Lipopolysaccharide Inhibits Virus-mediated Induction of Interferon Genes by Disruption of Nuclear Transport of Interferon Regulatory Factors 3 and 7
1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês
10.1074/jbc.274.25.18060
ISSN1083-351X
AutoresYuang-T. Juang, Wei-Chun Au, William Lowther, John Hiscott, Paula M. Pitha,
Tópico(s)Viral Infections and Vectors
ResumoWe have studied the effects of lipopolysaccharide (LPS) on the Newcastle disease virus (NDV)-mediated induction of cytokine genes expression. Raw cells treated with LPS before or after virus infection showed down-regulation in the expression of interferon A and, to a lesser extent, interferon B genes. In contrast, induction of the interleukin (IL)-6 gene was enhanced. The effects of LPS were not a result of the suppression of virus replication, because the transcription of viral nucleocapsid gene was not affected. Consistent with these findings, LPS also suppressed the NDV-mediated induction of chloramphenicol acetyltransferase reporter gene driven by murine interferon A4 promoter in a transient transfection assay. Furthermore, LPS inhibited virus-mediated phosphorylation of interferon regulatory factor (IRF)-3 and the consequent translocation of IRF-3 from cytoplasm to nucleus. The LPS-mediated inhibition of IFNA gene expression was much weaker in infected Raw cells that constitutively overexpressed IRF-3. The nuclear translocation of IRF-7 in infected cells was also inhibited by LPS. These data suggest that LPS down-regulates the virus-mediated induction of IFNA genes by post-translationally targeting the IRF-3 and IRF-7 proteins. We have studied the effects of lipopolysaccharide (LPS) on the Newcastle disease virus (NDV)-mediated induction of cytokine genes expression. Raw cells treated with LPS before or after virus infection showed down-regulation in the expression of interferon A and, to a lesser extent, interferon B genes. In contrast, induction of the interleukin (IL)-6 gene was enhanced. The effects of LPS were not a result of the suppression of virus replication, because the transcription of viral nucleocapsid gene was not affected. Consistent with these findings, LPS also suppressed the NDV-mediated induction of chloramphenicol acetyltransferase reporter gene driven by murine interferon A4 promoter in a transient transfection assay. Furthermore, LPS inhibited virus-mediated phosphorylation of interferon regulatory factor (IRF)-3 and the consequent translocation of IRF-3 from cytoplasm to nucleus. The LPS-mediated inhibition of IFNA gene expression was much weaker in infected Raw cells that constitutively overexpressed IRF-3. The nuclear translocation of IRF-7 in infected cells was also inhibited by LPS. These data suggest that LPS down-regulates the virus-mediated induction of IFNA genes by post-translationally targeting the IRF-3 and IRF-7 proteins. IFNs 1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; LPS, lipopolysaccharide; NDV, Newcastle Disease virus; IL, Interleukin; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; ISG, interferon-stimulated gene; CHX, cycloheximide; m.o.i., multiplicity of infection; GFP, green fluorescence protein; NF-κB, nuclear factor κB; Stat, signal transducer and activator of transcription; bp, base pair(s). are a family of natural proteins serving as part of the defense systems against infections. Cells can produce IFNs in response to virus infection, and the newly synthesized IFNs are secreted extracelluarly, bind to IFN receptors, and activate the Jak-Stat signaling pathway that leads to the stimulation of expression of cellular genes generally called ISGs (1Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. 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LPS is the major component of the outer membrane of Gram-negative bacteria (4Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Padova F.D. Schreier M. Brade H. FASEB J. 1994; 8: 217-224Crossref PubMed Scopus (1326) Google Scholar). Through activation of the target cells such as macrophages and B cells, LPS induces innate immune response and expression of cytokine genes which include IL-1, IFNB, and TNFα (5Cavaillon J.M. Haeffner-Cavaillon N. Immunol. Lett. 1985; 10: 35-41Crossref PubMed Scopus (19) Google Scholar,6Feist W. Ulmer A.J. Musehold J. Brade H. Kusumoto S. Flad H.D. Immunobiology. 1989; 179: 293-307Crossref PubMed Scopus (63) Google Scholar). These cytokines are responsible for most of the biological effects of LPS and deregulated production of these cytokines results in the generalized inflammation or endotoxic shock. 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Parmely T.J. Russell S.W. Murphy W.J. J. Biol. Chem. 1997; 272: 1226-1230Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 22Look D.C. Pelletier M.R. Holtzman M.J. J. Biol. Chem. 1994; 269: 8952-8958Abstract Full Text PDF PubMed Google Scholar). Considering that IFNA and IFNB promoters contain cis-elements which are highly homologous to the interferon stimulation responsible element, these findings indicate that LPS may potentially modulate the expression of IFN genes. The signal transduction pathway leading to the induction of IFN genes expression in virus-infected cells is largely unknown. The analysis of the virus responsive element (VRE) of both IFNA and IFNB promoters has identified highly conserved purine-rich sequence repeats that were shown to bind the transcription factors of the IRF family (23Au W.C. Su Y. Raj N.B. Pitha P.M. J. Biol. Chem. 1993; 268: 24032-24040Abstract Full Text PDF PubMed Google Scholar, 24Raj N.B. Au W.C. Pitha P.M. J. Biol. 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However, the virus-mediated induction of IFNA and IFNB genes was not impaired in mice or fibroblasts with homozygous deletion of the IRF-1 gene (36Matsuyama T. Kimura T. Kitagawa M. Pfeffer K. Kawakami T. Watanabe N. Kundig T.M. Amakawa R. Kishihara K. Wakeham A. Porter J. Furlonger C.L. Narendran A. Suzuki H. Ohashi P.S. Paige C.J. Taniguchi T. Mak T.W. Cell. 1993; 75: 83-97Abstract Full Text PDF PubMed Scopus (558) Google Scholar, 37Reis L.F. Ruffner H. Stark G. Aguet M. Weissmann C. EMBO J. 1994; 13: 4798-4806Crossref PubMed Scopus (167) Google Scholar). The identification of IRF-3 and characterization of its role as a signaling transducer in virus-infected cells has provided a major step toward the understanding of the virus-mediated signaling pathway leading to the expression of type I IFN genes (38Au W.C. Moore P.A. Lowther W. Juang Y.T. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11657-11661Crossref PubMed Scopus (349) Google Scholar, 39Pitha P.M. Au W.C. Lowther W. Juang Y.T. Schafer S.L. Burysek L. Hiscott J. Moore P.A. Biochimie (Paris). 1998; 80: 651-658Crossref PubMed Scopus (73) Google Scholar). It was shown that IRF-3 is expressed constitutively in a variety of tissues and cell lines and synergistically cooperates with virus in the induction of both IFNA and IFNB genes. Virus infection induces phosphorylation of IRF-3 at one threonine and several serine residues at the carboxyl-terminal end. The phosphorylated IRF-3 is then translocated from cytoplasm to nucleus where it forms a complex with the transcription coactivator, p300/CBP (29Lin R. Heylbroeck C. Pitha M.P. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, 30Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). Recently, another IRF member, IRF-7, was identified that seems to play a critical role in the induction of IFNA genes (33Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar, 34Au W.C. Moore P.A. LaFleur D.W. Tombal B. Pitha P.M. J. Biol. Chem. 1998; 273: 29210-29217Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 35Sato M. Hata N. Asagiri M. Nakaya T. Taniguchi T. Tanaka N. FEBS Lett. 1998; 441: 106-110Crossref PubMed Scopus (465) Google Scholar). IRF-7 is also phosphorylated in infected cells and transported from cytoplasm to nucleus. In contrast to IRF-3, IRF-7 is preferentially expressed in cells of lymphoid origin, and its transcription is stimulated by IFNA and virus infection (34Au W.C. Moore P.A. LaFleur D.W. Tombal B. Pitha P.M. J. Biol. Chem. 1998; 273: 29210-29217Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). While the role of IRF-3 and IRF-7 in the induction of IFN genes has been gradually unveiled, kinases that are responsible for the phosphorylation of IRF-3 and IRF-7 have not been identified yet. We have been interested in the influence of other pathogens such as bacteria, fungus, or parasites on the virus-mediated induction of IFNs. Mixed infection is a clinical condition that occurs with high frequency in the immune-compromised people as a consequence of HIV-1 infection, organ transplantation or cancer. As the initial step to address this question, we used LPS as the model to mimic the bacterial infection and examined whether it can influence the virus-mediated signaling pathway and induction of IFN gene expression. We have found that LPS is a potent suppressor of virus-mediated induction of IFN genes. Characterization of the underlying molecular mechanism has correlated the LPS-mediated suppression with the inhibition of the virus-mediated phosphorylation and nuclear translocation of IRF-3 and IRF-7. Furthermore, overexpression of IRF-3 partially reverted the LPS effect. Our study illustrates the scenario in which bacterial infection interferes with the virus-mediated induction of IFN genes expression. Raw cells were purchased from ATCC (American Type Culture Collection) and grown in RPMI medium supplemented with 10% fetal calf serum. NDV was propagated in the allantoic cavity of 10-day-old eggs. Sendai virus was purchased from Specific Pathogen-Free Avian Supply (Preston, CT). The antibody to mouse IRF-3 was a gift from Dr. T. Fujita (The Tokyo Metropolitan Institute of Medical Science). Antibodies to human IRF-3 were prepared by immunization of rabbit with GST-IRF-3 fusion protein (32Juang Y.-T. Lowther W. Kellum M. Au W.-C. Lin R. Hiscott J. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9837-9842Crossref PubMed Scopus (239) Google Scholar). CD14 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LPS (Escherichia coli, serotype 055B5) was purchased from Sigma (St. Louis, MO), and mouse IFN (mixture of IFNA and IFNB) was purchased from Lee Biomolecular Research (San Diego, CA). The Raw-CMV-IRF-3 cell line was established by transfecting the pcDNA-IRF-3 into Raw cells and selecting for cells resistant to G418. A pool of stably transfected colonies was used in the experiments. The huIRF-3 expression plasmid, pcDNA-IRF-3, was constructed by cloning the full-length huIRF-3 cDNA into the pcDNA3 (Invitrogen, San Diego, CA) at theHindIII/NotI sites immediately 3′ to the CMV promoter. The plasmids used for synthesis of IFNA, IFNB, and IL-6 riboprobes have been described previously (40Bisat F. Raj N.B. Pitha P.M. Nucleic Acids Res. 1988; 16: 6067-6083Crossref PubMed Scopus (35) Google Scholar, 41Pitha P.M. Biegel D. Yetter R.A. Morse III, H.C. J. Immunol. 1988; 141: 3611-3616PubMed Google Scholar). The plasmid containing the NDV-encoded nucleocapsid (NP) gene of NDV was obtained from Dr. T. Morrison (University of Massachusetts, Worchester, MA). The plasmids containing the murine IFNA4 promoter and its deletion mutants (IFNA4-(-464), IFNA4-(-118)) inserted in front of the CAT gene have been described before (42Au W.C. Raj N.B. Pine R. Pitha P.M. Nucleic Acids Res. 1992; 20: 2877-2884Crossref PubMed Scopus (50) Google Scholar). The GFP vector was obtained from CLONTECH (Palo Alto, CA), and the GFP-IRF-3 and GFP-IRF-7 expression plasmids were described previously (29Lin R. Heylbroeck C. Pitha M.P. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, 34Au W.C. Moore P.A. LaFleur D.W. Tombal B. Pitha P.M. J. Biol. Chem. 1998; 273: 29210-29217Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Total RNA was isolated with TRIzol reagent (Life Technologies, Gaithersburg, MD) and purified according to the protocol of the manufacturer. Ten micrograms of purified RNA was analyzed on 0.8% formaldehyde-agarose gel and followed by transfer to nitrocellulose paper. The filters were prehybridized in the hybridization buffer (50% formamide, 5× SSC, 150 μg/ml herring sperm DNA, 5× phosphatidylethanolamine) for 1 h and then hybridized with the 32P-labeled riboprobes in the same buffer in 65 °C (for cDNA probe, 50 °C) overnight. Blots were washed sequentially with the following buffers: 2× SSC,0.1% SDS; 0.5× SSC, 0.1%SDS; 0.1× SSC, 0.1%SDS until clear background was achieved. The ethidium bromide-stained gel was used as the loading control. Raw cells (3×10 6) were seeded on 60-mm dishes 1 day before transfection. Five micrograms of reporter IFNA4/CAT plasmids and 100 ng of β-galactosidase were transfected into Raw cells with Superfect reagent (Qiagen, Chatsworth, CA). When indicated, cells were infected with NDV or treated with LPS for the indicated time periods as described in the respective figure legend. The results from the CAT assay were normalized to an equal amount of β-galactosidase. Raw cells were treated with LPS or infected with Sendai virus as described in the figure legends. Whole cellular extracts were prepared by incubation of cell pellets in cell lysis buffer (20 mm HEPES, pH 7.9, 50 mm NaCl, 10 mm EDTA, 2 mm EGTA, 0.1% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, 50 mm sodium fluoride, 5 mm sodium orthovanadate) on ice for 30 min. The extracts were cleared by ultracentrifugation at 15,000 rpm for 30 min, and proteins (30 μg) were separated by electrophoresis in 7.5% acrylamide, SDS gel and transferred to nitrocellulose paper. The Western blotting was performed by following the protocols of the manufacturer (ECL method, Amersham Pharmacia Biotech) Raw cells (5×105 cells) were seeded in the chambered cover glass (Nunc, Naperville, IL) 24 h before transfection with 0.5 μg of GFP/IRF-3 (29Lin R. Heylbroeck C. Pitha M.P. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar) or GFP/IRF-7 (34Au W.C. Moore P.A. LaFleur D.W. Tombal B. Pitha P.M. J. Biol. Chem. 1998; 273: 29210-29217Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) expression plasmids. Transfection was done with Superfect reagent (Qiagen), and 16 h after transfection, cells were treated with LPS at the time points described in the figure legends and infected with Sendai virus for 6 h. Cells were examined under fluorescence microscope at the wavelength of 507 nm. The pictures presented were recorded under the same magnification power and the same exposure time. To examine the effect of LPS treatment on the virus-mediated induction of IFNA and IFNB genes, the mouse macrophage cell line Raw 264.7 was treated with LPS (1 μg/ml) for 1 h and then infected with NDV (m.o.i. 5) for 6 h. Analysis of the relative levels of IFNA and IFNB mRNA has shown that IFNA mRNA could only be detected in NDV-infected cells but not in cells that were pretreated with LPS (Fig.1 A). The relative levels of IFNB mRNA were also suppressed in LPS-treated cells, but the level of suppression was lower than that of IFNA. In contrast, induction of IL-6 gene expression by NDV was enhanced by LPS pretreatment. The dose response experiments (Fig. 1 B) demonstrated that as low as 10 ng/ml of LPS was sufficient to suppress the levels of IFNA mRNA by about 80%, whereas the same amount of LPS suppressed the levels of IFNB mRNA only by about 30%. The kinetics study shown in Fig.1 B demonstrated that the suppression of IFN genes expression by LPS was very fast; treatment with LPS for 10 min was already able to suppress virus-mediated induction of IFNA and IFNB genes. In an independent experiment, the effective dose of LPS to mediate the suppression of IFNA gene induction has been determined to be as low as 3 ng/ml (data not shown). It was shown that the effect of LPS can be divided into low (∼ 1 ng/ml) and high dose effects and that the low dose effect was CD14-dependent (7Gallay P. Heumann D. Le Roy D. Barras C. Glauser M.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9935-9938Crossref PubMed Scopus (119) Google Scholar). Because the inhibition of IFNA and IFNB genes expression could be observed with low levels of LPS, it seemed to indicate that LPS may employ a CD14-dependent pathway to suppress the virus-mediated induction of IFN. However, co-incubation of anti-CD14 antibody (Santa Cruz Biotechnology) with LPS neither blocked the LPS inhibition nor modulated the virus-mediated induction of IFNA and IFNB genes (data not shown). To determine whether the observed inhibition of IFN genes expression by LPS is a result of the inhibition of viral replication, we examined NDV replication in LPS-treated cells and untreated controls by analyzing the NP gene transcripts. An earlier study has observed that the levels of NDV NP transcripts could be correlated with the induction level of IFN mRNA (43Nickolaus P. Rammensee H.G. Zawatzky R. J. Interferon Cytokine Res. 1998; 18: 187-196Crossref PubMed Scopus (4) Google Scholar). As shown in Fig. 2, at 6 h post-NDV infection, the high levels of NP transcripts could be detected (Fig. 2 A, lane 2), which were not modulated by LPS treatment applied at 1, 2.5, or 3.5 h after NDV infection (Fig. 2 A, lane 3–5). The relative levels of NP transcripts were, however, lower in cells infected with NDV in the presence of CHX (5 μg/ml) (Fig. 2 A, lane 7). Only when cells were treated with high levels of LPS (1 μg/ml) 1 h before virus infection (Fig. 2 A,lane 6) was viral replication inhibited, and the relative levels of NP transcripts were lower than in the infected, untreated controls. Pretreatment with LPS at 10 ng/ml and lower has not affected NP synthesis. Therefore, in the following experiments, we have used LPS at the concentration of 10 ng/ml for pretreatment and 1 μg/ml for LPS treatments initiated after virus infection. LPS was shown to induce a low level of IFNB in the monocytes and macrophages (44Belardelli F. Gessani S. Proietti E. Locardi C. Borghi P. Watanabe Y. Kawade Y. Gresser I. J. Gen. Virol. 1987; 68: 2203-2212Crossref PubMed Scopus (55) Google Scholar, 45Gessani S. Belardelli F. Borghi P. Boraschi D. Gresser I. J. Immunol. 1987; 139: 1991-1998PubMed Google Scholar). A previous study has disclosed that some of the effects of LPS on the macrophages, such as induction of nitric oxide, is due to the production of IFNB (46Fujihara M. Ito N. Pace J.L. Watanabe Y. Russell S.W. Suzuki T. J. Biol. Chem. 1994; 269: 12773-12778Abstract Full Text PDF PubMed Google Scholar). Although LPS treatment (1 μg/ml, 4 h) was unable to induce IFNA gene expression in Raw cells, IFNB mRNA could be detected by reverse transcriptase-polymerase chain reaction under the same treatment (data not shown). While LPS pretreatment (1 h) totally suppressed the virus-mediated induction of the IFNA gene, the exogenously added IFN (250 units/ml, 1 h) slightly enhanced the virus-mediated induction of the IFNA gene (data not shown). Furthermore, LPS-mediated suppression of IFNA genes expression was not affected by the presence of CHX (5 μg/ml) (data not shown). This concentration of CHX blocked the transcription of viral nucleocapsid gene by 80% (Fig.2 A, lane 7). These data demonstrated that the effect of LPS pretreatment was not caused by IFN or any other LPS-induced protein. The study of the interaction between LPS and virus in the context of IFN induction was further extended by treating Raw cells with LPS at different time points after the virus infection to determine at which stages of viral infection the LPS-mediated inhibition is still effective. As shown in Fig. 2 B, the inhibition of IFNA and IFNB genes expression was less effective if LPS treatment was started later than 3 h after NDV infection. In contrast, the induction of IL-6 was enhanced by LPS treatment even when started as late as 3.5 h post-infection. The LPS-mediated inhibition was also not specific for NDV infection and also could be demonstrated in Raw cells infected with Sendai virus (data not shown). To determine whether the LPS-mediated inhibition of the IFN genes induction is regulated at the transcriptional level, we analyzed the effect of LPS on the inducible expression of a reporter gene in transiently transfected Raw cells. The cells were transfected with a plasmid containing the CAT gene under the control of the IFNA4 promoter, infected with NDV, and treated with LPS as indicated in Fig.3. It can be seen that LPS treatment, either before or after virus infection, suppressed virus-mediated induction of IFNA/CAT plasmid by 2–5-fold, indicating that LPS suppression occurs at the transcriptional level. The suppression was more effective in the cells treated with LPS before or 1 h after the infection than at a later time post-infection. This is consistent with the results obtained when the expression of the endogenous IFNA gene was analyzed. To determine whether the LPS inhibits the virus-mediated transcriptional activation of IFNA promoter by induction of phosphatases, we treated cells with okadaic acid, a known inhibitor of phosphatases (PP1 and PP2A) before and simultaneously with LPS treatment. However, the inhibition of virus-mediated induction of IFN/CAT plasmid was not affected by okadaic acid. To further define the cis-elements of the IFNA4 promoter that are critical for LPS-mediated suppression, plasmids containing deletion mutants of IFNA4 promoters linked to the CAT reporter gene were used for transfection. We have found that as short as −118 bp of the IFNA promoter, which contains a 35-bp long virus-inducible element (IE), is able to confer the LPS-mediated inhibition (data not shown). We and others have recently described that IRF-3 serves as a transducer of virus-mediated signaling from cytoplasm to nucleus and plays a critical role in the induction of IFNA and IFNB genes (29Lin R. Heylbroeck C. Pitha M.P. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, 30Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar, 34Au W.C. Moore P.A. LaFleur D.W. Tombal B. Pitha P.M. J. Biol. Chem. 1998; 273: 29210-29217Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). It was shown that IRF-3 is phosphorylated in infected cells and consequently translocated to the nucleus, where it interacts with the transcription coactivator CBP/p300. To determine whether the LPS-mediated inhibition of IFNA and IFNB genes expression is the result of interference with IRF-3 function, we analyzed the effect of LPS on the virus-mediated phosphorylation of IRF-3. IRF-3 is constitutively present in uninfected Raw cells and infection of Raw cells with Sendai virus for 6 h resulted in a decrease of relative levels of IRF-3 and the phosphorylation of IRF-3, which was reflected by the appearance of a slow migrating band (Fig. 4) on Western blot analysis. These results are in agreement with our previous findings in which we demonstrated that phosphorylated IRF-3 is targeted for degradation presumably by the ubiquitin pathway (29Lin R. Heylbroeck C. Pitha M.P. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar). In infected, LPS-treated cells, phosphorylation of IRF-3 was prevented as indicated by the absence of the slow migrating IRF-3 band. The absence of this slow migrating band was most clear in cells treated with LPS 1 h before or 1 h after the infection (Fig. 4). Thus, for both the suppression of IRF-3 phosphorylation as well as for the inhibition of expression of the IFN genes, LPS treatment has to be initiated before or soon after virus infection. LPS treatment alone has not induced phosphorylation of IRF-3 protein (data not shown). To determine whether the suppression of phosphorylation of IRF-3 by LPS results in the inhibition of translocation of cytoplasmic IRF-3 into nucleus, we analyzed the effect of LPS treatment on nuclear translocation of the GFP-tagged IRF-3. In a transfection experiment with IFN/CAT plasmid, the GFP-tagged IRF-3 was able to stimulate expres
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