ISRE3 sequences, respectively, were obtained by PCR also. Primers used for PCR amplification are those listed in Table 1
.
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RESULTS |
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Signalling through the IFN pathway
Recent studies have shown that nucleocapsid HBV protein could be involved in a deficient IFN response (Rosmorduc et al., 1999). Thus, we have tested for a possible interference of the HBc and HBe proteins with the IFN signal transduction pathway. Total cell extracts from HepG2, Chang and HeLa cells transfected with pCi-HBc or pCi-HBe were analysed for Jak1, Stat1 and Stat2 levels. Jak1 is a tyrosine kinase associated with the IFN-
/
receptor and is activated upon IFN binding. As shown in Fig. 4
, no apparently quantitative differences in the levels of Jak1 expression were found in the Chang cell line. Stat1 and Stat2 are the cytoplasmic signal transducers that are tyrosine-phosphorylated by Jak1 and they form an essential part of the ISGF3 complex. Total levels of both proteins did not seem to be affected by the expression of HBc or HBe protein (Fig. 4
, lanes 36). Similar results were obtained in the HepG2 and HeLa cell lines (data not shown). In addition, the levels of tyrosine phosphorylation of Stat1 and Stat2 remained unchanged, irrespective of whether or not HBc or HBe was present; similar results were observed in all cell lines tested (data not shown).
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DISCUSSION |
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Since neither HBc nor HBe appeared to affect the IFN-signalling pathway, we made an initial approach to investigate whether these proteins could be acting at a later step. In co-transfection experiments with the human MxA promoter and constructs expressing constitutively core (HBc) or precore (HBe) antigens, we have shown a clear inhibition of the reporter activity after IFN induction. This result was consistent in all cell lines tested, showing that the inhibition was not dependent on host cell factors and, thus, that it is not restricted to liver cells, which is in agreement with our previous observations in human mononuclear cells (Fernandez et al., 1997) and with the inhibition reported by others (Rosmorduc et al., 1999
) in similar experiments made with defective HBV genomes. The major characteristic of this defective genome is the accumulation of the core protein in the cytoplasm of transfected cells and the authors concluded that this core protein might be involved in the reduced capacity to respond to IFN. In addition, we have also demonstrated the capacity of preC/C proteins to inhibit the IFN-dependent cell response.
Several previous works have shown how the human MxA promoter is organized (Aebi et al., 1989; Horisberger et al., 1990
; Ronni et al., 1998
) and, by sequence analysis, it was found that the promoter contains three ISREs, although only ISRE2 and ISRE3 showed a functional capacity to respond to IFN-
(Horisberger et al., 1990
; Ronni et al., 1998
). Accordingly, in our study, the first deletion mutant, pMx1CAT, which contains only the ISRE1 sequence, did not exhibit any CAT activity. However, pMx2CAT, which also contains the ISRE2 sequence, showed a weak capacity to be induced by IFN. In addition, CAT activity was similar to the initial full-length cloned promoter represented by pMx4CAT, only when the ISRE3 sequence was added in pMx3CAT. These deletion mutants allowed us to confirm earlier reports in our system with several cell lines. Interestingly, when cells were co-transfected with the HBc or the HBe expression plasmids and treated with IFN-
, pMx2CAT and pMx3CAT showed a clear inhibition of CAT activity, as occurs with the full-length promoter. Together, these results suggested that a direct interaction of the core protein (HBc) and/or precore (HBe) might occur at the promoter region comprising ISRE2 and ISRE3 sequences. This hypothesis was confirmed by DNA-binding assays showing the formation of DNAprotein complexes with MxISRE2/3, but not with
MxISRE2/3 probes lacking ISRE, thus excluding a non-specific shift caused by the basic nature of both proteins (Heim et al., 1999
). Therefore, these results confirm a direct interaction between HBc and HBe and two regions of the human MxA promoter. In co-transfection experiments with the deletion mutants, HBV proteins were able to inhibit the IFN-induced CAT activity of pMx3CAT as well as with pMx2CAT. The latter does not share the sequence downstream of the ISRE2 element but it was also inhibited. This observation, together with the EMSA results, suggest that the targets of the interaction of the preC/C proteins are probably the ISRE sequences of the human MxA promoter. The direct interaction of HBe with the human MxA promoter is unclear at present because the HBe construct has the capacity to express preC/C-related proteins. However, because of the nature of recombinant HBe, it cannot be ruled out that such an interaction occurs with other components of the IFN system. In this sense, using a similar approach with the IFN-
promoter, Whitten et al. (1991)
concluded that HBc and HBe inhibit the activity of the IFN-
regulatory elements, although they failed to show any mechanism implicated in this effect.
HBc plays an important role in HBV maturation and its capacity to bind DNA sequences is not striking because there are several nucleic acid-binding motifs at the C-terminal region of the protein (Hatton et al., 1992). However, the secreted precore protein is not essential for replication or infection (Chang et al., 1987
; Schlicht et al., 1987
), although recent studies have implicated a non-secreted precore precursor in the regulation of HBV replication (Scaglioni et al., 1997
). Nevertheless, for HBe to mediate transcriptional repression, the precore protein needs to be localized in the nucleus. The majority of the protein buds into the endoplasmic reticulum and is processed before secretion as HBeAg (Bruss & Gerlich, 1988
). However, some of the precore protein may show a nuclear localization (Aiba et al., 1997
). A mutation in the precore sequence has been reported that prevents formation of HBe (Carman et al., 1989
). Collectively, these findings might explain differences in MxA inducibility among serum HBeAg-negative patients with the precore mutant (Fernandez et al., 1997
).
IFN- is efficacious in patients chronically infected with HBV but there are still some patients who do not respond to treatment or who relapse after having achieved an initial response (Torresi & Locarnini, 2000
; Lin & Keeffe, 2001
; Zuckerman & Zuckerman, 2000
). Little is known about the mechanism that mediates the IFN-induced elimination of HBV from a patient. However, if HBc and HBe bind to the ISRE sequences present in the IFN-induced genes, this effect could account for the interference observed in the IFN response in these patients and highlights the potential role of using combination therapies with other antiviral compounds that can evade this virus-evolved escape mechanism. Moreover, these interactions could explain why patients with a low level of viraemia (e.g. low synthesis of viral proteins in the infected cells), which is a recognized predictive factor of response to drug therapy (Brook et al., 1989
), respond better to IFN-
than patients with a high level of viraemia. The relevance of this downregulatory mechanism is substantiated further by the finding of Gordien et al. (2001)
, who have reported recently that induction of MxA expression in the human hepatoma cell line HuH7 followed by transient transfection with the full-length HBV genome leads to inhibition of HBV replication. However, it is possible that inhibition of HBV may also occur via an MxA-independent pathway, as shown recently for hepatitis C virus (Frese et al., 2001
). Interactions of HBV preC/C with other components of the IFN system could not be excluded and should be studied in the future.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aiba, N., McGarvey, M. J., Waters, J., Hadziyannis, S. J., Thomas, H. C. & Karayiannis, P. (1997). The precore sequence of hepatitis B virus is required for nuclear localization of the core protein. Hepatology 26, 13111317.[Medline]
Brook, M. G., Karayiannis, P. & Thomas, H. C. (1989). Which patients with chronic hepatitis B virus infection will respond to alpha-interferon therapy? A statistical analysis of predictive factors. Hepatology 10, 761763.[Medline]
Bruss, V. & Gerlich, W. H. (1988). Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein. Virology 163, 268275.[Medline]
Campillo, M. L., Quiroga, J. A., Bartolome, J., Moraleda, G., Castillo, I. & Carreño, V. (1992). Protein composition of the hepatitis B virus e antigen in the natural course of disease and following interferon therapy. J Clin Microbiol 30, 12561261.[Abstract]
Carman, W. F., Jacyna, M. R., Hadziyannis, S., Karayiannis, P., McGarvey, M. J., Makris, A. & Thomas, H. C. (1989). Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet ii, 588591.
Caselmann, W. H. (1996). Trans-activation of cellular genes by hepatitis B virus proteins: a possible mechanism of hepatocarcinogenesis. Adv Virus Res 47, 253302.[Medline]
Chang, C., Enders, G., Sprengel, R., Peters, N., Varmus, H. E. & Ganem, D. (1987). Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol 61, 33223325.[Medline]
Chang, K. C., Hansen, E., Foroni, L., Lida, J. & Goldspink, G. (1991). Molecular and functional analysis of the virus- and interferon-inducible human MxA promoter. Arch Virol 117, 115.[Medline]
Fernandez, M., Quiroga, J. A., Martin, J., Cotonat, T., Pardo, M., Horisberger, M. A. & Carreño, V. (1997). Impaired interferon induction of human MxA protein in chronic hepatitis B virus infection. J Med Virol 51, 332337.[CrossRef][Medline]
Frese, M., Pietschmann, T., Moradpour, D., Haller, O. & Bartenschlager, R. (2001). Interferon- inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway. J Gen Virol 82, 723733.
Gordien, E., Rosmorduc, O., Peltekian, C., Garreau, F., Brechot, C. & Kremsdorf, D. (2001). Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol 75, 26842691.
Haque, S. J. & Williams, B. R. (1998). Signal transduction in the interferon system. Semin Oncol 25, 1422.[Medline]
Hatton, T., Zhou, S. & Standring, D. N. (1992). RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol 66, 52325241.[Abstract]
Heim, M. H., Moradpour, D. & Blum, H. E. (1999). Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway. J Virol 73, 84698475.
Henkler, F., Hoare, J., Waseem, N., Goldin, R. D., McGarvey, M. J., Koshy, R. & King, I. A. (2001). Intracellular localization of the hepatitis B virus HBx protein. J Gen Virol 82, 871882.
Horisberger, M. A., McMaster, G. K., Zeller, H., Wathelet, M. G., Dellis, J. & Content, J. (1990). Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter. J Virol 64, 11711181.[Medline]
Ihle, J. N. (1996). STATs: signal transducers and activators of transcription. Cell 84, 331334.[Medline]
Keskinen, P., Nyqvist, M., Sareneva, T., Pirhonen, J., Melen, K. & Julkunen, I. (1999). Impaired antiviral response in human hepatoma cells. Virology 263, 364375.[CrossRef][Medline]
Lee, Y. H. & Yun, Y. (1998). HBx protein of hepatitis B virus activates Jak1-STAT signaling. J Biol Chem 273, 2551025515.
Lin, O. S. & Keeffe, E. B. (2001). Current treatment strategies for chronic hepatitis B and C. Annu Rev Med 52, 2949.[CrossRef][Medline]
Melen, K., Keskinen, P., Lehtonen, A. & Julkunen, I. (2000). Interferon-induced gene expression and signaling in human hepatoma cell lines. J Hepatol 33, 764772.[CrossRef][Medline]
Miller, D. M., Zhang, Y., Rahill, B. M., Waldman, W. J. & Sedmak, D. D. (1999). Human cytomegalovirus inhibits IFN--stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-
signal transduction. J Immunol 162, 61076113.
Muller, U., Steinhoff, U., Reis, L. F., Hemmi, S., Pavlovic, J., Zinkernagel, R. M. & Aguet, M. (1994). Functional role of type I and type II interferons in antiviral defense. Science 264, 19181921.[Medline]
Ono, Y., Onda, H., Sasada, R., Igarashi, K., Sugino, Y. & Nishioka, K. (1983). The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res 11, 17471757.[Abstract]
Pellegrini, S. & Dusanter-Fourt, I. (1997). The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs). Eur J Biochem 248, 615633.[Abstract]
Quiroga, J. A., Gonzalez, O., Carreño, V., Mora, I., Porres, J. C., Gutiez, J. & Hernandez-Guio, C. (1985). Radioimmunoassay for detecting hepatitis B core antigen in serum from patients with chronic hepatitis B infection. Clin Chem 31, 831834.
Revel, M. & Chebath, J. (1986). Interferon-activated genes. Trends Biochem Sci 11, 166170.[CrossRef]
Ronni, T., Matikainen, S., Lehtonen, A. & 7 other authors (1998). The proximal interferon-stimulated response elements are essential for interferon responsiveness: a promoter analysis of the antiviral MxA gene. J Interferon Cytokine Res 18, 773781.[Medline]
Rosmorduc, O., Sirma, H., Soussan, P., Gordien, E., Lebon, P., Horisberger, M., Brechot, C. & Kremsdorf, D. (1999). Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein. J Gen Virol 80, 12531262.[Abstract]
Scaglioni, P. P., Melegari, M. & Wands, J. R. (1997). Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J Virol 71, 345353.[Abstract]
Schlicht, H. J., Salfeld, J. & Schaller, H. (1987). The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. J Virol 61, 37013709.[Medline]
Sells, M. A., Chen, M. L. & Acs, G. (1987). Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84, 10051009.[Abstract]
Sen, G. C. & Ransohoff, R. M. (1993). Interferon-induced antiviral actions and their regulation. Adv Virus Res 42, 57102.[Medline]
Staeheli, P. (1990). Interferon-induced proteins and the antiviral state. Adv Virus Res 38, 147200.[Medline]
Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. & Schreiber, R. D. (1998). How cells respond to interferons. Annu Rev Biochem 67, 227264.[CrossRef][Medline]
Torresi, J. & Locarnini, S. (2000). Antiviral chemotherapy for the treatment of hepatitis B virus infections. Gastroenterology 118 (Suppl. 1), S83S103.[Medline]
von Wussow, P., Jakschies, D., Hochkeppel, H. K., Fibich, C., Penner, L. & Deicher, H. (1990). The human intracellular Mx-homologous protein is specifically induced by type I interferons. Eur J Immunol 20, 20152019.[Medline]
Whitten, T. M., Quets, A. T. & Schloemer, R. H. (1991). Identification of the hepatitis B virus factor that inhibits expression of the beta interferon gene. J Virol 65, 46994704.[Medline]
Williams, B. R. (1991). Transcriptional regulation of interferon-stimulated genes. Eur J Biochem 200, 111.[Abstract]
Zuckerman, J. N. & Zuckerman, A. J. (2000). Current topics in hepatitis B. J Infect 41, 130136.[CrossRef][Medline]
Received 8 November 2002;
accepted 23 April 2003.
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