Identification of a Novel Pathway Essential for th
http://www.100md.com
病菌学杂志 2006年第1期
Centre for Gene Therapeutics and Departments of Biochemistry and Biomedical Sciences
Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
ABSTRACT
Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.
INTRODUCTION
Viral infection elicits the induction of an innate immune response involving production of the soluble cytokine interferon (IFN), with the ultimate goal of protecting surrounding cells from being infected. The innate immune response plays an essential role in providing the first line of defense against invading pathogens. IFNs themselves do not directly cause an antiviral response in cells but instead induce numerous IFN-stimulated genes (ISGs) that collectively limit virus replication and spread (16).
The production of IFN leading to the expression of ISGs is mediated by IFN regulatory factor 3 (IRF-3). IRF-3 is part of a growing family of transcription factors that regulate multiple aspects of host defense (58). In eukaryotic cells, IRF-3 is constitutively expressed. During virus infection, IRF-3 is phosphorylated on serine residues in the carboxyl region by two recently identified members of the IB kinase (IKK)-related kinase family, IKK and TANK-binding kinase 1 (TBK-1) (20, 56). Phosphorylation of IRF-3 leads to its homodimerization, nuclear translocation, and association with other transcription factors like NFB, ATF-2/c-jun, and the coactivator CREB-binding protein to form a complex that binds to the IFN- promoter (60). IRF-3 can also directly bind to several DNA-binding motifs, including the IFN-stimulated response element, which is located in the promoter region of ISGs, indicating that IRF-3 also plays a role in the direct induction of ISGs in the absence of IFN production (23, 42).
Although the main inducing element of IFN in response to both RNA and DNA viruses is thought to be double-stranded RNA (dsRNA), a by-product of virus replication, it is becoming clear that cells differentially recognize and respond to the various stages and by-products produced during a viral infection. For example, Toll-like receptors (TLRs) mediate IFN and/or proinflammatory cytokine responses following recognition of either virion components (e.g., glycoproteins) or nucleic acid components (dsRNA or CpG DNA) (reviewed in references 5, 7, 41, and 57). In addition, we and others have demonstrated that a subset of ISGs can be induced in response to the entry of a diverse range of enveloped virus particles (9, 13, 42, 46, 65). Work with the human herpesviruses herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) has shown that this response requires both binding and penetration but is not reliant on the viral genome, its subsequent replication, or IFN production (6, 42, 45, 50).
Viruses have the ability to modulate various signal transduction pathways, including the extracellular regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK), the p38 and Jun N-terminal kinase (JNK) stress-activated protein kinases (SAPK), and the phosphoinositide 3-kinase (PI3 kinase) pathways, to favor their replication. Historically, research has focused on how viruses alter these pathways during a productive lytic infection. HSV-1 has been shown to induce the stress-activated kinases JNK and p38 in a process requiring viral gene expression (25, 31, 38, 63, 64). On the other hand, HCMV viral gene expression is required for sustained ERK 1/2 and p38 activation (10, 28, 51). The PI3 kinase pathway has been implicated in mediating the entry of and viral replication of several viruses in cells, including adenovirus, HCMV, and HSV-1 (11, 30, 35). HCMV particle binding and entry rapidly activate ERK 1/2 and PI3 kinase within 5 to 30 min postinfection (29, 30). Not only are these pathways implicated in optimizing virus replication, but they are also involved in the innate immune response to the infection (reviewed in references 17 and 33).
The prototypic class I PI3 kinase (herein referred to as PI3K) is the most-studied member of this vast family. PI3K is a dimeric protein composed of the adaptor subunit p85 and the catalytic subunit p110. The lipid products of PI3K activation act as second messengers to promote the activity of numerous downstream effectors, including the well-studied serine/threonine kinase Akt/PKB (21). Recently, Sarkar et al. showed that dsRNA-mediated phosphorylation of TLR-3 results in the activation of both TBK-1 and PI3K/Akt, leading to the induction of ISG56 (55). Their data suggest that full activation of IRF-3 in response to dsRNA requires two phosphorylation events, the first dependent on TBK-1 and the second on PI3K (55).
We are interested in identifying the signal transduction pathways that are activated following entry of the physical virus particle into a cell, prior to virus replication. Here we report that treatment of cells with the general PI3 kinase inhibitor LY294002 prevented the induction of ISG56 and a subsequent antiviral response without affecting dimerization or nuclear translocation of IRF-3 in human embryonic lung (HEL) fibroblasts upon entry of enveloped virus particles. The decrease in ISG56 mRNA and protein expression following LY294002 treatment was not due to inhibition of the prototypic PI3K or other LY294002-sensitive kinases implicated in IRF-3 activation. Taken together, these data imply the involvement of a novel kinase within the PI3 kinase family in the induction of an antiviral state in response to virus particle entry.
MATERIALS AND METHODS
Reagents. The inhibitors LY294002, U0126, SB202190, SP600125, and cycloheximide (Sigma) were reconstituted in dimethyl sulfoxide (DMSO). The concentration of LY294002 required to elicit a biological response varied from 25 to 50 μM, depending on the particular batch and lot number. U0126, SB202190, SP600125, and cycloheximide were used at 10 μM, 25 μM, 25 μM, and 50 μg/ml, respectively. The properties of each inhibitor used in this study are listed in Table 1. The dsRNA mimetic poly(I-C) (GE Healthcare) was reconstituted in phosphate-buffered saline (PBS) at a concentration of 5 mg/ml. Human IFN- (Sigma) was reconstituted in PBS at a concentration of 1,000 IU/μl. Human recombinant platelet-derived growth factor (PDGF; Oncogene Research Products) was reconstituted in PBS containing 0.1% bovine serum albumin (BSA) at a concentration of 50 ng/μl.
Cell culture and virus infections. HEL fibroblasts and Vero cells were maintained in Dulbecco modified Eagle medium containing 10% and 5% fetal calf serum (FCS), respectively. A549 human epithelial cells were maintained in alpha minimum essential medium (MEM) containing 10% FCS. Primary DNA-PK catalytic subunit (DNA-PKcs)+/– and DNA-PKcs–/– mouse embryonic fibroblasts (MEFs; generously provided by D. Chen) were maintained in alpha MEM containing 20% FCS. All cell lines were used between passages 2 and 18. HSV-1 (strain KOS) was propagated on Vero cells. HCMV (strain AD169; kindly provided by T. Compton) was propagated on HEL fibroblasts. Sendai virus (SeV; strain Cantell) was purchased from Charles River Laboratories. All herpesvirus infections were carried out in serum-free media at a multiplicity of infection (MOI) of 10 PFU/cell for 1 h at 37°C, unless otherwise specified. For SeV infections, 10 hemagglutinin units of virus were used per 106 cells. A vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP; kindly provided by B. Lichty) was propagated on Vero cells and used in the VSV plaque reduction assays. For experiments with the U0126, SB202190, SP600125, and LY294002 inhibitors, HEL cells were grown to confluence and serum starved for 2 h. Cells were pretreated for 1 h with the indicated inhibitor prior to infection. In addition, the inhibitor was present for the duration of the experiment. UV inactivation of viruses was performed with a UV Stratalinker 2400 (Stratagene) for the length of time required to prevent viral gene expression as measured by indirect immunofluorescence. Standard plaque reduction assays with replication-competent VSV were performed as previously described (11).
Replication-deficient adenovirus constructs containing either a dominant negative form of the regulatory subunit of PI3K (Addnp85) (24, 54) or a kinase-dead mutant form of the p21-activated kinase (PAK; AdPAKK299R) (22) were diluted in PBS supplemented with 0.9 mM MgCl2 and 0.5 mM CaCl2 and allowed to adsorb to the cells for 30 min at room temperature, with the subsequent addition of Dulbecco modified Eagle medium and incubation at 37°C for 24 h.
Preparation of cell extracts. Whole-cell extracts were prepared by washing the cells twice and harvesting them in cold PBS, followed by centrifugation at 200 x g for 5 min at 4°C. The cell pellets were resuspended in whole-cell extract buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM -glycerophosphate, 0.2% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 1x protease inhibitor cocktail [Sigma]) and lysed for 15 min on ice. The lysate was centrifuged at 13,000 x g for 10 min at 4°C, and protein quantification was performed with the Bradford assay kit (Bio-Rad Laboratories).
Native protein extracts were prepared as previously described (55). Briefly, nuclear or cytoplasm-enriched extracts were incubated with rehydration buffer containing 8 M urea, 2% (wt/vol) 3-([3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and 20 mM dithiothreitol. The cytoplasmic and nuclear extracts were sonicated for 5 s, and debris was removed by centrifugation at 13,000 x g for 10 min. Protein quantification was carried out with a 2-D Quant kit (GE Healthcare). Eight micrograms and 2 μg of the cytoplasm-enriched and nuclear extracts, respectively, were separated on a 7.5% nondenaturing gel in a 25 mM Tris-HCl (pH 8.4)-192 mM glycine buffer with 0.2% deoxycholate in the cathode chamber.
Western blot analysis. Polyacrylamide gels were transferred onto polyvinylidene difluoride membranes (Millipore) with a semidry transfer apparatus at 400 mA for 1.5 h. All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) at room temperature for 1 h. ERK 1/2 (Cell Signaling no. 9102), P-ERK 1/2 (Cell Signaling no. 9101), total Akt (Cell Signaling no. 9272), P-Akt (Ser473; Cell Signaling no. 9271), total p38 MAPK (Cell Signaling no. 9212), P-p38 MAPK (Cell Signaling no. 9211), total SAPK/JNK (Cell Signaling no. 9252), P-SAPK-JNK (Cell Signaling no. 9251), PI3K subunit p85 (Santa Cruz SC-1637), PAK (Santa Cruz SC-881), and anti-human IRF-3 (Immuno-Biological Laboratories Co. Ltd.) primary antibodies were all used at a dilution of 1:1,000 in 5% BSA in TBS-Tween (0.1%). The ISG56 (provided by G. Sen) and -actin (Santa Cruz SC-1616) primary antibodies were used at a dilution of 1:1,000 in TBS-Tween (0.1%). The anti-ISG15 antibody (provided by E. Borden) was used at a dilution of 1:500 in TBS-Tween (0.1%). Secondary antibodies were conjugated to horseradish peroxidase and visualized by chemiluminescence.
RNA extraction and reverse transcription (RT)-PCR analysis. Total cellular RNA was obtained with Trizol reagent (Invitrogen) according to the manufacturer's protocols. Two micrograms of total RNA was reverse transcribed with 100 U of Superscript II (Invitrogen) in a total reaction volume of 20 μl. A random hexamer primer was used to synthesize cDNA at 42°C for 50 min, followed by a 15-min incubation at 70°C. PCR was performed according to the manufacturer's specifications with previously described primer sets (13).
Indirect and direct immunofluorescence. HEL fibroblasts were grown to semiconfluence on glass coverslips overnight. Cells were treated with the indicated stimuli, and at the indicated times postinfection, coverslips were washed twice with PBS and fixed in a solution of 4% paraformaldehyde in PBS. Cells were permeabilized in 0.1% Triton X-100 and then blocked in a solution of 3% BSA, 3% goat serum, and 0.02% Tween 20. Polyclonal anti-HSV-1 (DAKO no. B0114) or monoclonal anti-IE1/IE2 (Rumbaugh-Goodwin Institute no. 1203) primary antibodies were used at a dilution of 1:1,000 to detect expression of HSV-1 or HCMV proteins, respectively. A polyclonal anti-IRF-3 primary antibody (a generous gift from M. David) was used at a dilution of 1:5,000. Alexa Fluor-conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) were used at a dilution of 1:500. Cells were also stained with the intercalating DNA dye Hoechst in order to determine the total number of cells in a field of view. Images were captured on a Leica DM-IRE2 inverted microscope. The percentage of cells positive for HSV-1, HCMV, or nuclear IRF-3 was calculated from three independent experiments with a total of 12 random fields of view for each sample.
A549 cells were grown to semiconfluence on glass coverslips. Cells were infected with AdE1/E3 or AdPAKK299R at an MOI of 50 PFU/cell for 24 h, washed twice in PBS, and fixed in a 4% solution of paraformaldehyde in PBS. Nuclei were then stained with Hoechst dye. Cells expressing the GFP-tagged AdPAKK299R constructs were visualized on a Leica DM-IRE2 inverted microscope to determine cellular morphology and infection efficiency.
Data analysis. Data were expressed as means ± the standard error of the mean. Statistical analysis was performed with either a t test or a one-way analysis of variance with the Tukey test post hoc with Sigma Stat 2.03.
RESULTS
Activation of the ERK 1/2 and p38 MAPK pathways is not required for induction of ISG56. Signal transduction pathways exhibit complex cross talk with each other in mammalian cells. Since replicating viruses activate numerous signal transduction pathways that mediate innate and adaptive immune responses, we tested whether the activity of these pathways is required for the immediate-early induction of ISGs following enveloped virus particle entry. The U0126 inhibitor has been shown to specifically inhibit MEK 1/2 activity, preventing phosphorylation of its downstream effector, ERK 1/2 (Table 1) (19). The SB202190 inhibitor, on the other hand, specifically inhibits p38 activity by preventing the transfer of the phosphate group from p38 to its downstream effector kinase(s) (Table 1) (62), leading to retention of phosphorylated p38 in the presence of the SB202190 inhibitor. As a control for ERK 1/2 and p38 MAPK phosphorylation, cells were treated with FCS or UV irradiation, respectively. To ensure maintenance of U0126 and SB202190 inhibitor activity throughout the experimental procedure, the positive control was administered 30 min prior to sample harvest (Fig. 1A and B, lanes 13 and 14). Poly(I-C), HSV-UV, and HCMV-UV were able to stimulate accumulation of ISG56 mRNA to similar levels regardless of the phosphorylation state of ERK 1/2 or p38 MAPK (Fig. 1A and B). These data suggest that activation of the ERK 1/2 and p38 MAPK pathways is not required for ISG56 induction in response to virus particle entry.
Inhibition of the JNK/SAPK and PI3 kinase pathways prevents induction of ISG56 in human fibroblast cells. Since it has previously been shown that herpesviruses activate the JNK/SAPK and PI3 kinase pathways during infection (25, 30, 38, 63), we determined whether activation of these pathways is necessary for the induction of ISG56 upon enveloped virus particle entry. As a control for JNK/SAPK and PI3K/Akt phosphorylation, cells were treated with UV irradiation or PDGF, respectively (Fig. 1C, lanes 13 and 14; see also Fig. 3, lanes 8, 16, and 24). To ensure maintenance of inhibitor activity, the positive control was administered 30 min prior to sample harvest. Poly(I-C), HSV-UV, and HCMV-UV showed a marked decrease in the level of ISG56 mRNA in the presence of SP600125 (Fig. 1C). In the presence of the LY294002 inhibitor, similar results were observed (Fig. 1D). Consistent with previous reports (13), replication-deficient, nonenveloped adenovirus particles were unable to induce the accumulation of ISG56 mRNA (Fig. 1D).
Inhibition of the PI3K pathway prevents the induction of an antiviral state in human fibroblasts. To confirm that the decrease in ISG56 mRNA accumulation correlated with the inability of the cells to induce an antiviral state, we performed a VSV plaque reduction assay as previously described (13). In the absence of ERK 1/2, p38, and JNK MAPK activities, enveloped virions were still able to induce an antiviral state in human fibroblasts (Table 2). In the presence of the LY294002 inhibitor, however, poly(I-C) and HSV-UV were unable to elicit an antiviral state in human fibroblasts. Interestingly, in the presence of high-multiplicity HCMV-UV infection (MOI, >0.1 PFU/cell), which we found induces IFN production (data not shown), the effect of the LY294002 inhibitor was less apparent and was unable to prevent the induction of an antiviral state. Although ISG56 mRNA was barely detectable following infection with HCMV-UV at a low MOI (0.1 PFU/cell), induction of an antiviral response sufficient to prevent VSV replication was sporadic. Since an increase in the MOI to 0.5 PFU/cell consistently induced an antiviral response whereas a decrease in the MOI to 0.05 PFU/cell failed to induce an antiviral response, we hypothesize that an MOI of 0.1 PFU/cell represents a threshold limit of virus capable of initiating an antiviral response.
Inhibition of the JNK/SAPK and PI3K pathways does not prevent entry and expression of HSV-1 and HCMV in human fibroblasts. Since HCMV and HSV entry is thought to require PI3K activity (30) and both binding and penetration of HSV-1 and HCMV virions are required for maximal ISG induction (45, 50), we tested whether the observed reduction of ISG56 upon inhibition of PI3 kinase and SAPK activity was due to a block in viral entry. Human fibroblasts were infected with replicating HSV-1 (Fig. 2A) or HCMV (Fig. 2B) in the presence or absence of LY294002 or SP600125. At 4 h postinfection, the percentage of cells staining positive for HSV-1 proteins was lower in the presence of SP600125, as determined by indirect immunofluorescence. By 8 h postinfection, no significant difference was observed and by 12 h postinfection, almost all of the cells stained positive for HSV-1 proteins regardless of inhibitor treatment. There were no differences in the frequency of cells staining positive for HCMV proteins upon treatment with DMSO, LY294002, or SP600125.
The prototypic class I PI3K signal transduction pathway is not involved in inhibition of the ISG56 protein. Although studies of virus-mediated PI3 kinase signaling almost exclusively focus on the prototypic class I PI3K, the LY294002 compound inhibits multiple PI3 kinase family members. To distinguish between PI3K and alternate PI3 kinase family members, fibroblasts were infected with an adenovirus expressing a dominant negative form of the PI3K p85 subunit (Addnp85) or an empty adenovirus (AdE1/E3) or treated with the LY294002 inhibitor (Fig. 3). Cells were then serum starved for 2 h prior to treatment with the indicated stimuli. Although Addnp85 and LY294002 were able to inhibit the phosphorylation of Akt in response to PDGF (Fig. 3, lanes 8, 16, and 24), only LY294002 was able to significantly reduce ISG56 protein production in response to poly(I-C) or virus particle entry. This reduction correlated with the absence of an antiviral state in cells pretreated with LY294002 but not Addnp85 (data not shown), with the exception of high-multiplicity HCMV infection, as noted in Fig. 1C. Moreover, although enveloped virions were able to elicit the transient phosphorylation of ERK1/2 at early times postentry (15 to 30 min), we failed to detect phosphorylation of Akt at any time postentry (data not shown). These data suggest that the prototypic PI3K enzyme is not involved in activating ISG56 in response to enveloped virus particle entry and instead suggests that an alternate member(s) of the PI3 kinase family is required.
DNA-PK and PAK, two protein kinases implicated in IRF-3 activation, are not essential for virus-mediated ISG induction. Recently, two protein kinases, DNA-PK and PAK, were implicated in IRF-3 activation (18, 32). The activities of both of these kinases are directly or indirectly blocked by inhibitors of PI3 kinase activity, including LY294002 (26, 47, 48). Low-passage, primary wild-type and DNA-PKcs–/– MEFs were treated with HSV for 6 h in the presence or absence of cycloheximide, RNA was harvested, and RT-PCR performed (Fig. 4). Since low-passage, nonimmortalized MEFs secrete IFN in response to various stimuli, including virus particle entry (Fig. 4, lanes 3 and 7), ISG56 mRNA induction was assayed in the presence of cycloheximide to prevent de novo synthesis of IFN. In the absence of the catalytic subunit of DNA-PK, cells were able to express similar levels of ISG56 mRNA compared to wild-type MEFs (Fig. 4, compare lanes 4 and 8), indicating that the catalytic activity of DNA-PK is not essential for the induction of ISG56 mRNA.
To investigate whether PAK activity was required for IRF-3-mediated ISG induction in response to the virion particles, we treated A549 epithelial cells with an adenovirus expressing a dominant-negative PAK construct (AdPAKK299R) tagged with GFP (Fig. 5A). Since we have previously shown that A549 cells and HEL fibroblasts are able to induce ISG56 protein to similar levels following SeV particle entry (13), we used A549 in place of HEL fibroblasts due to their enhanced permissiveness to adenovirus infection. Cells expressing the dominant negative PAK construct were significantly larger compared to uninfected cells and contained numerous lamellipodia, indicative of PAK inhibition (Fig. 5B) (22).
Cells preloaded with dominant negative PAK were treated with replicating and nonreplicating forms of SeV to investigate whether inhibition of PAK activity prevented induction of ISGs. At both 12 h and 24 h postinfection, protein extracts were harvested and Western blot analysis performed (Fig. 5C). Upon overexpression of dominant negative PAK, the levels of both ISG15 and ISG56 remained similar in cells treated with either SeV or SeV-UV, suggesting that PAK activity is not essential for the induction of ISGs in response to both replicating and nonreplicating enveloped virus particles.
The LY294002 inhibitor does not inhibit dimerization or nuclear localization of IRF-3 upon virus particle treatment. Given that the two LY294002-sensitive protein kinases DNA-PK and PAK, which have been implicated in IRF-3 activation, were shown to be nonessential for ISG induction in response to virion particles, we examined whether LY294002 inhibition had any effect on IRF-3 activation. We first investigated, by indirect immunofluorescence assay, whether the LY294002 inhibitor was capable of preventing nuclear translocation of IRF-3. HEL fibroblasts were treated with the LY294002 compound in combination with replication-deficient HSV-1 or HCMV. At various times postinfection, cells were fixed and stained with an antibody for IRF-3. As previously observed (13), the level of nuclear translocation of endogenous IRF-3 was minimal following induction of an IFN-independent antiviral response (HSV-1, MOI of 10 PFU/cell [Fig. 6A and B]; HCMV, MOI of 0.1 PFU/cell [data not shown]). Importantly, there was no difference in the amount of IRF-3 nuclear translocation when the LY294002 compound was present, indicating that the LY294002 inhibitor does not have an effect on the ability of IRF-3 to translocate to the nucleus upon virus particle stimulation. For enhanced detection, the experiment was repeated with elevated MOIs; results obtained with HCMV-UV (MOI, 10 PFU/cell) are outlined in Fig. 6C and D. Under these conditions, there was a significant increase in the number of cells that stained positive for nuclear IRF-3 at early times compared to the mock-treated sample. Interestingly, as the time course progressed, the number of nuclei positive for IRF-3 decreased significantly in the DMSO-treated samples compared to the LY294002-treated samples. One explanation is that LY294002 prevents virus-mediated IRF-3 degradation. However, consistent with previous observations (13), infection at low or high multiplicities with UV-inactivated HSV-1 or HCMV failed to affect the level of endogenous IRF-3 in whole-cell extracts as determined by standard denaturing gel electrophoresis (data not shown). Alternatively, the observed difference in the level of nuclear IRF-3 following high-multiplicity infection could result from either LY294002-mediated retention of IRF-3 in the nucleus or prevention of a conformational change leading to the inability of the antibody to recognize IRF-3 under the conditions used.
To differentiate between these possibilities and determine if LY294002 affects IRF-3 dimerization upon virus particle entry, we performed native gel electrophoresis on cytoplasmic and nuclear extracts following high-multiplicity HCMV-UV infection in the presence or absence of LY294002. Consistent with immunofluorescence microscopy, HCMV virions induced IRF-3 dimerization and nuclear translocation within 4 h of infection in the presence or absence of the inhibitor (Fig. 7, lanes 1 to 8). Furthermore, at late times of infection, an overall decrease in the level of IRF-3 was observed, which was reproducibly more enhanced in the DMSO samples relative to the LY294002 samples. However, there was no significant difference in the relative amounts of IRF-3 within the cytoplasm or nucleus within either sample. Taken together, these data suggest that LY294002 does not affect the ability of incoming virions to elicit the dimerization or nuclear translocation of IRF-3 but may modulate, to some degree, subsequent modifications of IRF-3 that alter the conformation of the protein.
DISCUSSION
During virus infection, cells recognize different viral structures and viral life cycle events, leading to the activation and modulation of a multitude of signal transduction pathways. For example, TLRs recognize different pathogen-associated molecular patterns, leading to the production of type I IFN and/or proinflammatory cytokines (39, 43). With respect to viruses, certain TLRs stimulate proinflammatory cytokines upon recognition of virion-associated proteins or genome-encoded CpG motifs while others stimulate IFN production in response to dsRNA (reviewed in references 5, 7, and 57). Cells are also able to recognize viral particle entry prior to uncoating or initiation of replication, with the nature of the response depending on the type of virus. For example, entry of nonenveloped adenovirus particles induces a proinflammatory cytokine cascade mediated by NF-B (37) whereas entry of enveloped virus particles induces an IFN-independent ISG response mediated by IRF-3 (5, 13, 46). Cellular antiviral responses also differ depending on the cell type, as observed with virion-associated glycoprotein B (gB) of HCMV. In fibroblasts, gB triggers IRF-3-mediated ISG induction (6, 8), whereas in peripheral blood mononuclear cells, gB triggers NF-B-mediated proinflammatory cytokine production (14). Finally, within a given cell type, the MOI can modulate the nature of the resulting cellular response. In primary human fibroblasts, enveloped virus particle entry at a low multiplicity elicits an IFN-independent antiviral response, whereas at elevated multiplicities, IFN production is observed (P. Paladino, R. Noyce, and K. Mossman, unpublished data).
Accumulation of dsRNA during lytic virus infection results in the activation of numerous cellular signal transduction pathways, leading to the activation of IRF-3, JNK, and p38 SAPKs (12, 27), TLR-dependent (1) and TLR-independent (3, 61) pathways, culminating in IFN production and antiviral state induction. As well, it has recently been appreciated that the prototypic class I PI3K is necessary for full activation of IRF-3 leading to the induction of ISG56 in response to dsRNA, via activation of Akt (55). Other signal transduction pathways become activated upon viral protein synthesis. For example, HCMV viral protein expression leads to PI3K (30), ERK 1/2 (51), and p38 (28) activation. Depending on the system studied, activation of these pathways has been shown to be critical both for the cell in mediating an antiviral response and for the virus in ensuring productive lytic infection.
The focus of this study was to determine whether signal transduction pathways other than IRF-3 are essential for the immediate-early, IFN-independent antiviral response to enveloped virus particle entry. Although virion entry rapidly and transiently induced the activation of some of the pathways studied (e.g., ERK1/2), inhibition of these pathways had little to no effect on ISG and antiviral state induction, with the exception of LY294002. Although the JNK/SAPK inhibitor caused a decrease in the accumulation of ISG56 at the time point tested, it failed to prevent induction of an antiviral state in fibroblasts in response to enveloped virus particles. The inhibitor did, however, block production of IFN in response to treatment with poly(I-C) (data not shown), suggesting that the JNK/SAPK pathway may play a role in mediating IFN production as opposed to the IFN-independent antiviral response. Given that the JNK/SAPK inhibitor utilized in these studies is relatively new and not yet fully characterized, it remains to be determined whether JNK/SAPK plays a role, albeit nonessential, in the cellular response to virus particle entry. Studies are ongoing to address this issue.
The PI3 kinase family constitutes a large family of protein kinases that mediate a diverse array of intracellular pathways. With respect to virus infection, the prototypic class I PI3K is almost exclusively studied, and found to be activated by and important for, the replication of multiple viruses. Here, we found that targeted inactivation of the adaptor subunit of PI3K failed to block ISG accumulation or antiviral state induction following virion entry. This observation suggests that an alternate member of the PI3 kinase family is essential for virion-mediated antiviral state induction. Two such members have been implicated in IRF-3 activation, DNA-PK and PAK1 (18, 32), both of which can be inhibited by LY294002 treatment (26, 47, 48). Of particular interest, DNA-PK has been shown to phosphorylate IRF-3 (32), and the HSV-1 immediate-early protein ICP0, which we have shown blocks IRF-3 and IRF-7 activity (36), induces the degradation of DNA-PK (49). With primary MEFs harvested from mice lacking the catalytic subunit of DNA-PK, however, we found that this kinase is not essential for antiviral state induction mediated by virus entry. This result is in agreement with that of Melroe et al., who investigated immortalized glioma cells deficient in DNA-PK (40).
Recently, PAK1 was shown to be upstream of TBK-1 and IKK in the viral activation of IRF-3 (18). Given that PAK1 is involved in modulating the cytoskeleton (22), in conjunction with reports that herpesvirus particle entry induces cytoskeletal rearrangements (44, 52, 53), we hypothesized that entry of enveloped virus particles may activate IRF-3 via PAK1. However, though expression of a dominant negative PAK1 construct caused cytoskeletal deregulation, there was no effect on ISG or antiviral state induction. It remains to be determined if cytoskeletal rearrangements mediated by virion particle entry play any role in activation of IRF-3 and an antiviral response.
Although dsRNA-mediated phosphorylation of IRF-3 requires PI3K and Akt activities, it is unclear whether the LY294002-sensitive PI3 kinase family member involved in antiviral state induction in response to virus particle entry functions within, or parallel to, the IRF-3 pathway. We have previously shown that induction of an antiviral response to enveloped particles in fibroblasts does not correlate with IRF-3 hyperphosphorylation and its subsequent degradation (13). Thus, we set out to determine if LY294002 affects IRF-3 dimerization and/or nuclear translocation. Although neither dimerization nor nuclear translocation was blocked in the presence of the inhibitor, it appeared that LY294002 may modify, to some degree, conformational changes that occur in IRF-3 following its translocation into the nucleus. Whether this constitutes an additional posttranslational modification in IRF-3 or the ability of IRF-3 to interact with a nuclear cofactor(s) remains to be determined.
Taken together, the results of this study show that cellular responses to viral infections differ dramatically during the course of infection. While activation of signaling cascades involving ERK1/2, p38, JNK/SAPK, and the prototypic PI3K has been shown to be important for cells to respond to lytic virus infection, these pathways are not essential for the immediate-early response to virion entry. Entry does, however, result in the activation of IRF-3, leading to the induction of ISGs and an antiviral response in the absence of IFN production. Here, we provide evidence that a novel member of the PI3 kinase pathway that is sensitive to LY294002 is essential for this immediate-early response. While this kinase does not appear to function upstream of IRF-3, it is unclear whether it mediates its effects on IRF-3 following nuclear translocation or instead functions in parallel with the IRF-3 pathway. Given that the early interactions between a virus and its host mediate the outcome of a virus infection, particularly under conditions of low multiplicity that likely represent the nature of most biological infections, it is critical to understand these interactions. Current studies are thus focused on identifying and characterizing the LY294002-sensitive kinase along with its potential effects on IRF-3 nuclear events.
ACKNOWLEDGMENTS
We thank E. Borden, D. Chen, T. Compton, B. Lichty, M. David, and G. Sen for reagents. We also thank D. Cummings for growing adenovirus stocks for use in this study.
This work was supported by a grant from the Canadian Institutes of Health Research. R.S.N. is supported by a Natural Sciences and Engineering Research Council (NSERC) postgraduate doctoral scholarship. K.L.M. is supported by an Rx&D-Health Research Foundation career award.
REFERENCES
Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732-738.
Bain, J., H. McLauchlan, M. Elliott, and P. Cohen. 2003. The specificities of protein kinase inhibitors: an update. Biochem. J. 371:199-204.
Balachandran, S., E. Thomas, and G. N. Barber. 2004. A FADD-dependent innate immune mechanism in mammalian cells. Nature 432:401-405.
Bennett, B. L., D. T. Sasaki, B. W. Murray, E. C. O'Leary, S. T. Sakata, W. Xu, J. C. Leisten, A. Motiwala, S. Pierce, Y. Satoh, S. S. Bhagwat, A. M. Manning, and D. W. Anderson. 2001. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98:13681-13686.
Boehme, K. W., and T. Compton. 2004. Innate sensing of viruses by Toll-like receptors. J. Virol. 78:7867-7873.
Boehme, K. W., J. Singh, S. T. Perry, and T. Compton. 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78:1202-1211.
Bowie, A. G., and I. R. Haga. 2005. The role of Toll-like receptors in the host response to viruses. Mol. Immunol. 42:859-867.
Boyle, K. A., R. L. Pietropaolo, and T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19:3607-3613.
Browne, E. P., B. Wing, D. Coleman, and T. Shenk. 2001. Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J. Virol. 75:12319-12330.
Chen, J., and M. F. Stinski. 2002. Role of regulatory elements and the MAPK/ERK or p38 MAPK pathways for activation of human cytomegalovirus gene expression. J. Virol. 76:4873-4885.
Cheshenko, N., B. Del Rosario, C. Woda, D. Marcellino, L. M. Satlin, and B. C. Herold. 2003. Herpes simplex virus triggers activation of calcium-signaling pathways. J. Cell Biol. 163:283-293.
Chu, W.-M., D. Ostertag, Z.-W. Li, L. Chang, Y. Chen, Y. Hu, B. Williams, J. Perrault, and M. Karin. 1999. JNK2 and IKK are required for activating the innate response to viral infection. Immunity 11:721-731.
Collins, S. E., R. S. Noyce, and K. L. Mossman. 2004. Innate cellular response to virus particle entry requires IRF3 but not virus replication. J. Virol. 78:1706-1717.
Compton, T., E. A. Kurt-Jones, K. W. Boehme, J. Belko, E. Latz, D. T. Golenbock, and R. W. Finberg. 2003. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J. Virol. 77:4588-4596.
Davies, S., H. Reddy, M. Caivano, and P. Cohen. 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351:95-101.
Der, S. D., A. Zhou, B. R. G. Williams, and R. H. Silverman. 1998. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:15623-15628.
Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55-72.
Ehrhardt, C., C. Kardinal, W. J. Wurzer, T. Wolff, C. von Eichel-Streiber, S. Pleschka, O. Planz, and S. Ludwig. 2004. Rac1 and PAK1 are upstream of IKK- and TBK-1 in the viral activation of interferon regulatory factor-3. FEBS Lett. 567:230-238.
Favata, M. F., K. Y. Horiuchi, E. J. Manos, A. J. Daulerio, D. A. Stradley, W. S. Feeser, D. E. Van Dyk, W. J. Pitts, R. A. Earl, F. Hobbs, R. A. Copeland, R. L. Magolda, P. A. Scherle, and J. M. Trzaskos. 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273:18623-18632.
Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, and T. Maniatis. 2003. IKK and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4:491-496.
Franke, T. F., D. R. Kaplan, L. C. Cantley, and A. Toker. 1997. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665-668.
Frost, J. A., A. Khokhlatchev, S. Stippec, M. A. White, and M. H. Cobb. 1998. Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation. J. Biol. Chem. 273:28191-28198.
Guo, J., K. L. Peters, and G. C. Sen. 2000. Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection. Virology 267:209-219.
Hara, K., K. Yonezawa, H. Sakaue, A. Ando, K. Kotani, T. Kitamura, Y. Kitamura, H. Ueda, L. Stephens, T. Jackson, M. Waterfield, and M. Kasuga. 1994. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc. Natl. Acad. Sci. USA 91:7415-7419.
Hargett, D., T. McLean, and S. L. Bachenheimer. 2005. Herpes simplex virus ICP27 activation of stress kinases JNK and p38. J. Virol. 79:8348-8360.
Hartley, K., D. Gell, G. Smith, H. Zhang, N. Divecha, M. Connelly, A. Admon, S. Lees-Miller, C. Anderson, and S. Jackson. 1995. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 82:849-856.
Iordanov, M. S., J. M. Paranjape, A. Zhou, J. Wong, B. R. G. Williams, E. F. Meurs, R. H. Silverman, and B. E. Magun. 2000. Activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways. Mol. Cell. Biol. 20:617-627.
Johnson, R. A., S.-M. Huong, and E.-S. Huang. 2000. Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38. J. Virol. 74:1158-1167.
Johnson, R. A., X.-L. Ma, A. D. Yurochko, and E.-S. Huang. 2001. The role of MKK1/2 kinase activity in human cytomegalovirus infection. J. Gen. Virol. 82:493-497.
Johnson, R. A., X. Wang, X.-L. Ma, S.-M. Huong, and E.-S. Huang. 2001. Human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (PI3-K) pathway: inhibition of PI3-K activity inhibits viral replication and virus-induced signaling. J. Virol. 75:6022-6032.
Karaca, G., D. Hargett, T. I. McLean, J. S. Aguilar, P. Ghazal, E. K. Wagner, and S. L. Bachenheimer. 2004. Inhibition of the stress-activated kinase, p38, does not affect the virus transcriptional program of herpes simplex virus type 1. Virology 329:142-156.
Karpova, A. Y., M. Trost, J. M. Murray, L. C. Cantley, and P. M. Howley. 2002. Interferon regulatory factor-3 is an in vivo target of DNA-PK. Proc. Natl. Acad. Sci. USA 99:2818-2823.
Koyasu, S. 2003. The role of PI3K in immune cells. Nat. Immunol. 4:313-319.
Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, J. E. Strickler, M. M. McLaughlin, I. R. Siemens, S. M. Fisher, G. P. Livi, J. R. White, J. L. Adams, and P. R. Young. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739-746.
Li, E., D. Stupack, R. Klemke, D. A. Cheresh, and G. R. Nemerow. 1998. Adenovirus endocytosis via alpha v integrins requires phosphoinositide-3-OH kinase. J. Virol. 72:2055-2061.
Lin, R., R. S. Noyce, S. E. Collins, R. D. Everett, and K. L. Mossman. 2004. The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J. Virol. 78:1675-1684.
Liu, Q., and D. A. Muruve. 2003. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 10:935-940.
McLean, T. I., and S. L. Bachenheimer. 1999. Activation of cJUN N-terminal kinase by herpes simplex virus type 1 enhances viral replication. J. Virol. 73:8415-8426.
Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394-397.
Melroe, G. T., N. A. DeLuca, and D. M. Knipe. 2004. Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production. J. Virol. 78:8411-8420.
Morrison, L. A. 2004. The Toll of herpes simplex virus infection. Trends Microbiol. 12:353-356.
Mossman, K. L., P. F. Macgregor, J. J. Rozmus, A. B. Goryachev, A. M. Edwards, and J. R. Smiley. 2001. Herpes simplex virus triggers and then disarms a host antiviral response. J. Virol. 75:750-758.
Muzio, M., G. Natoli, S. Saccani, M. Levrero, and A. Mantovani. 1998. The human Toll signaling pathway: divergence of nuclear factor kappa B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J. Exp. Med. 187:2097-2101.
Naranatt, P. P., H. H. Krishnan, M. S. Smith, and B. Chandran. 2005. Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus. J. Virol. 79:1191-1206.
Netterwald, J. R., T. R. Jones, W. J. Britt, S.-J. Yang, I. P. McCrone, and H. Zhu. 2004. Postattachment events associated with viral entry are necessary for induction of interferon-stimulated genes by human cytomegalovirus. J. Virol. 78:6688-6691.
Nicholl, M. J., L. H. Robinson, and C. M. Preston. 2000. Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1. J. Gen. Virol. 81:2215-2218.
Nobes, C. D., and, A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53-62.
Papakonstanti, E. A., and C. Stournaras. 2002. Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization. Mol. Biol. Cell 13:2946-2962.
Parkinson, J., S. P. Lees-Miller, and R. D. Everett. 1999. Herpes simplex virus type 1 immediate-early protein Vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase. J. Virol. 73:650-657.
Preston, C. M., A. N. Harman, and M. J. Nicholl. 2001. Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J. Virol. 75:8909-8916.
Rodems, S. M., and D. H. Spector. 1998. Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection. J. Virol. 72:9173-9180.
Rosenthal, K., R. Perez, and C. Hodnichak. 1985. Inhibition of herpes simplex virus type 1 penetration by cytochalasins B and D. J. Gen. Virol. 66:1601-1605.
Rue, C. A., and P. Ryan. 2003. A role for glycoprotein C in pseudorabies virus entry that is independent of virus attachment to heparan sulfate and which involves the actin cytoskeleton. Virology 307:12-21.
Sakaue, H., W. Ogawa, M. Takata, S. Kuroda, K. Kotani, M. Matsumoto, M. Sakaue, S. Nishio, H. Ueno, and M. Kasuga. 1997. Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes. Mol. Endocrinol. 11:1552-1562.
Sarkar, S. N., K. L. Peters, C. P. Elco, S. Sakamoto, S. Pal, and G. C. Sen. 2004. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nat. Struct. Mol. Biol. 11:1060-1067.
Sharma, S., B. R. ten Oever, N. Grandvaux, G.-P. Zhou, R. Lin, and J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300:1148-1151.
Takeda, K., and S. Akira. 2005. Toll-like receptors in innate immunity. Int. Immunol. 17:1-14.
Taniguchi, T., K. Ogasawara, A. Takaoka, and N. Tanaka. 2001. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19:623-655.
Vlahos, C., W. Matter, K. Hui, and R. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241-5248.
Wathelet, M. G., C. H. Lin, B. S. Parekh, L. V. Ronco, P. M. Howley, and T. Maniatis. 1998. Virus infection induces the assembly of coordinately activated transcription factors on the IFN- enhancer in vivo. Mol. Cell 1:507-518.
Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5:730-737.
Young, P. R., M. M. McLaughlin, S. Kumar, S. Kassis, M. L. Doyle, D. McNulty, T. F. Gallagher, S. Fisher, P. C. McDonnell, S. A. Carr, M. J. Huddleston, G. Seibel, T. G. Porter, G. P. Livi, J. L. Adams, and J. C. Lee. 1997. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272:12116-12121.
Zachos, G., B. Clements, and J. Conner. 1999. Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1. J. Biol. Chem. 274:5097-5103.
Zachos, G., M. Koffa, C. M. Preston, J. B. Clements, and J. Conner. 2001. Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication. J. Virol. 75:2710-2728.
Zhu, H., J.-P. Cong, and T. Shenk. 1997. Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs. Proc. Natl. Acad. Sci. USA 94:13985-13990.(Ryan S. Noyce, Susan E. C)
Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
ABSTRACT
Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.
INTRODUCTION
Viral infection elicits the induction of an innate immune response involving production of the soluble cytokine interferon (IFN), with the ultimate goal of protecting surrounding cells from being infected. The innate immune response plays an essential role in providing the first line of defense against invading pathogens. IFNs themselves do not directly cause an antiviral response in cells but instead induce numerous IFN-stimulated genes (ISGs) that collectively limit virus replication and spread (16).
The production of IFN leading to the expression of ISGs is mediated by IFN regulatory factor 3 (IRF-3). IRF-3 is part of a growing family of transcription factors that regulate multiple aspects of host defense (58). In eukaryotic cells, IRF-3 is constitutively expressed. During virus infection, IRF-3 is phosphorylated on serine residues in the carboxyl region by two recently identified members of the IB kinase (IKK)-related kinase family, IKK and TANK-binding kinase 1 (TBK-1) (20, 56). Phosphorylation of IRF-3 leads to its homodimerization, nuclear translocation, and association with other transcription factors like NFB, ATF-2/c-jun, and the coactivator CREB-binding protein to form a complex that binds to the IFN- promoter (60). IRF-3 can also directly bind to several DNA-binding motifs, including the IFN-stimulated response element, which is located in the promoter region of ISGs, indicating that IRF-3 also plays a role in the direct induction of ISGs in the absence of IFN production (23, 42).
Although the main inducing element of IFN in response to both RNA and DNA viruses is thought to be double-stranded RNA (dsRNA), a by-product of virus replication, it is becoming clear that cells differentially recognize and respond to the various stages and by-products produced during a viral infection. For example, Toll-like receptors (TLRs) mediate IFN and/or proinflammatory cytokine responses following recognition of either virion components (e.g., glycoproteins) or nucleic acid components (dsRNA or CpG DNA) (reviewed in references 5, 7, 41, and 57). In addition, we and others have demonstrated that a subset of ISGs can be induced in response to the entry of a diverse range of enveloped virus particles (9, 13, 42, 46, 65). Work with the human herpesviruses herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) has shown that this response requires both binding and penetration but is not reliant on the viral genome, its subsequent replication, or IFN production (6, 42, 45, 50).
Viruses have the ability to modulate various signal transduction pathways, including the extracellular regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK), the p38 and Jun N-terminal kinase (JNK) stress-activated protein kinases (SAPK), and the phosphoinositide 3-kinase (PI3 kinase) pathways, to favor their replication. Historically, research has focused on how viruses alter these pathways during a productive lytic infection. HSV-1 has been shown to induce the stress-activated kinases JNK and p38 in a process requiring viral gene expression (25, 31, 38, 63, 64). On the other hand, HCMV viral gene expression is required for sustained ERK 1/2 and p38 activation (10, 28, 51). The PI3 kinase pathway has been implicated in mediating the entry of and viral replication of several viruses in cells, including adenovirus, HCMV, and HSV-1 (11, 30, 35). HCMV particle binding and entry rapidly activate ERK 1/2 and PI3 kinase within 5 to 30 min postinfection (29, 30). Not only are these pathways implicated in optimizing virus replication, but they are also involved in the innate immune response to the infection (reviewed in references 17 and 33).
The prototypic class I PI3 kinase (herein referred to as PI3K) is the most-studied member of this vast family. PI3K is a dimeric protein composed of the adaptor subunit p85 and the catalytic subunit p110. The lipid products of PI3K activation act as second messengers to promote the activity of numerous downstream effectors, including the well-studied serine/threonine kinase Akt/PKB (21). Recently, Sarkar et al. showed that dsRNA-mediated phosphorylation of TLR-3 results in the activation of both TBK-1 and PI3K/Akt, leading to the induction of ISG56 (55). Their data suggest that full activation of IRF-3 in response to dsRNA requires two phosphorylation events, the first dependent on TBK-1 and the second on PI3K (55).
We are interested in identifying the signal transduction pathways that are activated following entry of the physical virus particle into a cell, prior to virus replication. Here we report that treatment of cells with the general PI3 kinase inhibitor LY294002 prevented the induction of ISG56 and a subsequent antiviral response without affecting dimerization or nuclear translocation of IRF-3 in human embryonic lung (HEL) fibroblasts upon entry of enveloped virus particles. The decrease in ISG56 mRNA and protein expression following LY294002 treatment was not due to inhibition of the prototypic PI3K or other LY294002-sensitive kinases implicated in IRF-3 activation. Taken together, these data imply the involvement of a novel kinase within the PI3 kinase family in the induction of an antiviral state in response to virus particle entry.
MATERIALS AND METHODS
Reagents. The inhibitors LY294002, U0126, SB202190, SP600125, and cycloheximide (Sigma) were reconstituted in dimethyl sulfoxide (DMSO). The concentration of LY294002 required to elicit a biological response varied from 25 to 50 μM, depending on the particular batch and lot number. U0126, SB202190, SP600125, and cycloheximide were used at 10 μM, 25 μM, 25 μM, and 50 μg/ml, respectively. The properties of each inhibitor used in this study are listed in Table 1. The dsRNA mimetic poly(I-C) (GE Healthcare) was reconstituted in phosphate-buffered saline (PBS) at a concentration of 5 mg/ml. Human IFN- (Sigma) was reconstituted in PBS at a concentration of 1,000 IU/μl. Human recombinant platelet-derived growth factor (PDGF; Oncogene Research Products) was reconstituted in PBS containing 0.1% bovine serum albumin (BSA) at a concentration of 50 ng/μl.
Cell culture and virus infections. HEL fibroblasts and Vero cells were maintained in Dulbecco modified Eagle medium containing 10% and 5% fetal calf serum (FCS), respectively. A549 human epithelial cells were maintained in alpha minimum essential medium (MEM) containing 10% FCS. Primary DNA-PK catalytic subunit (DNA-PKcs)+/– and DNA-PKcs–/– mouse embryonic fibroblasts (MEFs; generously provided by D. Chen) were maintained in alpha MEM containing 20% FCS. All cell lines were used between passages 2 and 18. HSV-1 (strain KOS) was propagated on Vero cells. HCMV (strain AD169; kindly provided by T. Compton) was propagated on HEL fibroblasts. Sendai virus (SeV; strain Cantell) was purchased from Charles River Laboratories. All herpesvirus infections were carried out in serum-free media at a multiplicity of infection (MOI) of 10 PFU/cell for 1 h at 37°C, unless otherwise specified. For SeV infections, 10 hemagglutinin units of virus were used per 106 cells. A vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP; kindly provided by B. Lichty) was propagated on Vero cells and used in the VSV plaque reduction assays. For experiments with the U0126, SB202190, SP600125, and LY294002 inhibitors, HEL cells were grown to confluence and serum starved for 2 h. Cells were pretreated for 1 h with the indicated inhibitor prior to infection. In addition, the inhibitor was present for the duration of the experiment. UV inactivation of viruses was performed with a UV Stratalinker 2400 (Stratagene) for the length of time required to prevent viral gene expression as measured by indirect immunofluorescence. Standard plaque reduction assays with replication-competent VSV were performed as previously described (11).
Replication-deficient adenovirus constructs containing either a dominant negative form of the regulatory subunit of PI3K (Addnp85) (24, 54) or a kinase-dead mutant form of the p21-activated kinase (PAK; AdPAKK299R) (22) were diluted in PBS supplemented with 0.9 mM MgCl2 and 0.5 mM CaCl2 and allowed to adsorb to the cells for 30 min at room temperature, with the subsequent addition of Dulbecco modified Eagle medium and incubation at 37°C for 24 h.
Preparation of cell extracts. Whole-cell extracts were prepared by washing the cells twice and harvesting them in cold PBS, followed by centrifugation at 200 x g for 5 min at 4°C. The cell pellets were resuspended in whole-cell extract buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM -glycerophosphate, 0.2% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 1x protease inhibitor cocktail [Sigma]) and lysed for 15 min on ice. The lysate was centrifuged at 13,000 x g for 10 min at 4°C, and protein quantification was performed with the Bradford assay kit (Bio-Rad Laboratories).
Native protein extracts were prepared as previously described (55). Briefly, nuclear or cytoplasm-enriched extracts were incubated with rehydration buffer containing 8 M urea, 2% (wt/vol) 3-([3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and 20 mM dithiothreitol. The cytoplasmic and nuclear extracts were sonicated for 5 s, and debris was removed by centrifugation at 13,000 x g for 10 min. Protein quantification was carried out with a 2-D Quant kit (GE Healthcare). Eight micrograms and 2 μg of the cytoplasm-enriched and nuclear extracts, respectively, were separated on a 7.5% nondenaturing gel in a 25 mM Tris-HCl (pH 8.4)-192 mM glycine buffer with 0.2% deoxycholate in the cathode chamber.
Western blot analysis. Polyacrylamide gels were transferred onto polyvinylidene difluoride membranes (Millipore) with a semidry transfer apparatus at 400 mA for 1.5 h. All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) at room temperature for 1 h. ERK 1/2 (Cell Signaling no. 9102), P-ERK 1/2 (Cell Signaling no. 9101), total Akt (Cell Signaling no. 9272), P-Akt (Ser473; Cell Signaling no. 9271), total p38 MAPK (Cell Signaling no. 9212), P-p38 MAPK (Cell Signaling no. 9211), total SAPK/JNK (Cell Signaling no. 9252), P-SAPK-JNK (Cell Signaling no. 9251), PI3K subunit p85 (Santa Cruz SC-1637), PAK (Santa Cruz SC-881), and anti-human IRF-3 (Immuno-Biological Laboratories Co. Ltd.) primary antibodies were all used at a dilution of 1:1,000 in 5% BSA in TBS-Tween (0.1%). The ISG56 (provided by G. Sen) and -actin (Santa Cruz SC-1616) primary antibodies were used at a dilution of 1:1,000 in TBS-Tween (0.1%). The anti-ISG15 antibody (provided by E. Borden) was used at a dilution of 1:500 in TBS-Tween (0.1%). Secondary antibodies were conjugated to horseradish peroxidase and visualized by chemiluminescence.
RNA extraction and reverse transcription (RT)-PCR analysis. Total cellular RNA was obtained with Trizol reagent (Invitrogen) according to the manufacturer's protocols. Two micrograms of total RNA was reverse transcribed with 100 U of Superscript II (Invitrogen) in a total reaction volume of 20 μl. A random hexamer primer was used to synthesize cDNA at 42°C for 50 min, followed by a 15-min incubation at 70°C. PCR was performed according to the manufacturer's specifications with previously described primer sets (13).
Indirect and direct immunofluorescence. HEL fibroblasts were grown to semiconfluence on glass coverslips overnight. Cells were treated with the indicated stimuli, and at the indicated times postinfection, coverslips were washed twice with PBS and fixed in a solution of 4% paraformaldehyde in PBS. Cells were permeabilized in 0.1% Triton X-100 and then blocked in a solution of 3% BSA, 3% goat serum, and 0.02% Tween 20. Polyclonal anti-HSV-1 (DAKO no. B0114) or monoclonal anti-IE1/IE2 (Rumbaugh-Goodwin Institute no. 1203) primary antibodies were used at a dilution of 1:1,000 to detect expression of HSV-1 or HCMV proteins, respectively. A polyclonal anti-IRF-3 primary antibody (a generous gift from M. David) was used at a dilution of 1:5,000. Alexa Fluor-conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) were used at a dilution of 1:500. Cells were also stained with the intercalating DNA dye Hoechst in order to determine the total number of cells in a field of view. Images were captured on a Leica DM-IRE2 inverted microscope. The percentage of cells positive for HSV-1, HCMV, or nuclear IRF-3 was calculated from three independent experiments with a total of 12 random fields of view for each sample.
A549 cells were grown to semiconfluence on glass coverslips. Cells were infected with AdE1/E3 or AdPAKK299R at an MOI of 50 PFU/cell for 24 h, washed twice in PBS, and fixed in a 4% solution of paraformaldehyde in PBS. Nuclei were then stained with Hoechst dye. Cells expressing the GFP-tagged AdPAKK299R constructs were visualized on a Leica DM-IRE2 inverted microscope to determine cellular morphology and infection efficiency.
Data analysis. Data were expressed as means ± the standard error of the mean. Statistical analysis was performed with either a t test or a one-way analysis of variance with the Tukey test post hoc with Sigma Stat 2.03.
RESULTS
Activation of the ERK 1/2 and p38 MAPK pathways is not required for induction of ISG56. Signal transduction pathways exhibit complex cross talk with each other in mammalian cells. Since replicating viruses activate numerous signal transduction pathways that mediate innate and adaptive immune responses, we tested whether the activity of these pathways is required for the immediate-early induction of ISGs following enveloped virus particle entry. The U0126 inhibitor has been shown to specifically inhibit MEK 1/2 activity, preventing phosphorylation of its downstream effector, ERK 1/2 (Table 1) (19). The SB202190 inhibitor, on the other hand, specifically inhibits p38 activity by preventing the transfer of the phosphate group from p38 to its downstream effector kinase(s) (Table 1) (62), leading to retention of phosphorylated p38 in the presence of the SB202190 inhibitor. As a control for ERK 1/2 and p38 MAPK phosphorylation, cells were treated with FCS or UV irradiation, respectively. To ensure maintenance of U0126 and SB202190 inhibitor activity throughout the experimental procedure, the positive control was administered 30 min prior to sample harvest (Fig. 1A and B, lanes 13 and 14). Poly(I-C), HSV-UV, and HCMV-UV were able to stimulate accumulation of ISG56 mRNA to similar levels regardless of the phosphorylation state of ERK 1/2 or p38 MAPK (Fig. 1A and B). These data suggest that activation of the ERK 1/2 and p38 MAPK pathways is not required for ISG56 induction in response to virus particle entry.
Inhibition of the JNK/SAPK and PI3 kinase pathways prevents induction of ISG56 in human fibroblast cells. Since it has previously been shown that herpesviruses activate the JNK/SAPK and PI3 kinase pathways during infection (25, 30, 38, 63), we determined whether activation of these pathways is necessary for the induction of ISG56 upon enveloped virus particle entry. As a control for JNK/SAPK and PI3K/Akt phosphorylation, cells were treated with UV irradiation or PDGF, respectively (Fig. 1C, lanes 13 and 14; see also Fig. 3, lanes 8, 16, and 24). To ensure maintenance of inhibitor activity, the positive control was administered 30 min prior to sample harvest. Poly(I-C), HSV-UV, and HCMV-UV showed a marked decrease in the level of ISG56 mRNA in the presence of SP600125 (Fig. 1C). In the presence of the LY294002 inhibitor, similar results were observed (Fig. 1D). Consistent with previous reports (13), replication-deficient, nonenveloped adenovirus particles were unable to induce the accumulation of ISG56 mRNA (Fig. 1D).
Inhibition of the PI3K pathway prevents the induction of an antiviral state in human fibroblasts. To confirm that the decrease in ISG56 mRNA accumulation correlated with the inability of the cells to induce an antiviral state, we performed a VSV plaque reduction assay as previously described (13). In the absence of ERK 1/2, p38, and JNK MAPK activities, enveloped virions were still able to induce an antiviral state in human fibroblasts (Table 2). In the presence of the LY294002 inhibitor, however, poly(I-C) and HSV-UV were unable to elicit an antiviral state in human fibroblasts. Interestingly, in the presence of high-multiplicity HCMV-UV infection (MOI, >0.1 PFU/cell), which we found induces IFN production (data not shown), the effect of the LY294002 inhibitor was less apparent and was unable to prevent the induction of an antiviral state. Although ISG56 mRNA was barely detectable following infection with HCMV-UV at a low MOI (0.1 PFU/cell), induction of an antiviral response sufficient to prevent VSV replication was sporadic. Since an increase in the MOI to 0.5 PFU/cell consistently induced an antiviral response whereas a decrease in the MOI to 0.05 PFU/cell failed to induce an antiviral response, we hypothesize that an MOI of 0.1 PFU/cell represents a threshold limit of virus capable of initiating an antiviral response.
Inhibition of the JNK/SAPK and PI3K pathways does not prevent entry and expression of HSV-1 and HCMV in human fibroblasts. Since HCMV and HSV entry is thought to require PI3K activity (30) and both binding and penetration of HSV-1 and HCMV virions are required for maximal ISG induction (45, 50), we tested whether the observed reduction of ISG56 upon inhibition of PI3 kinase and SAPK activity was due to a block in viral entry. Human fibroblasts were infected with replicating HSV-1 (Fig. 2A) or HCMV (Fig. 2B) in the presence or absence of LY294002 or SP600125. At 4 h postinfection, the percentage of cells staining positive for HSV-1 proteins was lower in the presence of SP600125, as determined by indirect immunofluorescence. By 8 h postinfection, no significant difference was observed and by 12 h postinfection, almost all of the cells stained positive for HSV-1 proteins regardless of inhibitor treatment. There were no differences in the frequency of cells staining positive for HCMV proteins upon treatment with DMSO, LY294002, or SP600125.
The prototypic class I PI3K signal transduction pathway is not involved in inhibition of the ISG56 protein. Although studies of virus-mediated PI3 kinase signaling almost exclusively focus on the prototypic class I PI3K, the LY294002 compound inhibits multiple PI3 kinase family members. To distinguish between PI3K and alternate PI3 kinase family members, fibroblasts were infected with an adenovirus expressing a dominant negative form of the PI3K p85 subunit (Addnp85) or an empty adenovirus (AdE1/E3) or treated with the LY294002 inhibitor (Fig. 3). Cells were then serum starved for 2 h prior to treatment with the indicated stimuli. Although Addnp85 and LY294002 were able to inhibit the phosphorylation of Akt in response to PDGF (Fig. 3, lanes 8, 16, and 24), only LY294002 was able to significantly reduce ISG56 protein production in response to poly(I-C) or virus particle entry. This reduction correlated with the absence of an antiviral state in cells pretreated with LY294002 but not Addnp85 (data not shown), with the exception of high-multiplicity HCMV infection, as noted in Fig. 1C. Moreover, although enveloped virions were able to elicit the transient phosphorylation of ERK1/2 at early times postentry (15 to 30 min), we failed to detect phosphorylation of Akt at any time postentry (data not shown). These data suggest that the prototypic PI3K enzyme is not involved in activating ISG56 in response to enveloped virus particle entry and instead suggests that an alternate member(s) of the PI3 kinase family is required.
DNA-PK and PAK, two protein kinases implicated in IRF-3 activation, are not essential for virus-mediated ISG induction. Recently, two protein kinases, DNA-PK and PAK, were implicated in IRF-3 activation (18, 32). The activities of both of these kinases are directly or indirectly blocked by inhibitors of PI3 kinase activity, including LY294002 (26, 47, 48). Low-passage, primary wild-type and DNA-PKcs–/– MEFs were treated with HSV for 6 h in the presence or absence of cycloheximide, RNA was harvested, and RT-PCR performed (Fig. 4). Since low-passage, nonimmortalized MEFs secrete IFN in response to various stimuli, including virus particle entry (Fig. 4, lanes 3 and 7), ISG56 mRNA induction was assayed in the presence of cycloheximide to prevent de novo synthesis of IFN. In the absence of the catalytic subunit of DNA-PK, cells were able to express similar levels of ISG56 mRNA compared to wild-type MEFs (Fig. 4, compare lanes 4 and 8), indicating that the catalytic activity of DNA-PK is not essential for the induction of ISG56 mRNA.
To investigate whether PAK activity was required for IRF-3-mediated ISG induction in response to the virion particles, we treated A549 epithelial cells with an adenovirus expressing a dominant-negative PAK construct (AdPAKK299R) tagged with GFP (Fig. 5A). Since we have previously shown that A549 cells and HEL fibroblasts are able to induce ISG56 protein to similar levels following SeV particle entry (13), we used A549 in place of HEL fibroblasts due to their enhanced permissiveness to adenovirus infection. Cells expressing the dominant negative PAK construct were significantly larger compared to uninfected cells and contained numerous lamellipodia, indicative of PAK inhibition (Fig. 5B) (22).
Cells preloaded with dominant negative PAK were treated with replicating and nonreplicating forms of SeV to investigate whether inhibition of PAK activity prevented induction of ISGs. At both 12 h and 24 h postinfection, protein extracts were harvested and Western blot analysis performed (Fig. 5C). Upon overexpression of dominant negative PAK, the levels of both ISG15 and ISG56 remained similar in cells treated with either SeV or SeV-UV, suggesting that PAK activity is not essential for the induction of ISGs in response to both replicating and nonreplicating enveloped virus particles.
The LY294002 inhibitor does not inhibit dimerization or nuclear localization of IRF-3 upon virus particle treatment. Given that the two LY294002-sensitive protein kinases DNA-PK and PAK, which have been implicated in IRF-3 activation, were shown to be nonessential for ISG induction in response to virion particles, we examined whether LY294002 inhibition had any effect on IRF-3 activation. We first investigated, by indirect immunofluorescence assay, whether the LY294002 inhibitor was capable of preventing nuclear translocation of IRF-3. HEL fibroblasts were treated with the LY294002 compound in combination with replication-deficient HSV-1 or HCMV. At various times postinfection, cells were fixed and stained with an antibody for IRF-3. As previously observed (13), the level of nuclear translocation of endogenous IRF-3 was minimal following induction of an IFN-independent antiviral response (HSV-1, MOI of 10 PFU/cell [Fig. 6A and B]; HCMV, MOI of 0.1 PFU/cell [data not shown]). Importantly, there was no difference in the amount of IRF-3 nuclear translocation when the LY294002 compound was present, indicating that the LY294002 inhibitor does not have an effect on the ability of IRF-3 to translocate to the nucleus upon virus particle stimulation. For enhanced detection, the experiment was repeated with elevated MOIs; results obtained with HCMV-UV (MOI, 10 PFU/cell) are outlined in Fig. 6C and D. Under these conditions, there was a significant increase in the number of cells that stained positive for nuclear IRF-3 at early times compared to the mock-treated sample. Interestingly, as the time course progressed, the number of nuclei positive for IRF-3 decreased significantly in the DMSO-treated samples compared to the LY294002-treated samples. One explanation is that LY294002 prevents virus-mediated IRF-3 degradation. However, consistent with previous observations (13), infection at low or high multiplicities with UV-inactivated HSV-1 or HCMV failed to affect the level of endogenous IRF-3 in whole-cell extracts as determined by standard denaturing gel electrophoresis (data not shown). Alternatively, the observed difference in the level of nuclear IRF-3 following high-multiplicity infection could result from either LY294002-mediated retention of IRF-3 in the nucleus or prevention of a conformational change leading to the inability of the antibody to recognize IRF-3 under the conditions used.
To differentiate between these possibilities and determine if LY294002 affects IRF-3 dimerization upon virus particle entry, we performed native gel electrophoresis on cytoplasmic and nuclear extracts following high-multiplicity HCMV-UV infection in the presence or absence of LY294002. Consistent with immunofluorescence microscopy, HCMV virions induced IRF-3 dimerization and nuclear translocation within 4 h of infection in the presence or absence of the inhibitor (Fig. 7, lanes 1 to 8). Furthermore, at late times of infection, an overall decrease in the level of IRF-3 was observed, which was reproducibly more enhanced in the DMSO samples relative to the LY294002 samples. However, there was no significant difference in the relative amounts of IRF-3 within the cytoplasm or nucleus within either sample. Taken together, these data suggest that LY294002 does not affect the ability of incoming virions to elicit the dimerization or nuclear translocation of IRF-3 but may modulate, to some degree, subsequent modifications of IRF-3 that alter the conformation of the protein.
DISCUSSION
During virus infection, cells recognize different viral structures and viral life cycle events, leading to the activation and modulation of a multitude of signal transduction pathways. For example, TLRs recognize different pathogen-associated molecular patterns, leading to the production of type I IFN and/or proinflammatory cytokines (39, 43). With respect to viruses, certain TLRs stimulate proinflammatory cytokines upon recognition of virion-associated proteins or genome-encoded CpG motifs while others stimulate IFN production in response to dsRNA (reviewed in references 5, 7, and 57). Cells are also able to recognize viral particle entry prior to uncoating or initiation of replication, with the nature of the response depending on the type of virus. For example, entry of nonenveloped adenovirus particles induces a proinflammatory cytokine cascade mediated by NF-B (37) whereas entry of enveloped virus particles induces an IFN-independent ISG response mediated by IRF-3 (5, 13, 46). Cellular antiviral responses also differ depending on the cell type, as observed with virion-associated glycoprotein B (gB) of HCMV. In fibroblasts, gB triggers IRF-3-mediated ISG induction (6, 8), whereas in peripheral blood mononuclear cells, gB triggers NF-B-mediated proinflammatory cytokine production (14). Finally, within a given cell type, the MOI can modulate the nature of the resulting cellular response. In primary human fibroblasts, enveloped virus particle entry at a low multiplicity elicits an IFN-independent antiviral response, whereas at elevated multiplicities, IFN production is observed (P. Paladino, R. Noyce, and K. Mossman, unpublished data).
Accumulation of dsRNA during lytic virus infection results in the activation of numerous cellular signal transduction pathways, leading to the activation of IRF-3, JNK, and p38 SAPKs (12, 27), TLR-dependent (1) and TLR-independent (3, 61) pathways, culminating in IFN production and antiviral state induction. As well, it has recently been appreciated that the prototypic class I PI3K is necessary for full activation of IRF-3 leading to the induction of ISG56 in response to dsRNA, via activation of Akt (55). Other signal transduction pathways become activated upon viral protein synthesis. For example, HCMV viral protein expression leads to PI3K (30), ERK 1/2 (51), and p38 (28) activation. Depending on the system studied, activation of these pathways has been shown to be critical both for the cell in mediating an antiviral response and for the virus in ensuring productive lytic infection.
The focus of this study was to determine whether signal transduction pathways other than IRF-3 are essential for the immediate-early, IFN-independent antiviral response to enveloped virus particle entry. Although virion entry rapidly and transiently induced the activation of some of the pathways studied (e.g., ERK1/2), inhibition of these pathways had little to no effect on ISG and antiviral state induction, with the exception of LY294002. Although the JNK/SAPK inhibitor caused a decrease in the accumulation of ISG56 at the time point tested, it failed to prevent induction of an antiviral state in fibroblasts in response to enveloped virus particles. The inhibitor did, however, block production of IFN in response to treatment with poly(I-C) (data not shown), suggesting that the JNK/SAPK pathway may play a role in mediating IFN production as opposed to the IFN-independent antiviral response. Given that the JNK/SAPK inhibitor utilized in these studies is relatively new and not yet fully characterized, it remains to be determined whether JNK/SAPK plays a role, albeit nonessential, in the cellular response to virus particle entry. Studies are ongoing to address this issue.
The PI3 kinase family constitutes a large family of protein kinases that mediate a diverse array of intracellular pathways. With respect to virus infection, the prototypic class I PI3K is almost exclusively studied, and found to be activated by and important for, the replication of multiple viruses. Here, we found that targeted inactivation of the adaptor subunit of PI3K failed to block ISG accumulation or antiviral state induction following virion entry. This observation suggests that an alternate member of the PI3 kinase family is essential for virion-mediated antiviral state induction. Two such members have been implicated in IRF-3 activation, DNA-PK and PAK1 (18, 32), both of which can be inhibited by LY294002 treatment (26, 47, 48). Of particular interest, DNA-PK has been shown to phosphorylate IRF-3 (32), and the HSV-1 immediate-early protein ICP0, which we have shown blocks IRF-3 and IRF-7 activity (36), induces the degradation of DNA-PK (49). With primary MEFs harvested from mice lacking the catalytic subunit of DNA-PK, however, we found that this kinase is not essential for antiviral state induction mediated by virus entry. This result is in agreement with that of Melroe et al., who investigated immortalized glioma cells deficient in DNA-PK (40).
Recently, PAK1 was shown to be upstream of TBK-1 and IKK in the viral activation of IRF-3 (18). Given that PAK1 is involved in modulating the cytoskeleton (22), in conjunction with reports that herpesvirus particle entry induces cytoskeletal rearrangements (44, 52, 53), we hypothesized that entry of enveloped virus particles may activate IRF-3 via PAK1. However, though expression of a dominant negative PAK1 construct caused cytoskeletal deregulation, there was no effect on ISG or antiviral state induction. It remains to be determined if cytoskeletal rearrangements mediated by virion particle entry play any role in activation of IRF-3 and an antiviral response.
Although dsRNA-mediated phosphorylation of IRF-3 requires PI3K and Akt activities, it is unclear whether the LY294002-sensitive PI3 kinase family member involved in antiviral state induction in response to virus particle entry functions within, or parallel to, the IRF-3 pathway. We have previously shown that induction of an antiviral response to enveloped particles in fibroblasts does not correlate with IRF-3 hyperphosphorylation and its subsequent degradation (13). Thus, we set out to determine if LY294002 affects IRF-3 dimerization and/or nuclear translocation. Although neither dimerization nor nuclear translocation was blocked in the presence of the inhibitor, it appeared that LY294002 may modify, to some degree, conformational changes that occur in IRF-3 following its translocation into the nucleus. Whether this constitutes an additional posttranslational modification in IRF-3 or the ability of IRF-3 to interact with a nuclear cofactor(s) remains to be determined.
Taken together, the results of this study show that cellular responses to viral infections differ dramatically during the course of infection. While activation of signaling cascades involving ERK1/2, p38, JNK/SAPK, and the prototypic PI3K has been shown to be important for cells to respond to lytic virus infection, these pathways are not essential for the immediate-early response to virion entry. Entry does, however, result in the activation of IRF-3, leading to the induction of ISGs and an antiviral response in the absence of IFN production. Here, we provide evidence that a novel member of the PI3 kinase pathway that is sensitive to LY294002 is essential for this immediate-early response. While this kinase does not appear to function upstream of IRF-3, it is unclear whether it mediates its effects on IRF-3 following nuclear translocation or instead functions in parallel with the IRF-3 pathway. Given that the early interactions between a virus and its host mediate the outcome of a virus infection, particularly under conditions of low multiplicity that likely represent the nature of most biological infections, it is critical to understand these interactions. Current studies are thus focused on identifying and characterizing the LY294002-sensitive kinase along with its potential effects on IRF-3 nuclear events.
ACKNOWLEDGMENTS
We thank E. Borden, D. Chen, T. Compton, B. Lichty, M. David, and G. Sen for reagents. We also thank D. Cummings for growing adenovirus stocks for use in this study.
This work was supported by a grant from the Canadian Institutes of Health Research. R.S.N. is supported by a Natural Sciences and Engineering Research Council (NSERC) postgraduate doctoral scholarship. K.L.M. is supported by an Rx&D-Health Research Foundation career award.
REFERENCES
Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732-738.
Bain, J., H. McLauchlan, M. Elliott, and P. Cohen. 2003. The specificities of protein kinase inhibitors: an update. Biochem. J. 371:199-204.
Balachandran, S., E. Thomas, and G. N. Barber. 2004. A FADD-dependent innate immune mechanism in mammalian cells. Nature 432:401-405.
Bennett, B. L., D. T. Sasaki, B. W. Murray, E. C. O'Leary, S. T. Sakata, W. Xu, J. C. Leisten, A. Motiwala, S. Pierce, Y. Satoh, S. S. Bhagwat, A. M. Manning, and D. W. Anderson. 2001. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98:13681-13686.
Boehme, K. W., and T. Compton. 2004. Innate sensing of viruses by Toll-like receptors. J. Virol. 78:7867-7873.
Boehme, K. W., J. Singh, S. T. Perry, and T. Compton. 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78:1202-1211.
Bowie, A. G., and I. R. Haga. 2005. The role of Toll-like receptors in the host response to viruses. Mol. Immunol. 42:859-867.
Boyle, K. A., R. L. Pietropaolo, and T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell. Biol. 19:3607-3613.
Browne, E. P., B. Wing, D. Coleman, and T. Shenk. 2001. Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J. Virol. 75:12319-12330.
Chen, J., and M. F. Stinski. 2002. Role of regulatory elements and the MAPK/ERK or p38 MAPK pathways for activation of human cytomegalovirus gene expression. J. Virol. 76:4873-4885.
Cheshenko, N., B. Del Rosario, C. Woda, D. Marcellino, L. M. Satlin, and B. C. Herold. 2003. Herpes simplex virus triggers activation of calcium-signaling pathways. J. Cell Biol. 163:283-293.
Chu, W.-M., D. Ostertag, Z.-W. Li, L. Chang, Y. Chen, Y. Hu, B. Williams, J. Perrault, and M. Karin. 1999. JNK2 and IKK are required for activating the innate response to viral infection. Immunity 11:721-731.
Collins, S. E., R. S. Noyce, and K. L. Mossman. 2004. Innate cellular response to virus particle entry requires IRF3 but not virus replication. J. Virol. 78:1706-1717.
Compton, T., E. A. Kurt-Jones, K. W. Boehme, J. Belko, E. Latz, D. T. Golenbock, and R. W. Finberg. 2003. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J. Virol. 77:4588-4596.
Davies, S., H. Reddy, M. Caivano, and P. Cohen. 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351:95-101.
Der, S. D., A. Zhou, B. R. G. Williams, and R. H. Silverman. 1998. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:15623-15628.
Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55-72.
Ehrhardt, C., C. Kardinal, W. J. Wurzer, T. Wolff, C. von Eichel-Streiber, S. Pleschka, O. Planz, and S. Ludwig. 2004. Rac1 and PAK1 are upstream of IKK- and TBK-1 in the viral activation of interferon regulatory factor-3. FEBS Lett. 567:230-238.
Favata, M. F., K. Y. Horiuchi, E. J. Manos, A. J. Daulerio, D. A. Stradley, W. S. Feeser, D. E. Van Dyk, W. J. Pitts, R. A. Earl, F. Hobbs, R. A. Copeland, R. L. Magolda, P. A. Scherle, and J. M. Trzaskos. 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273:18623-18632.
Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S.-M. Liao, and T. Maniatis. 2003. IKK and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4:491-496.
Franke, T. F., D. R. Kaplan, L. C. Cantley, and A. Toker. 1997. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665-668.
Frost, J. A., A. Khokhlatchev, S. Stippec, M. A. White, and M. H. Cobb. 1998. Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation. J. Biol. Chem. 273:28191-28198.
Guo, J., K. L. Peters, and G. C. Sen. 2000. Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection. Virology 267:209-219.
Hara, K., K. Yonezawa, H. Sakaue, A. Ando, K. Kotani, T. Kitamura, Y. Kitamura, H. Ueda, L. Stephens, T. Jackson, M. Waterfield, and M. Kasuga. 1994. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc. Natl. Acad. Sci. USA 91:7415-7419.
Hargett, D., T. McLean, and S. L. Bachenheimer. 2005. Herpes simplex virus ICP27 activation of stress kinases JNK and p38. J. Virol. 79:8348-8360.
Hartley, K., D. Gell, G. Smith, H. Zhang, N. Divecha, M. Connelly, A. Admon, S. Lees-Miller, C. Anderson, and S. Jackson. 1995. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 82:849-856.
Iordanov, M. S., J. M. Paranjape, A. Zhou, J. Wong, B. R. G. Williams, E. F. Meurs, R. H. Silverman, and B. E. Magun. 2000. Activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways. Mol. Cell. Biol. 20:617-627.
Johnson, R. A., S.-M. Huong, and E.-S. Huang. 2000. Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38. J. Virol. 74:1158-1167.
Johnson, R. A., X.-L. Ma, A. D. Yurochko, and E.-S. Huang. 2001. The role of MKK1/2 kinase activity in human cytomegalovirus infection. J. Gen. Virol. 82:493-497.
Johnson, R. A., X. Wang, X.-L. Ma, S.-M. Huong, and E.-S. Huang. 2001. Human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (PI3-K) pathway: inhibition of PI3-K activity inhibits viral replication and virus-induced signaling. J. Virol. 75:6022-6032.
Karaca, G., D. Hargett, T. I. McLean, J. S. Aguilar, P. Ghazal, E. K. Wagner, and S. L. Bachenheimer. 2004. Inhibition of the stress-activated kinase, p38, does not affect the virus transcriptional program of herpes simplex virus type 1. Virology 329:142-156.
Karpova, A. Y., M. Trost, J. M. Murray, L. C. Cantley, and P. M. Howley. 2002. Interferon regulatory factor-3 is an in vivo target of DNA-PK. Proc. Natl. Acad. Sci. USA 99:2818-2823.
Koyasu, S. 2003. The role of PI3K in immune cells. Nat. Immunol. 4:313-319.
Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, J. E. Strickler, M. M. McLaughlin, I. R. Siemens, S. M. Fisher, G. P. Livi, J. R. White, J. L. Adams, and P. R. Young. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739-746.
Li, E., D. Stupack, R. Klemke, D. A. Cheresh, and G. R. Nemerow. 1998. Adenovirus endocytosis via alpha v integrins requires phosphoinositide-3-OH kinase. J. Virol. 72:2055-2061.
Lin, R., R. S. Noyce, S. E. Collins, R. D. Everett, and K. L. Mossman. 2004. The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes. J. Virol. 78:1675-1684.
Liu, Q., and D. A. Muruve. 2003. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 10:935-940.
McLean, T. I., and S. L. Bachenheimer. 1999. Activation of cJUN N-terminal kinase by herpes simplex virus type 1 enhances viral replication. J. Virol. 73:8415-8426.
Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394-397.
Melroe, G. T., N. A. DeLuca, and D. M. Knipe. 2004. Herpes simplex virus 1 has multiple mechanisms for blocking virus-induced interferon production. J. Virol. 78:8411-8420.
Morrison, L. A. 2004. The Toll of herpes simplex virus infection. Trends Microbiol. 12:353-356.
Mossman, K. L., P. F. Macgregor, J. J. Rozmus, A. B. Goryachev, A. M. Edwards, and J. R. Smiley. 2001. Herpes simplex virus triggers and then disarms a host antiviral response. J. Virol. 75:750-758.
Muzio, M., G. Natoli, S. Saccani, M. Levrero, and A. Mantovani. 1998. The human Toll signaling pathway: divergence of nuclear factor kappa B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J. Exp. Med. 187:2097-2101.
Naranatt, P. P., H. H. Krishnan, M. S. Smith, and B. Chandran. 2005. Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus. J. Virol. 79:1191-1206.
Netterwald, J. R., T. R. Jones, W. J. Britt, S.-J. Yang, I. P. McCrone, and H. Zhu. 2004. Postattachment events associated with viral entry are necessary for induction of interferon-stimulated genes by human cytomegalovirus. J. Virol. 78:6688-6691.
Nicholl, M. J., L. H. Robinson, and C. M. Preston. 2000. Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1. J. Gen. Virol. 81:2215-2218.
Nobes, C. D., and, A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53-62.
Papakonstanti, E. A., and C. Stournaras. 2002. Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization. Mol. Biol. Cell 13:2946-2962.
Parkinson, J., S. P. Lees-Miller, and R. D. Everett. 1999. Herpes simplex virus type 1 immediate-early protein Vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase. J. Virol. 73:650-657.
Preston, C. M., A. N. Harman, and M. J. Nicholl. 2001. Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J. Virol. 75:8909-8916.
Rodems, S. M., and D. H. Spector. 1998. Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection. J. Virol. 72:9173-9180.
Rosenthal, K., R. Perez, and C. Hodnichak. 1985. Inhibition of herpes simplex virus type 1 penetration by cytochalasins B and D. J. Gen. Virol. 66:1601-1605.
Rue, C. A., and P. Ryan. 2003. A role for glycoprotein C in pseudorabies virus entry that is independent of virus attachment to heparan sulfate and which involves the actin cytoskeleton. Virology 307:12-21.
Sakaue, H., W. Ogawa, M. Takata, S. Kuroda, K. Kotani, M. Matsumoto, M. Sakaue, S. Nishio, H. Ueno, and M. Kasuga. 1997. Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes. Mol. Endocrinol. 11:1552-1562.
Sarkar, S. N., K. L. Peters, C. P. Elco, S. Sakamoto, S. Pal, and G. C. Sen. 2004. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nat. Struct. Mol. Biol. 11:1060-1067.
Sharma, S., B. R. ten Oever, N. Grandvaux, G.-P. Zhou, R. Lin, and J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300:1148-1151.
Takeda, K., and S. Akira. 2005. Toll-like receptors in innate immunity. Int. Immunol. 17:1-14.
Taniguchi, T., K. Ogasawara, A. Takaoka, and N. Tanaka. 2001. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19:623-655.
Vlahos, C., W. Matter, K. Hui, and R. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241-5248.
Wathelet, M. G., C. H. Lin, B. S. Parekh, L. V. Ronco, P. M. Howley, and T. Maniatis. 1998. Virus infection induces the assembly of coordinately activated transcription factors on the IFN- enhancer in vivo. Mol. Cell 1:507-518.
Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5:730-737.
Young, P. R., M. M. McLaughlin, S. Kumar, S. Kassis, M. L. Doyle, D. McNulty, T. F. Gallagher, S. Fisher, P. C. McDonnell, S. A. Carr, M. J. Huddleston, G. Seibel, T. G. Porter, G. P. Livi, J. L. Adams, and J. C. Lee. 1997. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272:12116-12121.
Zachos, G., B. Clements, and J. Conner. 1999. Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1. J. Biol. Chem. 274:5097-5103.
Zachos, G., M. Koffa, C. M. Preston, J. B. Clements, and J. Conner. 2001. Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication. J. Virol. 75:2710-2728.
Zhu, H., J.-P. Cong, and T. Shenk. 1997. Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs. Proc. Natl. Acad. Sci. USA 94:13985-13990.(Ryan S. Noyce, Susan E. C)