Lipopolysaccharide Inhibits Virus-mediated Induction of Interferon Genes by Disruption of Nuclear Transport of Interferon Regulatory Factors 3 and 7*

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.


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 virusmediated 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 1 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 (1)(2)(3). Some of these genes encode proteins that can inhibit viral replication, thus conferring the antiviral state to the cells (3). While the molecular mechanism involved in the induction of IFN and ISG genes has been studied in great detail in vitro, it is largely unknown how much this system is affected by other stress factors. Of special concern is the potential impact of the bacterial infection on this system because concomitant infection with both bacteria and virus is a common clinical situation.
LPS is the major component of the outer membrane of Gramnegative bacteria (4). 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 TNF␣ (5,6). 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. The signal transduction pathway for LPS is initiated by its binding to LBP (LPS binding protein) (7), an acute phase reactant produced by the liver, followed by the binding to CD14, a glycosylphosphatidylinositol-anchored membrane protein (8,9) and Toll-like receptor (TLR2) (10). This receptor is activated by LPS and the response depends on the binding of LPS to LBP and is enhanced by CD14. After binding to the receptor, LPS activates a number of tyrosine and serine kinases, including Raf-1, p42 and p44 isoforms of the MAP kinase, the p38 kinase, c-Jun kinase, ceramide activated kinase (CAK) and CD14 receptor-coupled kinase p56 lyn (11)(12)(13), with consequent activation of nuclear transcription factors such as NF-B, Stat1 (signal transducer and activator of transcription), Stat3, and NF-IL6 (C/EBP) (14 -18). LPS was also shown to activate nuclear factors binding to the interferon stimulation responsible element although the identity of these factors has not been elucidated (19,20). Furthermore, functional cooperation between LPS and IFNs, especially IFN␥, was shown to result in expression of a set of pro-inflammatory genes (21,22). 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 (23)(24)(25)(26)(27)(28). In addition, IFNB gene promoter also contains a functional NF-B site. Three of the IRF factors, IRF-1, IRF-3, and IRF-7, were shown to activate the promoters of IFNA or IFNB gene in transient transfection assays (23, 29 -35). 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 (36,37). 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 * This work was supported by Grant AI19737 from the National Institutes of Health (to P. M. P.). This study is part of the Ph.D. thesis requirement for Y. T. Juang. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom reprint requests and correspondence should be addressed: The Johns Hopkins University, Oncology Center, 418 N. Bond St., Baltimore, MD 21231-1001. Tel.: 410-955-8871; Fax: 410-955-0840. 1 The 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-B, nuclear factor B; Stat, signal transducer and activator of transcription; bp, base pair(s). (38,39). 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 (29,30). Recently, another IRF member, IRF-7, was identified that seems to play a critical role in the induction of IFNA genes (33)(34)(35). 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 (34). 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.

EXPERIMENTAL PROCEDURES
Cells, Virus, and Reagents-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 (32). 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.
Northern Blot Hybridization-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, 5ϫ SSC, 150 g/ml herring sperm DNA, 5ϫ phosphatidylethanolamine) for 1 h and then hybridized with the 32 P-labeled riboprobes in the same buffer in 65°C (for cDNA probe, 50°C) overnight. Blots were washed sequentially with the following buffers: 2ϫ SSC,0.1% SDS; 0.5ϫ SSC, 0.1%SDS; 0.1ϫ SSC, 0.1%SDS until clear background was achieved. The ethidium bromide-stained gel was used as the loading control.
Transfection and CAT Assay-Raw cells (3ϫ10 6 ) were seeded on 60-mm dishes 1 day before transfection. Five micrograms of reporter IFNA 4 /CAT plasmids and 100 ng of ␤-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 ␤-galactosidase.
Western Blot Analysis-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 g) 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) Fluorescence Microscopic Study-Raw cells (5ϫ10 5 cells) were seeded in the chambered cover glass (Nunc, Naperville, IL) 24 h before transfection with 0.5 g of GFP/IRF-3 (29) or GFP/IRF-7 (34) 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.

Pretreatment of Raw Cells with LPS Differentially
Modulates the NDV-mediated Induction of Cytokines-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 g/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. 1A). 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. 1B)  Raw cells were either untreated or treated with different concentrations of LPS as indicated for 1 h, cells were then washed and infected with NDV for 6 h. Total RNA was isolated as described under "Experimental Procedures," was size-separated on 0.8% formaldehydeagarose gel, and was analyzed by Northern hybridization with IFNA, IFNB, and IL-6 specific probes. The ethidium bromide-stained RNA on agarose gel before transfer to the nitrocellulose filter is shown. onstrated 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. 1B 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 (ϳ 1 ng/ml) and high dose effects and that the low dose effect was CD14dependent (7). 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).

The LPS-mediated Suppression of IFN Induction by Virus Is Not a Consequence of Suppression of Viral Replication-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 (43). As shown in Fig. 2, at 6 h post-NDV infection, the high levels of NP transcripts could be detected (Fig. 2A, lane 2), which were not modulated by LPS treatment applied at 1, 2.5, or 3.5 h after NDV infection (Fig. 2A, lane 3-5). The relative levels of NP transcripts were, however, lower in cells infected with NDV in the presence of CHX (5 g/ml) (Fig. 2A, lane 7). Only when cells were treated with high levels of LPS (1 g/ml) 1 h before virus infection (Fig. 2A, 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 g/ml for LPS treatments initiated after virus infection.
LPS was shown to induce a low level of IFNB in the monocytes and macrophages (44,45). 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 (46). Although LPS treatment (1 g/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 g/ml) (data not shown). This concentration of CHX blocked the transcription of viral nucleocapsid gene by 80% ( Fig. 2A, 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. 2B, 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).
LPS Suppresses the Virus-mediated Activation of IFNA Promoter-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

FIG. 2. Comparison of the relative levels of the NDV nucleocapsid (NP) mRNA and the respective cytokine mRNA in LPStreated cells and NDV-infected Raw cells.
A, Raw cells were infected with NDV for 6 h, and LPS was added either 1 h before (designated as Ϫ1) or at 1, 2.5, or 3.5 h post-infection (designated as ϩ1, ϩ 2.5, and ϩ3.5, respectively). When indicated, CHX (5 g/ml) was added to the medium 80 min before NDV infection. Total RNA (10 g) was analyzed by Northern hybridization with the NP probe. B, LPS can suppress virus-mediated induction of the IFN genes when LPS was added shortly after virus infection. Raw cells were infected with NDV for 6 h, and LPS was added at the indicated time points post-infection. Ten micrograms of total RNA was then analyzed by Northern hybridization with the cytokine probes as indicated. 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 IFNA 4 promoter that are critical for LPS-mediated suppression, plasmids containing deletion mutants of IFNA 4 promoters linked to the CAT reporter gene were used for transfection. We have found that as short as Ϫ118 bp of the IFNA promoter, which contains a 35-bp long virus-inducible element (IE), is able to confer the LPSmediated inhibition (data not shown).
LPS Suppresses the Virus-mediated Phosphorylation of IRF-3 and Impedes Its Translocation from Cytoplasm to Nucleus-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 (29,30,34). 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 (29). 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 GFPtagged IRF-3 was able to stimulate expression of this plasmid in infected cells with the efficiency of about 45% of the wild type IRF-3 (data not shown). Raw cells were transfected with the expression plasmid encoding the GFP/IRF-3 fusion protein (29), and 24 h later, transfected cells were infected with Sendai virus for 6 h. It can be seen that, in uninfected cells, GFP/IRF-3 is located only in cytoplasm (Fig. 5a), whereas it is efficiently translocated into the nucleus at 6 h post-infection (Fig. 5b). Treatment with LPS 1 h before or 1 h after Sendai virus infection efficiently suppressed the nuclear translocation of GFP/IRF-3 (Fig. 5, c and d). When treatment with LPS was initiated 4 h after virus infection (5e), some cells showed the presence of GFP/IRF-3 only in cytoplasm, whereas others had the GFP/IRF-3 dispersed both in the cytoplasm and nucleus. These data clearly show that LPS treatment of the cells initiated either before or within the first hour after viral infection interferes with the phosphorylation of IRF-3 and its transportation from the cytoplasm to the nucleus.
Overexpression of IRF-3 Reverts the LPS-mediated Suppression of IFN Induction-To further determine whether IRF-3 is the target for LPS suppression of the virus-mediated induction of IFNA and IFNB genes expression, we generated a Raw cell line, Raw-CMV-IRF3, which constitutively overexpressed human IRF-3. Using Western blot analysis with antibodies to human IRF-3, we could detect expression of human IRF-3 in Raw-CMV-IRF3 cells but not in the parental Raw cells (Fig.  6A). Infection of the Raw-CMV-IRF3 cells with Sendai virus resulted in the expression of IFNA genes, as determined by the presence of IFNA mRNA. As shown in Fig. 6B, treatment of Raw cells with LPS initiated 2 or 4 h after virus infection resulted in a decrease in the relative levels of IFNA mRNA (70 and 50% suppression, respectively), while no LPS-mediated suppression could be seen in the infected CMV-IRF-3 cells. These results indicate that overexpression of IRF-3 can overcome the LPS-mediated inhibition of IFNA genes expression in infected cells.
LPS Interferes with the Nuclear Transport of IRF-7 in Infected Cells-We and others have recently shown that IRF-7 plays an important role in the expression of IFNA genes (33)(34)(35). Similarly as IRF-3, IRF-7 is transported from cytoplasm to nucleus in infected cells, and the phosphorylation of serine residues in the carboxyl terminus of this protein is important for its transactivating activity. We have, therefore, examined whether LPS treatment also affects the nuclear transport of GFP/IRF-7 fusion protein in infected cells. The results in Fig.  7a show that, in transfected Raw cells, GFP/IRF-7 can be detected predominantly in the cytoplasm. In contrast, at 7-h post-infection, GFP/IRF-7 starts to accumulate in the nucleus although the transport is not completed yet (Fig. 7b). In cells which were either pretreated with LPS or treated with LPS 1 h after the infection, GFP/IRF-7 is localized only in cytoplasm (Fig. 7, c and d). When LPS treatment was started at 4-h post-infection, some cells showed the presence of GFP/IRF-7 in both nucleus and cytoplasm, while others showed only the cytoplasmic localization (Fig. 7e). These data indicate that LPS treatment, when started before or early after infection, prevents transport of IRF-7 from cytoplasm to nucleus. However, these data also show that when overexpressed, low levels of GFP/IRF-7 can be detected in the nucleus even in the absence of viral infection. DISCUSSION Two families of transcription factors play a critical role in the virus-mediated induction of IFNA and IFNB gene expression. The IRFs factors, especially IRF-3 and IRF-7, can serve as direct transducers of virus-mediated signaling from cytoplasm into the nucleus and activate transcription of IFNA and IFNB genes. In addition, viral infection also leads to the phosphorylation and degradation of IB and subsequent transport of NF-B (p50/p65 heterodimer) into the nucleus where it participates in the activation of transcription of the IFNB gene. In the present study, we have demonstrated that LPS is a potent suppressor of virus-mediated stimulation of IFN genes expression. The suppression of IFN genes expression in Raw cells could be demonstrated when cells were treated with LPS before or soon after virus infection at concentrations as low as 3 ng/ml. Although LPS suppressed both the induction of IFNA and IFNB genes expression in infected cells, the suppression of IFNA gene expression was stronger than that of IFNB gene. One reason for the lower sensitivity of IFNB to the LPS-mediated inhibition could be the presence of functional NF-B site in the promoter of the IFNB gene. In macrophages and monocytes, LPS treatment was shown to activate IKK kinases (47), resulting in the phosphorylation of IB and release of NF-B factor (p50/p65 heterodimer) into nucleus. Indeed, we have found that treatment of Raw cells with LPS for 15 min led to the accumulation of p65/RelA in the nucleus and stimulated the binding of NF-B to the positive regulatory domain II (PRDII) of the IFNB promoter (data not shown). The role of NF-B in virus-mediated induction of the IFNB gene has been well demonstrated (48 -50). Thus, these data indicate that the LPS-mediated modulation of the transcriptional activity of IFNB gene involves both positive and negative regulations.
The observation that the LPS-mediated suppression of IFN genes expression is most effective at the early stages of viral infection indicates that LPS interferes with the initial stages of virus-induced signaling pathway. The suppression of IFN induction by LPS is not a result of an inhibition of virus replication because LPS can suppress IFN induction without affecting the transcription of the nucleocapsid genes. While LPS efficiently suppressed virus-mediated induction of IFNA and IFNB genes, induction of the IL-6 gene in infected cells was stimulated by LPS. LPS alone was found to induce expression of several early inflammatory genes in macrophages, including TNF␣, TNFR-2, IP-10, IL-1, IFNB, and IRF-1. This activation is dependent on the LPS-induced tyrosine phosphorylation of the MAP kinases (51,52). Thus, the selective susceptibility of IFN induction to LPS may indicate that LPS suppresses activation of factors crucial for the induction of IFNs, but not for IL-6 and other cytokines.
As discussed above, IRF-3 and IRF-7 were recently identified as transducers of virus-mediated signaling pathway (29,30,34,53). IRF-3 was shown to be post-translationally phosphorylated at Ser-385 and Ser-386 (30) and at Ser-396 and Ser-398 (29). Phosphorylation was shown to facilitate translocation of IRF-3 into the nucleus and stimulate transcription of both IFNA and IFNB genes in infected cells. Our data clearly show that LPS suppresses virus-mediated phosphorylation of IRF-3 and its consequent translocation to the nucleus. Complete inhibition of nuclear transport of IRF-3 in infected cells was observed in cells that were pretreated or treated with LPS within 1 h after the infection. When the LPS treatment was initiated 4 h after virus infection, IRF-3 could be detected both in cytoplasm and in the nucleus. These data correlate the inhibition of virusmediated IRF-3 phosphorylation and nuclear translocation with the inhibition of IFNA and IFNB gene expression in LPS-treated cells. Further evidence of the involvement of IRF-3 in the LPS-mediated inhibition came from experiments using a cell line constitutively overexpressing IRF-3. In these cells, suppression of virus-mediated induction of the IFNA gene by LPS was partially reverted when cells were pretreated with LPS (data not shown) and no suppression was observed when the cells were treated with LPS at 2 or 4 h after virus infection. These data indicate that LPS interferes with the function of IRF-3 which results in the impairment of IFNA gene induction by virus. In addition to IRF-3, IRF-7 was recently demonstrated to stimulate expression of IFNA genes in infected cells (33)(34)(35) and together with IRF-3 to be a part of the transcriptional enhansosome binding to the promoter region of IFNB gene in infected cells (31). Similarly as IRF-3, IRF-7 is transported from cytoplasm to nucleus (34,35) in infected cells. Phosphorylation of IRF-7 on two carboxyl-terminal serines was shown to be required for induction of the IFNA genes (33). Our data indicate that LPS treatment also interferes with the nuclear transport of GFP/IRF-7 in infected cells. Additional experiments have to determine whether LPS also inhibits virusmediated phosphorylation of IRF-7.
While the signaling pathway(s) activated in NDV or Sendai virus-infected cells is being unfolded, it is still unclear which serine/threonine kinase(s) phosphorylates IRF-3 or IRF-7. The similarity in the phosphorylation sites present on IRF-3 and IRF-7 as well as similar kinetics of the transport of these two factors to the nucleus in infected cells suggests that a similar kinase is phosphorylating IRF-3 and IRF-7. It was shown previously that double-stranded RNA-dependent protein kinase (PKR) can be activated in infected cells; however, it is unlikely that this kinase phosphorylates IRF-3 or IRF-7 because homozygous deletion of the PKR gene does not abolish virus-mediated induction of the IFN genes (54). LPS induces a complex signaling pathway which includes tyrosine phosphorylation of several targets, activation of G protein (55), PKC (56), as well as activation of Stat3 (17). LPS was also found to post-transcriptionally regulate the transcriptional transactivator C/EBP (15).Which one of these pleiotropic responses to endotoxin abrogates the virus-induced signaling and consequent phosphorylation of IRF-3 and IRF-7 remains to be established.