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Journal of Virology, October 1999, p. 8415-8426, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Activation of cJUN N-Terminal Kinase by Herpes Simplex Virus Type 1 Enhances Viral Replication

T. I. McLean1 and S. L. Bachenheimer1,2,*

Curriculum in Genetics and Molecular Biology1 and Department of Microbiology and Immunology,2 University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Received 19 February 1999/Accepted 19 July 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Signal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a program of gene expression that is characteristic for particular stimuli. Here we present evidence that infection by herpes simplex virus type 1 activated the two terminal kinases, cJUN N-terminal kinase (JNK) and p38, of stress-activated signal transduction kinase cascades. By using a solid-phase kinase assay, a phospho-specific antibody, and extracts prepared from a variety of infected cell types, we determined that activation of both kinases began 3 to 4 h postinfection (p.i.) and remained elevated out to 14 h p.i. Through the use of UV-irradiated or antibody-neutralized wild-type virus and the temperature-sensitive mutant tsB7, the high level of JNK activation was shown to be dependent on viral gene expression. Activation of JNK following infection by vi13, an ICP4 mutant virus that does not express early or late genes, suggested that only virus entry and immediate-early gene expression were necessary for JNK activation. The activation of JNK and p38 correlated with increased chloramphenicol acetyltransferase (CAT) activity in reporter assays dependent upon the activity of cJUN and ATF2 trans-activation domains. Increased CAT activity dependent on TRE and CRE promoter sites was also observed in response to herpes simplex virus infection. The activities of ERK and ERK-dependent transcription factors were unchanged or depressed following infection, showing that activation of JNK and p38 was a specific event. Finally, the activation of JNK was important for the efficiency of viral replication. The yield of virus in NIH 3T3 cells stably expressing JIP-1, an inhibitor of JNK translocation to the nucleus, was reduced 70% compared to that of control cells, in single-step growth experiments.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The cell's ability to sense external stimuli and to react by initiating a program of gene expression often involves propagation of a cell surface-initiated signal along a specific pathway(s) of protein kinases whose ultimate targets are nuclear-acting transcription factors. Conditions such as the type of stimulus, duration of stimulation, and cell type can all play roles in whether the transduced signals are interpreted as growth stimulatory (proliferative), growth inhibitory (differentiating), or apoptotic (death inducing) (40, 52, 75, 92). The prototypical signal transduction pathway is the mitogenic pathway. This pathway is initiated when growth factors bind to and activate their cognate growth factor receptors on the surface of the cell. Activation of the receptor leads to the membrane-bound G-protein, RAS, adopting an active GTP-bound state (18, 48, 75). RAS is critical for coordinating the activation of a serine/threonine kinase called RAF (5, 18). RAF then initiates a kinase cascade by phosphorylating and activating two highly related dual-specific kinases, MEK1 and MEK2, which in turn phosphorylate two highly related serine/threonine kinases, ERK1 and ERK2 (11, 12, 15, 75). Upon activation, ERKs can migrate into the nucleus and activate transcription factors such as cMYC and ELK1 by phosphorylating their trans-activation domains (TADs) (2, 7, 9, 28, 29, 32). These transcription factors can induce the expression of cdc25 and c-fos, respectively, for example, which are important for promoting cell cycle progression into S phase (3, 7, 25, 28, 47).

Kinases analogous to the ERKs and pathways parallel to the RAF-MEK-ERK pathway have been described. The related terminal serine/threonine kinases, which include the ERKs, are collectively referred to as mitogen-activated protein kinases (MAPKs). MEK1/2 now belongs to a class of proteins known as MAPK kinases (MAPKKs), and RAF and related kinases are called MAPKK kinases (MAPKKKs). The most well characterized of these analogous pathways are called the stress-activated protein kinase (SAPK) pathways, because stresses such as UV exposure and high osmolarity lead to their activation (40, 52, 92). JNK1-3 (cJUN N-terminal kinase) and p38, sometimes collectively referred to as SAPKs, are the MAPKs for these pathways, and corresponding MAPKKs and MAPKKKs have been characterized that specifically activate JNKs and/or p38 (40, 52, 92). The transcription factor ATF2 is a target for both p38 and JNKs, while the JNKs can also phosphorylate cJUN (33, 55, 86).

Viruses are ultimately dependent upon the host cell for their replication. Because they reorganize or utilize various cellular functions, it seems likely that viruses would take advantage of the preexisting signaling pathways to induce cellular and/or viral gene expression to promote viral replication. Indeed, studies characterizing the effects of infection by other viruses, such as bovine papillomavirus (60), vaccinia virus (50), simian virus 40 (83), human immunodeficiency virus type 1 (41), and herpesvirus saimiri (44), have shown a dependence upon ERK cascade signaling for replication, and in some cases, viral proteins have been identified that induce activation of ERKs. Additionally, other studies have described activation of different MAPKs, but the purposes for their activation following infection remain unclear (20, 71, 81, 100).

During lytic infection by herpes simplex virus (HSV) in permissive cells, there are increases in activities of AP-1 (42) and NF-kappa B (30, 68, 77), both of which are downstream targets of SAPK pathways (53, 57, 92), suggesting that these pathways may be activated. Additionally, there is evidence that infection by HSV inhibits cell cycle progression, raising the possibility that ERK activity, which promotes progression, is inhibited (14, 19). We present data here to support these predictions. We show that not only are SAPK pathways activated following infection, but activation of one SAPK, JNK, promotes efficient viral replication. ERK and ERK-dependent activities are not activated following infection. In agreement with our data is a recent report which also showed the activation of JNK and p38 following HSV infection (100).


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cells and virus. NIH 3T3, U2OS, C33, and Vero cells were grown and maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 2 mM L-glutamine. For growth of NIH 3T3 cells (originally obtained from Channing Der, UNC---Chapel Hill), the medium was supplemented with 10% bovine calf serum, while that for the Vero cells contained 5% bovine calf serum. DMEM for U2OS and C33 cells was supplemented with the addition of 10% fetal calf serum. BHK-21 cells were grown and maintained in alpha minimum essential media supplemented with L-glutamine as described above and 10% fetal calf serum. The G13.5 and G14.1 cell lines were established via stable transfection of NIH 3T3 cells with either a pcDNA3 empty vector or a pcDNA3 JIP-1-expressing vector, respectively. The JIP-1 vector (17) was provided by Roger Davis (University of Massachusetts Medical Center). These cells were selected and maintained in media supplemented with 600 µg of G418 (Geneticin; Gibco BRL) per ml. All experiments were performed with the KOS strain of HSV-1, unless otherwise noted. tsB7, provided by David Knipe (Harvard University), is a temperature-sensitive mutant derived from the HFEM strain of HSV-1 (4, 51). The virus mutant vi13, provided by Neal DeLuca (University of Pittsburgh), encodes an ICP4 containing a small insertion at amino acid (aa) 338, which reduces ICP4's affinity for DNA, prevents early and late gene expression, and results in overexpression of immediate-early (IE) proteins (reference 82 and this report).

Antibodies. ERK1 (C-16) and JNK1 (FL) antibodies were purchased from Santa Cruz Biotechnology, Inc. p38 (no. 9212) and phospho-p38 (no. 9211) antibodies (New England Biolabs) were a gift from Eng-Shang Huang. Mouse monoclonal antibodies to viral IE proteins ICP0 (1083) and ICP4 (H943) were obtained from Lenore Pereira (University of California at San Francisco). Rabbit polyclonal antibody to viral replication protein ICP8 was originally obtained from Ken Powell (Burroughs Wellcome). Rabbit polyclonal antibody against viral glycoprotein gC (Ab47) and monoclonal antibody against viral glycoprotein gB (SS10) were obtained from Roselyn Eisenberg and Gary Cohen (University of Pennsylvania). Monoclonal antibodies against viral glycoproteins gD (III 174-1.2), gH (52S), and gC (VII 13-7) were provided by Patricia Spear (Northwestern University). To neutralize HSV, the amount of gB, gD, and gH antibody added was enough to reduce virus titer by at least 2 orders of magnitude. Based on the relative quantity of antibody used for each of the neutralization experiments, an equal amount of antibody protein to gC was used in control experiments. Antimouse and antirabbit secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

CAT Assays. Each effector plasmid used encodes a fusion protein containing the yeast GAL4 DNA-binding domain and the TAD of the respective transcription factor. The 5×GAL-CAT reporter plasmid (54) encodes the bacterial chloramphenicol acetyltransferase (CAT) protein and contains GAL4 DNA-binding sites in the promoter. The 2AP1CAT reporter plasmid (90) contains two consensus AP-1 sites upstream of the CAT gene. The 3×CRECAT reporter plasmid contains three copies of the CRE consensus sequence upstream of the adenovirus E1b promoter and the CAT gene.

For each assay, 5 × 105 NIH 3T3 or BHK-21 cells in 60-mm-diameter dishes were transfected by the calcium phosphate precipitation procedure (31) with 2.5 µg of a 5×GAL-CAT reporter and the indicated amount of one of the following effectors: 150 ng of GAL-ELK1 (58), 500 ng of GAL-cFOS, 500 ng of GAL-cMYC (2), 500 ng of GAL-cJUN (37), 500 ng of GAL-ATF2, or 250 ng of GAL-RELA (80). In experiments using the pZIP vectors, 250 ng of pZIP-RAS15A (8), 200 ng (unless stated otherwise) of CDC25X (72), or equivalent amounts of the empty pZIP vector were cotransfected with 5×GAL-CAT and either GAL-cJUN or GAL-ATF2. Other dishes were transfected with 2.5 µg of 2AP1CAT or 3×CRECAT reporter. Eighteen hours after transfection, the cells were refed, and 12 h later, the cells were either mock infected or infected with HSV KOS (multiplicity of infection [MOI] = 5). Eighteen hours postinfection (p.i.) the cells were harvested. CAT assays were performed (31) with equivalent amounts of protein from each sample. Results were quantitated on an AMBIS scanner and were the average of at least three separate experiments, except in Fig. 2A, which was performed only once.

Lipofectamine reagent (GIBCO BRL) was also used to transfect cells in some experiments as indicated. Opti-Mem (GIBCO BRL) (400 µl) was mixed with the previously described amount of DNA, and another 400 µl of Opti-Mem was mixed with 8 µl of Lipofectamine. The two solutions were mixed and allowed to sit at room temperature for 30 min. An additional 2.2 ml of Opti-Mem was added to each solution prior to overlaying the cells that had been rinsed with Opti-Mem. Cells were refed with fresh medium at 5 and 18 h posttransfection and were then infected and harvested as described above.

Kinase assays. Cells were mock infected or infected with KOS (MOI = 5) and harvested at the indicated times p.i. to produce whole-cell lysates. Kinase assays were performed as described in reference 37. Cells were washed in phosphate-buffered saline and frozen in lysis buffer (25 mM HEPES [pH 7.7], 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100 plus inhibitors: 0.5 mM dithiothreitol, 20 mM beta -glycerophosphate, 0.1 mM Na3VO4, 2 µg of leupeptin/ml, 100 g of phenylmethylsulfonyl fluoride/ml, 1 mM NaF, and 1 µg of aprotinin/ml). Cells were allowed to thaw and lyse at 4°C for 4 to 5 h. Samples were then clarified by spinning at 13,000 × g for 10 min at 4°C. For each sample, a volume of the resulting supernatant, representing an equivalent number of cells, was diluted (final concentrations: 20 mM HEPES, 75 mM NaCl, 2.5 mM MgCl2, 0.01 mM EDTA, 0.05% Triton X-100 plus inhibitors). To precipitate JNK, 10 µg of glutathione S-transferase (GST)-cJUN (for JNK assays) was conjugated to GST-Sepharose beads and incubated overnight with lysates. In the assays utilizing GST-ATF2, 6 µg of that substrate was added to each sample. After extensive washing (wash buffer: 20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100), the beads were incubated in kinase buffer (20 mM HEPES [pH 7.6] and 20 mM MgCl2 plus 20 mM beta -glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na3VO4, 2 mM dithiothreitol, 1 mM NaF, 20 M ATP) with 5 µCi of [gamma -32P]ATP per reaction for 30 min at 30°C. Samples were diluted 1:2 in 2× sample buffer, boiled, and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels. Experiments using the viral glycoprotein antibodies were performed after the virus had been incubated with the antibodies for 1 h at 37°C prior to infecting the cells. For the ERK assays, cells were placed in 0.5% serum for 24 h prior to infection. The diluted extracts were first precleared with normal rabbit serum and protein A beads. ERK1 antibody was added and allowed to incubate for at least 5 h, and then protein A beads were added for an overnight incubation. After washing, the beads were resuspended in kinase buffer with [gamma -32P]ATP and 10 µg of myelin basic protein as a substrate and incubated for 30 min at 30°C. Samples were diluted 1:2 in 2× sample buffer, boiled, and fractionated by SDS-PAGE on 15% polyacrylamide gels.

Immunoblotting. Fractionated extracts were prepared as described by Hilton et al. (39). Cell equivalent amounts of cellular lysates prepared for JNK kinase assays were run on 10 or 12% polyacrylamide-SDS gels. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed for JNK, ERK, p38, or activated p38 or the indicated viral proteins. Membranes were blocked in TBST (150 mM NaCl, 20 mM Tris [pH 7.5], 0.05% Tween 20) with 3% milk, and all probing and washing was done in TBST with 0.5% milk. The secondary antibody was detected with the Renaissance chemiluminescence reagent (DuPont NEN).

Single-step growth assays. NIH 3T3 cells (G13.5 and G14.1) were seeded (2.5 × 105) into 60-mm-diameter dishes and infected the next day with HSV-1 KOS (MOI = 5). Cells and supernatant were harvested at the indicated times p.i. and frozen and thawed four times. Virus titers were determined by plaque assays on Vero cells. Each titration was done in triplicate, and each point represents three independent experiments.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

HSV induces the trans-activation function of cJUN and ATF2 via a RAS-independent mechanism. As a first means of investigating how HSV may be affecting cellular signal transduction pathways, we assessed the activities of the endpoints of several of these pathways by determining the activity of various transcription factors known to be phosphorylated and activated by different MAPKs. In these experiments, NIH 3T3 cells were cotransfected with a CAT reporter plasmid containing five GAL4 DNA-binding sites upstream of the CAT coding sequence and one of a set of plasmids that encodes a fusion protein containing the GAL4 DNA-binding domain and the TAD of a transcription factor. This experimental design negates any differences in DNA binding and focuses solely on the trans-activation ability of the various recombinant transcription factors. We utilized NIH 3T3 cells because the response of transcription factors to signals mediated by MAPK pathways and the methodology using these constructs in similar experiments to study these responses have been well characterized in this cell line (10, 35, 49, 70, 90, 94). Figure 1A shows the results of the CAT assays. Infection of transfected cells by HSV repressed the level of CAT activity when the effector was GAL-cMYC, GAL-ELK1, or GAL-RELA. GAL-cFOS-dependent CAT activity was relatively unaffected by infection. In contrast, CAT activity in extracts from HSV-infected cells transfected with GAL-cJUN or GAL-ATF2 was increased 20-fold over that detected in mock-infected extracts. (Note differences in scale among the assays for different effectors.) No CAT activity was detected in any extracts, mock or infected, in the absence of a transfected effector construct (negative data not shown).


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  		FIG. 1.   HSV activates the TAD of cJUN and ATF2 and stimulates AP-1 activity. (A) NIH 3T3 cells (5 × 105) were transfected with 2.5 µg of a 5×GAL-CAT reporter and 150 to 500 ng of the various effectors, each of which encodes a fusion protein containing the GAL4 DNA-binding domain and the TAD of the respective transcription factors. Under each effector heading is the reported MAPK, if known, that activates the TAD of that transcription factor. (B) NIH 3T3 or BHK-21 cells (5 × 105) were transfected with 2.5 µg of 2AP1CAT or 3×CRECAT reporter. 2AP1CAT contains two consensus AP-1 sites upstream of the CAT gene. 3×CRECAT contains three copies of the CRE in front of the adenovirus E1b promoter upstream of the CAT gene. For all CAT experiments, cells were refed 18 h after transfection, and 12 h later, the cells were either mock infected or infected with KOS (MOI = 5). Eighteen hours p.i., the cells were harvested. CAT assays were performed with equivalent amounts of protein from each sample. Results were quantitated on an AMBIS scanner and are the average of at least three separate experiments.

It has been reported that HSV infection causes an increase in AP-1 activity (42). AP-1 is a dimeric transcription factor composed either of one JUN family member and one FOS family member or of two JUN family members (3, 46). The cognate binding site for AP-1, the tetradecanoyl phorbol acetate (TPA)-responsive element (TRE), is TGACTCA. JUN can also heterodimerize with CREB family members such as ATF2, and these complexes bind to a related sequence called the cyclic AMP-responsive element (CRE), TGACGTCA (3, 46). We confirmed that HSV infection increased AP-1 activity in both NIH 3T3 and BHK-21 cells by using a CAT reporter, 2AP1CAT, containing two consensus AP-1 binding sites (TRE sequences) in the upstream region (Fig. 1B). Since we observed increased GAL-ATF2-dependent CAT activity, CRE-dependent promoter activity was also measured with 3×CRECAT. In this case, we observed no change in CRE-dependent activity in NIH 3T3, where the constitutive level was high. In BHK-21 cells, however, where constitutive activity was low, we observed a two-and-a-half-fold increase in CRE-dependent CAT activity after infection (Fig. 1B).

The presence of growth factors and mitogens upregulates RAS activity. Activated RAS in turn coordinates the induction of the RAF-MEK-ERK pathway, which upregulates the activity of some transcription factors, such as cMYC and Ets (6, 87). Growth factors only weakly activate JNK, and hence cJUN, in a RAS-dependent manner (46, 95). cJUN can also be activated by pathways that are not RAS dependent (5, 6, 61, 62, 87). These pathways, however, are inhibited by activated RAS, since the absence of activated RAS results in modest increases in cJUN activity (5, 6, 61, 62, 87). To ascertain whether the activation of cJUN and ATF2 detected after infection was dependent upon RAS, we repeated the CAT assays and included an expression vector that encoded a dominant-negative version of RAS, RAS15A, or a constitutively active version of CDC25, CDC25X. RAS15A can still bind to guanine exchange factors (GEFs), but it is unable to release GDP in exchange for GTP (8). RAS15A, therefore, sequesters GEFs into nonproductive complexes and inhibits the activation of wild-type RAS. CDC25X is a truncated, membrane-localized form of CDC25, a RAS GEF. Due to its membrane localization, it can constitutively interact with and activate RAS (72).

In NIH 3T3 cells, cJUN can be regulated by a RAS-dependent pathway, since constitutively activated CDC25 increased GAL-cJUN-dependent CAT activity (Fig. 2A, lanes 1 to 4), while dominant-negative RAS (acting downstream of CDC25) reversed this increase (Fig. 2A, lanes 5 to 7). In HSV-infected cells, however, neither a dominant-negative RAS nor a constitutively active CDC25 had any effect on the observed increase in GAL-cJUN-dependent activity (Fig. 2B, lanes 2, 4, and 6), while both these effectors caused an increase in GAL-cJUN activity in mock-infected cells (Fig. 2B, lanes 1, 3, and 5). Thus while cJUN can be weakly activated by a RAS-dependent mechanism in uninfected NIH 3T3 cells, this pathway does not significantly contribute to GAL-cJUN activation in HSV-infected cells. Inhibition of RAS activity (e.g., expression of RAS15A) can derepress pathways that also modestly activate cJUN as seen when comparing mock-infected pZIP- and pZIP-RAS15A-transfected cells (Fig. 2B, lanes 1 and 3). In contrast, there is no difference between HSV-infected pZIP- and pZIPRAS15A-transfected cells (Fig. 2B, lanes 2 and 4). When RAS15A was cotransfected with CDC25X, the level of CAT activity was restored to that seen in cells transfected with the reporter and GAL-cJUN alone (Fig. 2B, compare lanes 1 and 2 to lanes 7 and 8). Expression of the dominant-negative RAS had no effect on GAL-ATF2-dependent CAT activity in mock- or virus-infected cells (Fig. 2B, lanes 9 to 12). Furthermore, none of the pZIP vectors had any effect on the level of activity expressed from 5×GAL-CAT in the absence of GAL-cJUN or GAL-ATF2, because these samples had no detectable CAT activity (negative data not shown). Thus, even RAS-independent cellular pathways are not contributing to virus-induced cJUN activity.


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  		FIG. 2.   Activation of cJUN and ATF2 TADs is RAS independent. (A) NIH 3T3 cells (5 × 105) were transfected with 2.5 µg of a 5×GAL-CAT reporter, 500 ng of GAL-c-JUN, and various amounts of pZIP-CDC25X, with or without 250 ng of pZIP-RAS15A. An adjusted amount of pZIP vector was added accordingly to equalize the amount of transfected DNA between the samples. (B) NIH 3T3 cells (5 × 105) were transfected with 2.5 µg of a 5×GAL-CAT reporter and 500 ng of either GAL-c-JUN or GAL-ATF-2 and either 250 ng of pZIP-RAS15A, 200 ng of pZIP-CDC25X, or equivalent amounts of empty pZIP vector. For all CAT experiments, cells were refed 18 h after transfection, and 12 h later, the cells were either mock infected or infected with KOS (MOI = 5). Eighteen hours p.i., the cells were harvested. CAT assays were performed with equivalent amounts of protein from each sample. Results were quantitated on an AMBIS scanner and are the average of at least three separate experiments, except in panel A, which was performed only once.

HSV activates SAPKs but not ERK. We next focused our study on the possible activation of JNK and p38 as suggested by increased GAL-cJUN and GAL-ATF2 activity in the CAT assays. We also assayed the activity of another MAPK, ERK, that the CAT data suggested either decreased or remained unchanged following infection. We first performed in vitro solid substrate and immune complex kinase assays for JNK and ERK, respectively. The results from five different cell types are shown in Fig. 3. We used NIH 3T3 cells to directly correlate any differences we saw in kinase activation with the CAT data (Fig. 1). We also chose to study Vero and BHK-21 cells because they are commonly used for studies of HSV replication, and C33-A and U2OS cells to show that the effects also occurred in human cells. Although there was some variability in the degree to which JNK was activated, all cell types tested demonstrated upregulation of JNK following infection with HSV, as indicated by phosphorylation of a wild-type GST-cJUN substrate (Fig. 3A, upper panel of each cell type set). The induction was greater than 20-fold in all but NIH 3T3 cells (6-fold), comparable to the level seen when the cells were treated with the protein synthesis inhibitor, anisomycin, a potent inducer of JNK activity (22, 36, 96). JNK phosphorylates serines 63 and 73 in the amino terminus of cJUN (16, 37). When a GST-cJUN substrate was used in which Ser63 and Ser73 were mutated to alanines (AA), phosphorylation of GST-cJUN was abolished. Coomassie blue staining of the gels prior to autoradiography showed that all lanes contained equal amounts of substrate (data not shown).


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  		FIG. 3.   HSV activates JNK but not ERK. (A) BHK-21, U2OS, NIH 3T3, Vero, and C33-A cells were mock infected, infected with KOS (MOI = 5) for 10 h, or treated with anisomycin (20 µg/ml) for 20 min. Cells were then harvested to produce lysates. Equivalent numbers of cells were assayed in each sample. The top panel for each cell type is the result of an in vitro solid substrate kinase assay using 10 µg of GST-cJUN (aa 1 to 79) conjugated to GST-Sepharose beads, as described in Materials and Methods. Two different GST-cJUN substrates were used: a wild-type (WT) GST-cJUN and a mutant (AA) GST-cJUN. In the mutant construct, the serines that are phosphorylated to cause cJUN trans-activation (serines 63 and 73) have been mutated to alanines. The bottom panel for each cell type is an immunoblot, probed with an antibody to JNK, performed on a fraction of the pull down. (B) The same cell types used in panel A were used to compare the level of ERK activity in mock-infected cells (M) versus infected cells at 10 h p.i. (I). The top panel shows results from an in vitro immune complex kinase assay for ERK on extracts from serum-starved cells, as described in Materials and Methods. The bottom panel is an immunoblot from a fraction of the immunoprecipitation probed with an antibody against ERK.

The lower panel for each cell type set is an immunoblot for JNK demonstrating the amount of JNK which was precipitated in each experiment was constant. These results provide evidence that infection was activating preexisting JNK rather than inducing JNK expression. Because the kinase whose activity we detected must be precipitable by binding to the amino terminus of cJUN, and because the specificity of the phosphorylation of GST-cJUN in infected cells was identical to that seen in anisomycin-induced control cells, we conclude that we are detecting bona fide JNK activity. In support of this conclusion, we have obtained identical results when using an antibody against JNK to immunoprecipitate the kinase for use in immune complex kinase assays with GST-cJUN as the substrate (data not shown). Thus, activation of JNK upon HSV infection is a universal response, regardless of the species origin or cell type assayed.

Similar assays to detect ERK activity indicated no change in the level of ERK activity as a result of infection (Fig. 3B). The experiment in Fig. 3B was performed on serum-starved cells to reduce the ERK kinase activity background so as to more easily detect any increases in activity that may have been induced by infection. In all cell types tested, no change in ERK activity (Fig. 3B, upper panel) was detected in comparing mock-infected cells (M) to cells infected with KOS for 10 h (I). In the few cases in which differences were detected, the change could be accounted for by variability in the amount of ERK protein that was brought down in individual immunoprecipitations (Fig. 3B, lower panel). Kinase assays detecting ERK activity were also performed with cells grown under normal culture conditions. In these experiments, the background ERK activity was high, and no increases in activity could be detected. In fact, slight decreases were often detected (data not shown). Thus, the CAT assay results were predictive of how HSV affected the activity of various MAPKs responsible for phosphorylating and activating the TADs of the assayed transcription factors. Upregulation of JNK correlated well with increased GAL-cJUN-dependent CAT activity, while ERK was unaffected or slightly downregulated, as mirrored by the downregulation, for example, of GAL-cMYC-dependent CAT activity.

The data in Fig. 3 were from cells that had been infected for 10 h. To determine the kinetics and magnitude of JNK activation as well as to detect any transient activation of the MAPKs, we repeated the kinase assays over a time course of infection in U2OS cells (Fig. 4). Using GST-cJUN in solid substrate kinase assays (top two panels of Fig. 4A), we detected little or no JNK activity in mock-infected cells, but increased JNK activity was detected between 3 and 5 h p.i. in virus-infected cells. The activity increased 20-fold by 9 h p.i. when it appeared to plateau. It should be noted that great care had to be taken during infection to minimize cell exposure to air, because this situation could induce a transient JNK activation (data not shown).


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  		FIG. 4.   Activation of JNK occurs with early kinetics. U2OS cells were mock infected or infected with KOS (MOI = 5) and harvested to produce cellular lysates at the indicated times p.i. An equivalent number of cells was assayed in each sample. (A) The top two panels show results from an experiment using 10 µg of GST-cJUN (aa 1 to 79) as the precipitating agent and substrate in kinase reactions performed as described in Materials and Methods. The bottom two panels are the results with 6 µg of GST-ATF2 (aa 1 to 254) as the precipitating agent and substrate. (B) Cell-equivalent amounts of mock-infected and wild-type KOS-infected U2OS cellular lysates prepared for JNK kinase assays and collected at the indicated times p.i. were run on 10% polyacrylamide gels. Proteins were transferred to a PVDF membrane and probed for activated p38 (p-p38) or total p38 (p38). One-sixth the amount of protein was loaded onto gels to be used for detecting total p38 as was loaded onto gels to be used for detecting activated p38. (C) Samples of lysate were first precleared with normal rabbit serum and protein A beads. ERK1 antibody was then added to precipitate the kinase. Myelin basic protein (10 µg) was included in the kinase reaction as a substrate, as described in Materials and Methods.

Repeating the assay with GST-ATF2 as the substrate, we saw similar kinetics for kinase activation, but the activation was much reduced in magnitude (lower two panels of Fig. 4A). Both JNK and p38 are capable of binding to and phosphorylating ATF2, although ATF2 is a better substrate for p38 than it is for JNK (33, 73). To determine if p38 was playing a role in the phosphorylation of GST-ATF2 or whether JNK is solely activated, we decided to investigate p38's activation state as a result of infection. We infected U2OS cells over a similar time course and harvested samples to use for immunoblotting. We probed the immunoblots with a phospho-specific antibody that recognizes p38 phosphorylated on threonine 180 and tyrosine 182. Phosphorylation of these two residues correlates with activation of p38 (73). The use of an antibody that recognizes these two residues when dually phosphorylated, therefore, should be equivalent to doing direct kinase assays for determining p38 activation, as has been determined by other laboratories (64, 71). Figure 4B shows that over a time course, an activated form of p38 was detected in the infected samples (HSV p-p38 panel) with similar kinetics as was seen for the rise in JNK activation (Fig. 4A, top HSV panel). No activation of p38 was seen in the mock samples (Mock p-p38 panel). Despite some fluctuations in total p38 in the mock samples of this representative experiment (Mock p38 panels), infection by HSV induced activation of p38 without affecting the level of total p38 (HSV p-p38 and p38 panels). This result confirmed that both JNK and p38 are activated at relatively the same time p.i., and their activities remain high out to 11 h p.i.

Immune complex kinase assays for ERK showed no change in activity over the time course of infection (Fig. 4C). The failure to detect an induction in ERK was not due to limiting substrate or antibody in immunoprecipitation or kinase reactions, because increases in both did not result in any higher ERK activity (data not shown). Identical results were obtained in time course experiments using BHK-21, Vero, and C33 cells (data not shown).

IE gene expression is necessary for JNK activation. The absence of JNK activity at early times p.i. argued against the possibility that virions binding to cell surface receptors was initiating a receptor-mediated signal cascade. In order to fully negate the possibility of a delayed cell surface signal mediating the induction of JNK, we performed kinase assays using virus that had been neutralized with antibodies specific for different viral envelope glycoproteins. Antibodies which bind to glycoproteins B, D, and H have been shown to neutralize virus by blocking the virions' ability to fuse with the cellular membrane following initial attachment (23, 24, 66, 67). The glycoprotein C monoclonal antibody, VII 13-7, does not prevent binding or penetration, so that infection proceeds just as it would for virus not incubated with neutralizing antibody (84).

The amount of each antibody (except that for gC) used to neutralize virus reduced the titer by >2 logs as determined by plaque reduction assays. A roughly equivalent protein amount, compared to monoclonal antibodies against gB, gD, and gH, was used in experiments involving gC antibody. Additionally, cells infected with virus which had been treated with the empirically derived amounts of gB, gD, and gH neutralizing antibody described above did not express viral proteins, while cells infected with virus incubated with gC antibody expressed representative viral proteins (immunoblot data not shown). Virus treated with gB, gD, and gH neutralizing antibodies prior to infection did not induce JNK activity in U2OS cells out to 11 h p.i. (Fig. 5A). Nor did incubation of virus with gC antibody prevent JNK induction. We conclude that virus binding to the surface of the cell is not sufficient for JNK activation, nor is JNK activation seen in nonneutralized viral infections due to a delayed signal cascade initiated at the cell surface.


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  		FIG. 5.   Events prior to viral gene expression are not sufficient for JNK activation. (A) U2OS cells were mock infected, infected with KOS (MOI = 5), or infected with KOS (MOI = 5) previously incubated with an antibody specific for viral gB (ss10), gC (VII 13-7), gD (III 174-1.2), or gH (52S). Cells were harvested to produce cellular lysates at the indicated times p.i. Solid substrate kinase assays were performed with GST-cJUN as described in Materials and Methods. (B) U2OS cells were mock infected, infected with KOS (MOI = 5), or infected with KOS (MOI = 5) that had been irradiated with UV light sufficiently to reduce the effective titer by 3 logs. Cellular lysates were prepared at the indicated times p.i., and in vitro solid substrate kinase assays were performed with GST-cJUN as described in Materials and Methods. (C) U2OS cells preincubated at 33 or 39.5°C were mock infected, infected with tsB7 (MOI = 5 based on titer at 33°C), or infected with HFEM, the strain from which tsB7 was derived. Infections then proceeded at the indicated temperatures, and cellular lysates were prepared at the indicated times p.i. In vitro solid substrate kinase assays were performed with GST-cJUN as described in Materials and Methods.

Several virion proteins have additional functions upon entering a newly infected cell. To determine if any of these proteins were responsible for inducing JNK, we compared mock-infected cell extracts to extracts from cells infected with either wild-type virus or UV-irradiated virus over a time course of infection. The inoculum of irradiated virus used in this experiment had a titer that was reduced greater than 3 logs relative to the control inoculum (data not shown). The experiment presented in Fig. 5B shows that in U2OS cells, irradiated virus, at best, minimally activated JNK. The magnitude of activation was much less than that seen in infections with unirradiated virus. Identical results were seen in repeat experiments using Vero and C33 cells (data not shown). It is believed that UV-irradiated virus is impaired in its ability to express detectable viral proteins upon infection due to thymine dimers in the viral DNA impeding transcription of the genome (38). We cannot, however, exclude the possibility that UV irradiation may also cross-link some virion proteins and that this action may be responsible for the inability of UV-irradiated virus to induce high levels of JNK activity. To address this concern, we used a temperature-sensitive viral mutant, tsB7. At 33°C, tsB7 grows normally, albeit more slowly than the parental wild-type strain, HFEM. At 39.5°C, tsB7 virions can penetrate cells, and the capsids move to the nuclear pores, but the viral DNA is not released from the capsids into the nucleus (4). No viral genes, therefore, are expressed at the restrictive temperature (4, 51). tsB7 has been demonstrated to deliver functional VP16 to the nucleus of infected cells at the restrictive temperature (3a). The left panels of Fig. 4C show that at 33°C, mock-infected cells do not activate JNK, but tsB7- and HFEM-infected cells do with kinetics similar to that seen in KOS-infected cells. The right-hand panels show that at 39.5°C, only HFEM-infected cells significantly activate JNK. Activation actually occurs earlier at the higher temperature, for reasons we do not understand at this time. tsB7-infected cells at 39.5°C only induce a minimal level of JNK activity. From this result, we conclude that de novo viral gene expression is necessary for full JNK activation while virion proteins may activate JNK to low levels.

A temporally regulated program of IE, early, and late viral gene expression occurs following HSV infection (76). To determine which virally encoded proteins were responsible for activating JNK, we first compared the timing of JNK activation with the expression and accumulation of representative proteins of each of the viral kinetic classes of gene expression (Fig. 6A). By immunoblot analysis of the same extracts used in the kinase assays, we could detect ICP4, an IE protein, within 1 h p.i. and a rapid increase in expression peaking at 5 h p.i. Expression of an early protein, ICP8, was first detected at 5 h and increased out to 11 h. A late protein, glycoprotein C, was not detected until 9 h p.i. Comparing the patterns of protein expression (Fig. 6A) and JNK activation (Fig. 4A) revealed a rise in kinase activity following a significant accumulation of ICP4, but concurrently with the accumulation of ICP8 (Fig. 6A). This result could be interpreted in two ways. First, the expression of an early protein(s) leads to activation of JNK. Second, one or more IE proteins are responsible for JNK activation, so that the results of IE protein activity, i.e., early protein expression and JNK activation, occur simultaneously.


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  		FIG. 6.   JNK activation is dependent upon viral IE gene expression. (A) Cell-equivalent amounts of wild-type KOS-infected U2OS cellular lysates prepared for JNK kinase assays were run on 12% polyacrylamide gels. Proteins were transferred to a PVDF membrane and probed for viral proteins ICP4, ICP8, and gC. (B) An in vitro solid substrate kinase assay was performed with GST-cJUN as described in Materials and Methods with lysates prepared at the indicated times p.i. from wild-type KOS- and vi13-infected cells (MOI = 5). The kinase data points were derived by scanning the autorads on an LKB laser densitometer. (C) Immunoblot analysis of wild-type and vi13 ICPs was with the same antibodies as in panel A, with the addition of ICP0.

To distinguish between the two possibilities presented above, we assayed JNK activity following infection by vi13, a virus expressing an ICP4 with a 2-aa insertion at residue 338. The insertion reduces the DNA binding affinity of ICP4 and prevents early and late gene expression, while causing overexpression of IE proteins (82) (Fig. 6C). Infection of U2OS cells with vi13 resulted in a twofold increase in JNK activity compared to wild-type virus (Fig. 6B), indicating that expression of early or late proteins was not necessary for JNK activation. Based on this result and the fact that the timing of JNK activation was delayed relative to the binding and penetration of the virus into the cell (Fig. 4A), the activation of JNK is likely a result of the expression of one or more IE proteins. Additionally, if one or more IE proteins are responsible for activation, then the increased JNK activation observed in vi13-infected cells could be explained by the overexpression of IE proteins. Immunoblot analysis confirmed that vi13-infected cells did not express representative early or late proteins, but did overexpress IE proteins, e.g., ICP0 and ICP4 (Fig. 6C).

Activation of JNK enhances viral replication. Having documented activation of JNK following infection, we wished to know if its activation was important for viral replication. To address this question, we relied on the recently described inhibitor of JNK, JIP-1 (JNK-interacting protein-1). JIP-1 acts as a cytoplasmic scaffolding protein that coordinates the transduction of a signal through the SAPK pathway from the level of MAPKKKs to JNK (93). JIP-1 binds exclusively to JNK and not to other MAPKs, and it binds with a 100-fold greater affinity to JNK than JNK binds to any of its substrates (17). Overexpression of JIP-1 has been shown to effectively inhibit JNK from translocating into the nucleus (independent of JNK's activation state) which prevents JNK-mediated gene expression (93).

We created stable NIH 3T3 cell lines containing the pcDNA3-JIP-1 expressing vector by G418 selection (see Materials and Methods). In the absence of an antibody to JIP-1 to determine the expression level of JIP-1, we performed two functional assays to prove that JIP-1 was acting to inhibit JNK. First we performed GAL-cJUN-dependent CAT assays under the premise that since JIP-1 inhibits JNK-mediated gene expression, GAL-cJUN-dependent CAT activity should be decreased in JIP-1-overexpressing cells compared to that in control cells. In one such cell line that stably contains the pcDNA3-JIP-1 vector, G14.1, GAL-cJUN-dependent CAT activity was decreased in both mock (basal) and infected (induced) extracts compared to that in extracts from an empty vector control cell line, G13.5 (Fig. 7A). To verify that JNK was inhibited from translocating into the nucleus, we fractionated mock- and virus-infected G13.5 and G14.1 cells and ran extracts on gels to be used in immunoblotting for JNK. JNK was primarily found in the cytoplasm of mock-infected (MC) G13.5 and G14.1 cells, while very little JNK was detected in the nucleus (MN). Upon infection, JNK was found in both the cytoplasm (IC) and nucleus (IN) of G13.5 cells, but JNK was still retained in the cytoplasm of G14.1 cells (Fig. 7B). Solid-phase GST-cJUN-dependent kinase assays of extracts from G13.5 and G14.1 cells indicated that JNK activation occurred equally in both cell lines following infection (data not shown). These results are consistent with the known properties of JIP-1. They also confirm that G14.1 cells are expressing JIP-1 so that JNK is unable to translocate into the nucleus and activate its targets following infection. Single-step growth curves for G13.5 and G14.1 cell lines demonstrated that inhibiting the ability of JNK to activate its targets decreased viral yield by approximately 70% (Fig. 7C).


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  		FIG. 7.   Preventing JNK from activating nuclear targets decreases the efficiency of viral replication. (A) Two stably transfected NIH 3T3 cell lines, G13.5 (control cell line) and G14.1 (JIP-1 overexpressing cell line), were isolated as described in Materials and Methods and compared for their ability to support GAL-cJUN-dependent CAT activity. Cells were transfected with Lipofectamine reagent with 2.5 µg of 5×GAL-CAT and 500 ng of GAL-cJUN and then infected with KOS (MOI = 5) 24 h later. Eighteen hours p.i., the cells were harvested, and CAT assays were performed as described in Materials and Methods. (B) G13.5 and G14.1 cells were mock infected or infected with KOS (MOI = 5) for 8 h. Cells were harvested, and fractionated extracts were prepared. Equal amounts of protein from each fraction were run on 12% polyacrylamide gels. Proteins were transferred to a PVDF membrane and probed for JNK. MC, mock-infected cytoplasm; MN, mock-infected nucleus; IC, KOS-infected cytoplasm; IN, KOS-infected nucleus. (C) The two cell lines were infected with HSV at an MOI of 5. Cells and medium were harvested at the times indicated and frozen and thawed four times. Titers of aliquots were determined on Vero cell monolayers.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

HSV induces cJUN and ATF2 trans-activation. Utilizing a GAL-CAT reporter system, we have shown that the TADs of cJUN and ATF2 were substantially activated following infection by HSV. This experimental approach eliminated any differences in DNA binding that the transcription factors might exhibit and allowed us to indirectly assess the activity of upstream kinases. cJUN and ATF2 TADs are activated by SAPKs---cJUN by JNK and ATF2 by a p38 family member (strongly) and by JNK (weakly) (33, 55, 73, 86). Although ELK1 transcriptional activity can be induced by JNK or p38 as well as ERK (7, 9, 28, 29, 32, 94, 96-98), it is clear that JNK is not activating ELK1 under our experimental conditions. HSV may be activating pathways that specifically target certain JNK and p38 isoforms which in turn activate specific TADs. Three JNK genes have been identified, giving rise to at least 10 alternatively spliced mRNAs, and different JNK isoforms have different specificities and affinities for their respective substrates (34). Additionally, there are at least four different p38 isoforms which are differentially activated by stress stimuli (13, 21, 73, 88). The activities of all other cellular transcription factors we assayed decreased or were, at best, unchanged. The decrease in GAL-RELA-dependent CAT activity following infection agrees with a previous report that showed that NF-kappa B, while translocated to the nucleus following infection, did not demonstrate an increased ability to induce trans-activation (68). At this time, we do not know if HSV is specifically inhibiting the activity of these factors or if the decreases are an indirect effect of infection.

HSV infection has been shown to result in increased AP-1 activity (42). We used a CAT reporter containing two consensus AP-1 binding sites in the promoter region to confirm this finding (Fig. 1B). AP-1 can be composed of several different JUN and FOS family members that all bind to consensus AP-1 sites, called TREs (3, 46). Since cFOS is not activated following infection, but cJUN and ATF2 are, it is likely that increased TRE-mediated trans-activation is a result of activated cJUN in homodimers or in heterodimers with existing cFOS. Also of note, the study by Tognon et al. (85) demonstrated downregulation of cFOS expression by ICP4 which would result in decreased JUN-cFOS activity in favor of JUN-JUN and JUN-ATF2 activity. Additionally, cJUN is capable of forming heterodimers with ATF2 (3, 46). This heterodimer binds to sites called CREs that differ from TREs by the addition of 1 bp in the intervening sequence between the dyad repeats (3, 46). We observed a cell-type-specific increase in CRE-dependent CAT activity after HSV infection (Fig. 1B).

Activation of both cJUN and ATF2 could synergistically activate to very high levels the expression of genes that contain CREs in their promoters, such as the c-jun gene itself. It has been shown that c-jun transcripts are induced following infection consistent with cJUN-ATF2 activation (42, 47). In the study of Jang et al. (42) it was not possible to determine what kind of probe was used to study AP-1 activation, but since most of the assays used sequences derived from the c-jun promoter which contains CREs, we conclude that their gel shifts reflect increased CRE binding following infection. This interpretation, however, is in conflict with a recent report which demonstrated that TRE-binding proteins but not CRE-binding proteins accumulated (100). The latter study also showed that while TRE-binding complexes increased, the relative proportion of those complexes that contain cJUN decreased. These results are not in contradiction to our results, since they assayed DNA-binding activity of these transcription factors, while we assayed their trans-activation property. AP-1 is a complex transcription factor---it can be composed of several different proteins that can bind to TREs and can differ in their ability to activate transcription (46). Additionally, phosphorylation at specific sites on these proteins can enhance trans-activation without changing DNA-binding or dimerization properties (46). An important consideration in this regard is that changes in DNA-binding activity do not mirror the regulation of transcriptional activity (46). In order to measure the ability of AP-1 (and its constituents) to activate transcription, the trans-activation ability of those complexes must be measured with an appropriate reporter construct (46). Therefore, even though the population of cJUN-containing AP-1 complexes may be decreasing, the remaining cJUN is being activated by JNK. Likewise, even though CRE-binding proteins do not increase after infection of BHK cells (100), the activity of these proteins can be induced (Fig. 1B). Additionally, we cannot rule out the possibility that other JUN family members, such as JunB and JunD, which can bind to or be phosphorylated by JNK, respectively (45), are phosphorylated or mediate the phosphorylation of dimerization partners after infection and may be the true relevant targets in the infected cell.

RAS is a G-protein shown to be a key mediator of signaling in many pathways (6, 87). There are pathways, however, that do not involve RAS, and some downstream pathways are regulated by RAS only under certain conditions (5, 6, 61, 62, 87). cJUN-dependent transcription can be weakly upregulated either directly by activated RAS or indirectly by derepression of pathways normally downregulated by active RAS, as seen in Fig. 2B for the mock-infected extracts. The inability of either dominant-negative RAS or constitutively active CDC25 to affect the level of GAL-cJUN- or GAL-ATF2-dependent CAT activity in HSV-infected cells suggests that HSV is capable of stimulating cJUN and ATF2 TADs by a RAS-independent mechanism(s). Bypassing any requirement for the upregulation of RAS is in line with a study which showed that activated RAS actually antagonized efficient viral replication. Infection of RAS-transformed cells yielded less virus than nontransformed cells of the same cell type (26).

HSV activation of JNK is dependent on IE gene expression. To test the activities of different MAPKs, we performed immune complex kinase assays to detect ERK activity and solid-phase kinase assays to detect JNK activity. For the latter, we have used a GST-cJUN (1 to 79 aa) construct to bind endogenous JNK and act as its substrate. Results from these experiments indicated a 6- to 20-fold elevation, depending on the cell type, in JNK kinase activity compared to that of mock samples (Fig. 3A). We found no evidence for induction of JNK at any time within the first hour of infection, corresponding to when virus is binding to cell surface receptors. Activation typically began 3 to 4 h p.i., increased out to 8 h p.i., and remained elevated out to at least 14 h p.i. (Fig. 4A, 5, and 6B). Since we obtained identical results if we specifically immunoprecipitated for JNK, in addition to the fact that the responsible kinase must be able to bind to the N terminus of cJUN and only phosphorylate Ser63 and Ser73, we conclude that we were detecting bona fide JNK activity and not another cellular kinase or a viral kinase with JNK-like properties.

Using a GST-ATF2 construct in solid-phase kinase assays, we detected kinase activity that occurred with identical kinetics but a lower magnitude of activity than that detected with GST-cJUN as a substrate (Fig. 4A). Since the ATF2 TAD can be phosphorylated by both JNK and p38, we attempted to determine if p38 was activated by HSV. We took advantage of an antibody that recognizes a phosphorylated form of p38 that correlates with its being in an active state. We observed that p38 is activated with identical kinetics to that seen for the activation of JNK (compare Fig. 4B to 4A). MKK4 is activated by HSV infection (100). Since MKK4 is a MAPKK that can activate both JNK and p38, it is possible that HSV is activating MKK4 to coordinate the activation of both SAPKs.

In contrast, over an 11-h-time-course experiment, we did not detect any change in ERK activity (Fig. 4B). The binding of growth factors to their cognate receptors and other growth-promoting signals typically leads to ERK activation, which is critical for inducing the expression of certain G1 and G1/S phase-promoting proteins such as cyclin D (1a, 56, 67, 69, 75). There are many examples drawn from studies of DNA viruses, some of which are known to cause cell cycle progression, where viral proteins cause activation of the ERK pathway. The E5 protein of bovine papillomavirus inhibits epidermal growth factor (EGF) receptor (EGFR) and colony-stimulating factor receptor down-regulation, leading to a persistence of activated receptors at the cell surface (60). Vaccinia virus encodes a virus growth factor that mimics EGF's ability to stimulate EGFR (50). ERK molecules are actually incorporated into the human immunodeficiency virus type 1 virion, and ERK activity is necessary for the disengagement of the reverse transcription complex from the cell membrane and its translocation into the nucleus (41). The herpesvirus saimiri protein STP-C488 associates with RAS (43) and increases its GTP/GDP ratio, leading to constitutive activation of ERK (44). Small T antigen of simian virus 40 binds to and prevents protein phosphatase 2A from dephosphorylating ERK2 and MEK, which prolongs their activities (83). The inability to detect ERK activation following HSV infection correlates with other evidence that HSV infection fails to promote cell cycle progression (14, 19). Whether the failure to promote progression is related to the ability of HSV to encode most of its DNA replication machinery, thus precluding the necessity to establish a true S phase, remains to be determined.

Signals that lead to MAPK activation are often generated at the cell surface when receptors bind to their appropriate ligand. Some studies have shown that when virus binds to a cell, it may do so by binding to a cell surface receptor, and that interaction may mimic normal ligand-receptor events to activate downstream pathways. Binding of simian immunodeficiency virus (SIV) to cells activates ERK, JNK, and/or p38 depending upon the cell type, and hence, the receptor to which SIV binds (71). Cytomegalovirus has been shown to generate intracellular signals upon binding to the surface of permissive cells that induce the translocation and activation of NF-kappa B (79, 99). One reported cell surface receptor for HSV, HVEM or HveA, is a member of the tumor necrosis factor receptor (TNFR) family (63, 91). Activation of this receptor in overexpression studies leads to the generation of a signal which upregulates AP-1 activities (59). This upregulation, though not directly shown, is presumably due to induction of JNK, as many TNFR family members have been shown to induce JNK leading to upregulated AP-1 activity (65, 74, 78). Even though the cells we used do not express HVEM, HSV does bind to other cellular receptors (27), and we predicted that binding to one or more of these receptors may lead to MAPK activation. However, both the timing of JNK activation and the absence of induction following infection by antibody-neutralized HSV, which can bind but not enter cells, argue against this mechanism.

Infection with UV-irradiated virus failed to induce JNK to high levels. We believe the inability to activate JNK was due to the inability of irradiated virus to express viral genes; however, we could not discount potential cross-linking of virion proteins as being responsible for the lack of JNK activity. We therefore turned to a temperature-sensitive mutant which infects cells at the restrictive temperature, delivers functional VP16 to the nucleus (3a), but does not release viral DNA from capsids into the nucleus (4). Viral genes, therefore, are not expressed in a tsB7 infection at the restrictive temperature (4, 51), but tegument proteins are free to function normally. We failed to detect high levels of JNK activity after infection by tsB7 at the restrictive temperature. We did note that in UV-irradiated virus extracts and tsB7 samples from 39.5°C, there was a slight elevation in JNK activity over mock samples (approximately twofold). It is possible that this slight activation was due to the function of a tegument protein, such as VP16 (100). Alternatively, it could reflect a low level of activation in the small population of cells infected by less-damaged virus in UV-irradiated stocks or leaky tsB7 virus at the restrictive temperature, mimicking an infection at a low MOI. We have noted that activation is MOI sensitive, so that at an MOI of 0.5, the level of JNK activation is very low (unpublished observation). These results suggest that in order for HSV to significantly induce JNK activity, the virus must enter the cell and express viral genes.

Recently, JNK and p38 were reported to be activated after HSV infection by a VP16-dependent mechanism (100). Comparisons of mock- and VP16 mutant in1814-infected cells in that report were made difficult because of the very high background (mock-infected) levels of JNK activity. While we interpret these results as at best arguing for a role for VP16 in JNK activation, it is also possible that the defects in IE gene expression in in1814-infected cells may contribute to the failure to detect JNK activation (1). Although the experiments with in1814 were performed at a high MOI, it has not been directly demonstrated that this condition of infection restores IE gene expression to normal levels. The delayed activation kinetics of JNK (3 to 4 h p.i.) observed by us and Zachos et al. (100) also argue for at best an indirect role for VP16 and more directly a role for one or more IE proteins in the mechanism of activation. An additional experimental approach by these authors, in which transfection of wild-type VP16 and TAD-deleted VP16 expression vectors resulted in elevated JNK activity, did not take into account the possibility that overexpressed protein may in and of itself have resulted in a cellular stress response.

We were able to determine that expression of one or more IE proteins was necessary for JNK activation. Infection with vi13, an ICP4 mutant virus that only expresses IE proteins (reference 82 and this report), led to activation of JNK with identical kinetics to but greater magnitude than that of wild-type virus, indicating that neither expression of early and late genes nor viral DNA replication is necessary for activating JNK. Supporting this conclusion was the observation that vi13-infected cells overexpress IE proteins, which likely explains the increased level of JNK activity compared to that of wild-type-infected cells. Some of our current studies are attempting to determine the level of JNK activity following infection with viruses that are deleted for each viral IE gene. Due to functional and physical interactions among different IE proteins, we may also resort to using viruses that have multiple deletions of IE genes in order to determine all of the relevant viral proteins.

The above result is somewhat at odds with a similar experiment reported by Zachos et al. (100) in which infection with the ICP4 mutant tsK at the restrictive temperature did not induce increased JNK activation over wild-type virus. The inability to detect a difference by using tsK may relate to the small fold induction in JNK activity that these authors were able to detect with wild-type virus. While they noted a maximum 4-fold increase in JNK activity over mock, we were able to detect a 20-fold induction. We have been careful to modify our infection and harvest protocols to minimize transient activation of JNK which could have the effect of increasing background levels. Regardless, small differences in the level of JNK activation, such as the 2-fold increase by vi13 over KOS would be much easier to detect when KOS is activating JNK 20-fold.

Activation of JNK provides a beneficial activity for viral replication. HSV infection causes many changes to the infected cell, such as halting cellular macromolecular synthesis, reorganizing the nucleus, and causing cells to round up before they are ultimately lysed as a result of the release of viral progeny (76). In this report, we describe an additional event, the activation of SAPKs. Neither the mechanism for activation nor its role in the infectious process of HSV is currently known. The effect of JNK activation after infection could be complex and cell-type specific, since activation of JNK under different conditions in different cell types correlates with proliferation, oncogenic transformation, or apoptosis (40, 92). Previously, only four reports have described induction of JNK activity following virus infection, and the significance of these inductions is also unknown (20, 71, 81, 100).

JNK activation could represent a nonspecific or even an antiviral response by the cell, but several factors argue against this interpretation. First, our control experiments using mock-infected cells or cells infected with neutralized, UV-irradiated virus or tsB7 at the restrictive temperature demonstrate that JNK activation is not a nonspecific response to the mechanics of our infection procedure, due to growth factors or cytokines in the inoculum, or related to early events occurring in lytic infection. Additionally, the onset of JNK activation occurs 3 to 4 h p.i. By this time, virion components, such as VP16, have had ample time to perform their ascribed functions, and IE proteins have already reached high levels of accumulation. If the cells were responding to the presence, accumulation, or function of one or more of these proteins, a response would likely have occurred sooner. The failure of UV-irradiated virus or tsB7 at the restrictive temperature to induce JNK activity to high levels demonstrated that virion components are incapable of, or at least not sufficient for, activating JNK (Fig. 5). It is possible though that VP16, for example, could minimally induce JNK, as seen in UV-irradiated samples and tsB7 samples at 39.5°C. Additionally, no early or late gene expression nor any activity involved in viral DNA replication is necessary for the activation of JNK in HSV-infected cells, as judged by the results with the ICP4 mutant, vi13 (Fig. 6B).

In principle, virally encoded proteins could directly target and thus alter the activity of cellular transcription factors (e.g., cJUN and ATF2). However, it appears that HSV is targeting the upstream regulators of these factors, since increases in their trans-activation function are accompanied by increased JNK and p38 activity. We propose that expression of one or more IE proteins leads to the activation of the JNK pathway. Once activated, JNK would phosphorylate cellular transcription factors that could enhance the expression of IE and possibly early viral genes. Promoters of IE, early, and late genes contain cognate binding sites for a variety of cellular transcription factors, e.g., Oct-1, Sp1, AP-1, CAAT, and NF-kappa B. Mutation of these sites affects the efficiency of promoter function, although no cellular transcription factors per se have been demonstrated to serve an essential role in viral gene expression (76). We also cannot rule out the possibility that JNK could be targeting viral trans-activators ICP0, -4, and -27 to regulate their roles in the temporal regulation of viral gene expression. Alternatively, activation of SAPKs could be targeting cellular activities and upregulating cellular genes that would lead to a more conducive intracellular environment for viral replication.

By blocking the ability of JNK to act on its nuclear substrates, we showed that virus yield is decreased by 70% (Fig. 7C), suggesting that HSV requires the action of this cellular activity for efficient replication. While the decrease in viral yield in JIP-1 expressing G14.1 cells is modest, several points should be noted. First, JIP-1 overexpression did not completely abrogate the ability of JNK to activate cJUN-dependent trans-activation (Fig. 7A). Additionally, the activation of JNK in NIH 3T3 cells following infection was weaker than in other cell types, so viral replication may not be as dependent upon JNK activity in NIH 3T3 cells as in other cell types, where JNK is activated to higher levels. Finally much of the viral genome is devoted to increasing the efficiency of replication in different cell types (76, 89), and we would not expect that inhibiting one cellular activity would completely block viral replication. A key finding reported here, however, is that blocking a specific cellular activity (the ability of JNK to activate its substrates) has had a detrimental effect on replication. Patel et al. (68) reported that blocking the infection-mediated translocation into the nucleus of another cellular transcription factor, NF-kappa B, reduced viral yield by 90%. The activity of E2F transcription factors is reduced following infection by HSV as a result of an increase in the association of these factors with regulatory pocket proteins, such as pRB and p107 (39). Our findings in conjunction with these reports suggest that a number of different cellular activities which target transcription factors are being differentially manipulated in the infected cell and that these activities, in total, are significant for the overall efficiency of virus replication.


    ACKNOWLEDGMENTS

T.I.M. was supported by NIGMS T32 GM 07092. These studies were supported by PHS Program Project grant CA 19014 to S.L.B.

We thank Albert Baldwin for the GAL-RELA expression construct; Shannon Kenney for the GAL-ATF2 expression construct and the 3×CRECAT reporter construct; Laura Licota for GST-cJUN(AA); and Channing Der for all of the other GAL effector and CAT reporter constructs, GST-cJUN(WT), and GST-ATF2. We thank Roger Davis for helpful discussions and the gift of the JIP-1-expressing construct. We also thank Ginger L. Ehmann for critical review of the manuscript and Robert Johnson for assistance in probing immunoblots for total and activated p38.


    FOOTNOTES

* Corresponding author. Mailing address: The Department of Microbiology and Immunology, 837 Jones, CB 7290, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Phone: (919) 966-2445. Fax: (919) 962-8103. E-mail: bachlab{at}med.unc.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, October 1999, p. 8415-8426, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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