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J Virol. 2006 Nov; 80(21): 10624–10633.
Published online 2006 Aug 23. doi:  10.1128/JVI.00390-06
PMCID: PMC1641755

Rotavirus Activates JNK and p38 Signaling Pathways in Intestinal Cells, Leading to AP-1-Driven Transcriptional Responses and Enhanced Virus Replication


Rotavirus infection is known to regulate transcriptional changes in many cellular genes. The transcription factors NF-κB and AP-1 are activated by rotavirus infection, but the upstream processes leading to these events are largely unidentified. We therefore studied the activation state during rotavirus infection of c-Jun NH2-terminal kinase (JNK) and p38, which are kinases known to activate AP-1. As assessed by Western blotting using phospho-specific antibodies, infection with rhesus rotavirus (RRV) or exposure to UV-psoralen-inactivated RRV (I-RRV) resulted in the activation of JNK in HT-29, Caco-2, and MA104 cells. Activation of p38 during RRV infection was observed in Caco-2 and MA104 cells but not in HT-29 cells, whereas exposure to I-RRV did not lead to p38 activation in these cell lines. Rotavirus strains SA11, CRW-8, Wa, and UK also activated JNK and p38. Consistent with the activation of JNK, a corresponding increase in the phosphorylation of the AP-1 component c-Jun was shown. The interleukin-8 (IL-8) and c-jun promoters contain AP-1 binding sequences, and these genes have been shown previously to be transcriptionally up-regulated during rotavirus infection. Using specific inhibitors of JNK (SP600125) and p38 (SB203580) and real-time PCR, we showed that maximal RRV-induced IL-8 and c-jun transcription required JNK and p38 activity. This highlights the importance of JNK and p38 in RRV-induced, AP-1-driven gene expression. Significantly, inhibition of p38 or JNK in Caco-2 cells reduced RRV growth but not viral structural antigen expression, demonstrating the potential importance of JNK and p38 activation for optimal rotavirus replication.

Rotavirus, a segmented double-stranded RNA (dsRNA) virus of the Reoviridae family, is a leading cause of severe infantile gastroenteritis in humans and animals worldwide. Rotavirus infection is primarily targeted to intestinal epithelial cells and leads to transcriptional regulation of a large number of cellular genes, probably as a result of induction of innate cellular defense mechanisms or viral strategies to aid replication (12). The cellular events leading to rotavirus-induced changes in transcriptional activity are not well characterized. An understanding of these events will provide important insights into rotavirus-cell interactions, intestinal cell biology, and potential targets for development of novel antiviral therapies.

The c-Jun NH2-terminal kinase (JNK) and p38, often referred to as stress-activated protein kinases, are members of the mitogen-activated protein kinase (MAPK) family that also includes ERK (extracellular signal-regulated kinase). JNK and p38 can be activated by a number of stimuli, including genotoxic agents, proinflammatory cytokines, osmotic shock, and bacterial lipopolysaccharide (33). The activation of JNK and p38 can have a profound impact on cell fate. Depending on the nature and context of the signal, downstream effects include apoptosis, differentiation, and growth and inflammatory responses (33, 45). JNK and p38 signaling cascades are also induced by viruses, including the Reoviridae family member reovirus, human immunodeficiency virus type 1, echovirus 1, Sindbis virus, encephalomyocarditis virus, coxsackievirus B3, hepatitis C virus, herpes simplex virus 1, and the severe acute respiratory syndrome coronavirus (8, 20, 22, 26, 28, 29, 32, 39, 40, 51). The diverse effects of JNK and p38 activation by these viruses include induction of apoptosis in infected cells and enhancement of viral replication (8, 22).

Up to 12 JNK isoforms have been identified, which are the products of differential splicing of three different genes, jnk1, jnk2, and jnk3 (18, 33). JNK1 and JNK2 are expressed in most cell types, whereas JNK3 is found only in brain and testis (31). Splicing leads to expression of JNK proteins that have distinct molecular masses of approximately 46 kDa and 54 kDa. Four isoforms of p38 have been identified, which are referred to as p38α, -β, -γ, and -δ (33). All MAPKs, including JNK and p38, are activated by dual Thr and Tyr phosphorylation by MAPK kinases, also known as MAPK or ERK kinases (MEK). The residues phosphorylated during activation are Thr183/Tyr185 of JNK and Thr180/Tyr182 of p38. A major downstream target of JNK and p38 is the activator protein 1 (AP-1) transcription factor, which is a dimeric complex composed of members of the Jun, Fos, Maf, and activating transcription factor (ATF) protein subfamilies (45). After activation in the cytoplasm, JNK and p38 translocate to the nucleus, where they phosphorylate Ser and Thr residues on specific AP-1 subunits to augment transcriptional activity. Both JNK and p38 target ATF2 (ATF subfamily), while JNK also targets c-Jun and JunD (Jun subfamily) (33, 45). The expression of c-jun is strongly influenced by the presence of two AP-1 sites within its promoter, providing a positive autoregulatory mechanism for enhanced AP-1 activation (1). Transcription of junD is also positively autoregulated (3).

Rotavirus infection induces activation of transcription factors NF-κB and AP-1 in the intestinal cell lines HT-29 and Caco-2 (6, 34, 41). Inactivated virions of rhesus rotavirus (RRV) and recombinant virus-like particles lacking RNA also activate NF-κB, leading to the suggestion that replication is not required for this response and that virion proteins may be involved (41). A number of chemokines, including interleukin-8 (IL-8), are produced in response to rotavirus infection of intestinal cell lines and in vivo models (5, 6, 41, 46). IL-8 is a strong chemoattractant and activator of neutrophils, macrophages, T lymphocytes, and intraepithelial lymphocytes and therefore may play an important role in enhancing immune responses to rotavirus (2, 15). The IL-8 gene promoter contains AP-1 and NF-κB binding sequences, which are both required for maximal transcriptional activity following rotavirus infection (6). There is an implied role for rotavirus spike protein VP8* in directing NF-κB activation through direct binding to tumor necrosis factor (TNF) receptor-associated factor (TRAF) domains (34). However, little else is understood about upstream events leading to transcriptional regulation in rotavirus-infected cells.

In this study, we aimed to assess the activation state of cellular kinases linked to AP-1 activation, as well as any subsequent influence on AP-1-directed transcription, during rotavirus infection of intestinal and other permissive cells. It is shown here for the first time that rotaviruses from several species induce the activation of JNK and p38. It was determined that the activities of these kinases are necessary for maximal RRV-induced transcription of IL-8 and c-jun mRNA. In addition, the activity of JNK was shown to be required for optimal RRV replication in Caco-2 and monkey kidney cells, while the activity of p38 was also required in Caco-2 cells.


Antibodies and kinase inhibitors.

Rabbit polyclonal antibodies to phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), phospho-c-Jun (Ser63), and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal antibody to JNK (JNK1) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibodies to RRV were raised by immunization with purified RRV, as described previously (36). The antiserum showed a fluorescent focus reduction neutralization titer of 1:80,000 against RRV and reacted with all of the structural proteins of RRV by Western blotting (P. Halasz and B. S. Coulson, unpublished data). Kinase inhibitors SP600125 (JNK inhibitor [JNKi]), SB203580 (p38 inhibitor [p38i]), and PD98059 (MEK inhibitor [MEKi]) were purchased from Calbiochem (La Jolla, CA) and dissolved in dimethyl sulfoxide (DMSO) to give stock solutions of 10 mM.

Cell lines and viruses.

The monkey kidney (MA104) and Caco-2 cell lines were obtained and maintained as described elsewhere (37), except for the addition of nonessential amino acids (Gibco, Grand Island, NY) to the medium used to propagate Caco-2 cells. The HT-29 cell line was obtained from the ATCC and kindly provided by Carl Kirkwood, Murdoch Children's Research Institute, Victoria, Australia. HT-29 cells were passaged in McCoy's 5A medium (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (JRH Biosciences, Lenexa, KS), 2 mM l-glutamine (Gibco), 20 mM HEPES (Boehringer Mannheim, Mannheim, Germany), gentamicin (26.6 μg/ml; Pharmacia, Bentley, WA, Australia) and amphotericin B (Fungizone; 2 μg/ml [Apothecon, Bristol-Myers Squibb, Princeton, NJ]). The origins, propagation in MA104 cells following trypsin activation, and characterization of monkey rotavirus strains RRV and SA11 (P serotype 5B, G serotype 3), human rotavirus strain Wa (P1A, G1), porcine rotavirus strain CRW-8 (P9, G3), and bovine rotavirus strain UK (P7, G6) have been described previously (10, 11, 37). Complete RRV virions were purified by glycerol gradient ultracentrifugation (27). Briefly, virus was propagated in spinner flasks containing MA104 cells grown on Cytodex 3 microcarrier beads (Amersham, Arlington Heights, IL). Culture supernatant fluid was treated with methoxynonafluorobutane (Sigma, St. Louis, MO) as a substitute for the Arklone used in the original protocol (27) to free cell membrane-associated virus. Following removal of the methoxynonafluorobutane layer by centrifugation, virus was pelleted by ultracentrifugation. The virus pellet was subjected to glycerol gradient ultracentrifugation, and the visible band corresponding to triple-layered infectious rotavirus was extracted using a syringe and needle. Extracted virus was pelleted by ultracentrifugation; diluted in a buffer consisting of 30 mM Tris-HCl, pH 7.4, 6.6 mM CaCl2, and 150 mM NaCl; and stored at −70°C. Purified RRV was inactivated by treatment with psoralen and UV as previously reported (17). Inactivation was verified by infectivity titration in MA104 cells as previously described (21), and no infectious virus was detected at a dilution of 1 in 100. Inactivated purified RRV is herein referred to as I-RRV. Purified RRV and I-RRV bound similar levels of rabbit polyclonal antibodies to RRV (36) in an enzyme-linked immunosorbent assay (ELISA) carried out as previously described (10), demonstrating the antigenic integrity of I-RRV (P. Halasz and B. Coulson, unpublished data). RRV inactivated by treatment with UV-psoralen has previously been shown to be antigenically intact in an ELISA using monoclonal antibodies to outer capsid protein VP4 or VP7 or hyperimmune sera and a viral hemagglutination assay (17). Purified RRV was used for all experiments except those requiring multiple rotavirus strains. In those cases, clarified harvests of MA104 cells infected with RRV, SA11, Wa, CRW-8, or UK rotaviruses were employed.

Inhibitor toxicity assay.

Potential toxic effects of inhibitors were assayed using the MTS (tetrazolium salt)-based Celltiter 96 assay (Promega, Madison, WI) of cellular metabolic activity (9). For this, JNKi, p38i, MEKi, or DMSO in serum-free medium was added to confluent MA104, Caco-2, and HT-29 cells in 96-well plates. The DMSO level was maintained at 0.5% (vol/vol) in all media containing DMSO or inhibitors, irrespective of inhibitor concentration. After incubation for 17 h (h) at 37°C with 5% (vol/vol) CO2, 20 μl of CellTiter solution was added to each well, plates were incubated at 37°C for between 30 min and 2 h, and absorbance was read at 492 nm. Absorbance readings from wells containing medium and Celltiter solution without cells were subtracted as background from all other readings. For each inhibitor dose, a decrease in cellular activity of ≤10%, as compared to that in DMSO controls, was considered to be nontoxic in this study. In HT-29 cells, JNKi and p38i concentrations of ≤10 μM were nontoxic. In Caco-2 and MA104 cells, p38i was nontoxic at ≤50 μM and ≤20 μM, respectively, and JNKi was nontoxic at ≤20 μM. Concentrations of MEKi of ≤30 μM were nontoxic in all three cell lines. Nontoxic inhibitor concentrations were used in subsequent experiments.

Inoculation of cells with rotavirus.

Virus and mock inocula were treated for 20 min at 37°C with porcine trypsin (10 μg/ml; Sigma). Confluent cell monolayers in 24-well trays (Nunc, Wiesbaden, Germany) were washed twice with phosphate-buffered saline (PBS) and either mock infected or infected with purified RRV or I-RRV at the indicated multiplicity of infection (MOI) at 37°C. Based on previous findings an MOI of 10 was required to reach the maximal number of infected cells (approximately 85 to 95%, depending on the cell line; P. Halasz and B. Coulson, unpublished data). Therefore, to reduce the masking effects of active kinase in uninfected cells, an MOI of 10 was used for time course experiments analyzing JNK and p38 activity. The volume of I-RRV required was calculated from the infectivity titer of purified RRV measured prior to inactivation. Mock inocula for experiments using purified RRV and I-RRV were comprised of aliquots of a 30 mM Tris-HCl buffer, pH 7.4, containing 6.6 mM CaCl2 and 150 mM NaCl. For unpurified viruses, mock inocula consisted of mock-infected, clarified MA104 cell harvests used at the same dilution as virus. After 1 h, the inoculum was removed and replaced by serum-free medium in the presence or absence of kinase inhibitors or DMSO at the indicated concentrations for the remainder of the infection. The DMSO level was maintained at 0.5% (vol/vol) in all media containing DMSO or inhibitors, irrespective of inhibitor concentration.

Virus growth assays.

Cells were infected with RRV at an MOI of 0.1 and treated with inhibitors as described above. At 17 h postinfection (PI), all virus produced was harvested by two cycles of freezing and thawing of the combined supernatant and cell fractions. To collect cell-associated virus, the culture medium was removed and the cells were washed before the addition of fresh serum-free medium and the freeze-thawing process. Infectious virus titers were determined by indirect immunofluorescent staining of MA104 cells inoculated with serial dilutions, as described previously (21).

Western blot analysis.

At various times PI, cells were lysed on ice for 10 min in 100 μl of 50 mM Tris-HCl buffer, pH 8.0, containing 1% (vol/vol) NP-40, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride (Sigma), aprotinin (10 μg/ml; Sigma), and leupeptin (5 μg/ml; Sigma). Lysates were vortex mixed thoroughly and centrifuged at 1,300 × g for 20 min at 4°C. An aliquot (15 μl) of lysate supernatant was mixed with 5 μl of sample buffer, consisting of 350 mM Tris-HCl, pH 6.8, 10.3% (wt/vol) sodium dodecyl sulfate (SDS), 36% (vol/vol) glycerol, 5% (vol/vol) β-mercaptoethanol (Sigma), and 0.06% (wt/vol) bromophenol blue. Samples were electrophoresed in a 12% (wt/vol) SDS-polyacrylamide gel at 100 V. Separated proteins were electrophoretically transferred for 1 h at 100 V to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). Membranes were blocked in 20 mM Tris-HCl buffer, pH 7.4, containing 37 mM NaCl (TBS), with 2% (wt/vol) bovine serum albumin (Sigma) and reacted overnight at 4°C with primary antibodies diluted 1:500 to 1:1,000 in TBS containing 0.1% (vol/vol) Tween 20 (MP Biomedicals, Irvine, CA) and 2% (vol/vol) bovine serum albumin. After being washed with TBS containing 0.1% (vol/vol) Tween 20, membranes were incubated with horseradish peroxidase-conjugated sheep anti-rabbit antibody diluted 1:4,000 (Chemicon, Temecula, CA). Bound antibodies were detected using enhanced chemiluminescence (Amersham). When reprobing was required, membranes were stripped in 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% (wt/vol) SDS and 100 mM β-mercaptoethanol for 45 min at 37°C.

Real-time PCR.

Confluent HT-29 cells in 96-well trays were mock infected or infected with purified RRV or I-RRV at an MOI of 10. Serum-free medium containing JNKi, p38i, or DMSO at the indicated concentrations, each with a final DMSO concentration of 0.5% (vol/vol), was added after removal of the virus inoculum. At 8 h PI, total cellular RNA was collected using the RNeasy mini kit (QIAGEN, Hilden, Germany) with the additional RNase-free DNase step, according to the manufacturer's instructions. Oligonucleotide primers were designed with the aid of Primer Express software (v1.5; Applied Biosystems, Foster City, CA). Primer sequences were as follows: IL-8 forward, 5′-TCTGCAGCTCTGTGTGAAGG-3′, and reverse, 5′-AGTGTGGTCCACTCTCAATC; c-jun forward, 5′-ACGGCCAACATGCTCAGG-3′, and reverse 5′-TGTTTGCAACTGCTGCGTTAG-3′; 18S rRNA forward, 5′-CGGCTACCACATCCAAGGAA-3′, and reverse 5′-GCTGGAATTACCGCGGCT-3′. Single-step reactions of 25 μl were performed in triplicate using Multiscribe reverse transcriptase, RNase inhibitor, and SYBR green PCR master mix (Applied Biosystems). Temperature cycling and detection were carried out using the ABI Prism 7700 sequence detector and data were analyzed with Sequence detector software (v1.9; Applied Biosystems). The specificity of amplified products was verified using dissociation curve analysis software (v1.0; Applied Biosystems). Relative gene expression was calculated using the comparative Ct (ΔΔCt) method, employing 18S rRNA as the reference. All calculated expression levels were within predetermined dynamic ranges for each RNA species.

Flow cytometric analysis of RRV-infected cells.

Caco-2 cell monolayers in 24-well trays were mock infected or infected with RRV at an MOI of 0.1 or 1.0 as described above. Following removal of inoculum, cells were incubated in the presence of serum-free medium containing DMSO alone, 20 μM JNKi, or 50 μM p38i. The DMSO level was maintained at 0.5% (vol/vol) for all samples irrespective of inhibitor presence. Levels of viral antigen and the proportion of cells infected were determined by flow cytometry using an adaptation of a previously reported method (16). In brief, cells were washed with PBS at 16 h PI and detached by incubation at 37°C for 5 min in PBS containing 0.1% (wt/vol) trypsin (DIFCO Laboratories, Detroit, MI). Detached cells were washed in medium containing 1% (vol/vol) fetal bovine serum and permeabilized by methanol fixation. Cell-associated rotavirus antigen was detected with rabbit polyclonal antibodies to RRV and secondary staining with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G (Chemicon). Cells were analyzed in a FACSCalibur instrument with CellQuest software (Becton Dickinson, Cockeysville, MD).


Activation of JNK and c-Jun by RRV and I-RRV in HT-29, Caco-2, and MA104 cells.

The activation states of JNK and c-Jun following rotavirus infection of permissive cell lines were examined by Western blotting using phospho-specific antibodies (Fig. (Fig.1).1). In HT-29 cells, activation of JNK was observed from 2 h PI with RRV (Fig. (Fig.1A).1A). JNK activation levels continued to increase to a peak at 8 h PI and remained high until 16 h PI. Total levels of JNK were similar at all times. JNK activation was not assessed after 16 h PI due to increasing viral cytopathic effects. Consistent with the activation of JNK, a concomitant increase in c-Jun phosphorylation (p-c-Jun) was observed, which peaked at 8 to 12 h PI and then declined by 16 h PI. Levels of β-actin, used as a loading control for p-c-Jun blots, remained constant. Interestingly, exposure of HT-29 cells to I-RRV also led to activation of JNK with a similar profile to that of replicating RRV, although activation appeared to be transient and was diminished at 12 h PI and 16 h PI. As for replicating RRV, JNK activation by I-RRV was reflected in a similar increase in p-c-Jun levels. No activation of JNK or c-Jun was detected in mock-infected HT-29 cells. Infection with RRV or I-RRV also induced activation of JNK and c-Jun in Caco-2 cells (Fig. (Fig.1B).1B). This was observed from 2 h PI, peaked at 12 to 24 h PI, and did not occur in mock-infected cells, although some variability was observed in the (low) level of p-c-Jun seen in mock-infected cells. In the MA104 kidney cell line, activation of JNK was also observed following RRV and I-RRV infection, although not until 6 h PI (Fig. (Fig.1C).1C). Some activation of lower-molecular-weight forms of JNK (p46) was observed in mock-infected MA104 cells at 2 to 8 h PI that may have been induced by removal of serum during the infection protocol. As certain conditions can induce a biphasic activation of JNK and p38 (43), time points early after RRV infection were also examined. No activation of JNK or p38 was observed between 5 and 60 min PI in each cell type, compared to mock-infected cells (Fig. (Fig.1D,1D, ,1E,1E, and and1F).1F). Inactivated virus was not tested at early time points, as it would be unlikely to elicit a response when live virus had no effect.

FIG. 1.
Kinetics of JNK and c-Jun activation following RRV infection. Lysates prepared from HT-29 (A and D), Caco-2 (B and E), and MA104 (C and F) cells that had been mock infected or infected with RRV or I-RRV at an MOI of 10 for the indicated times were analyzed ...

Activation of p38 by RRV in Caco-2 and MA104 cells.

The activation of the related MAPK p38 in response to rotavirus was examined (Fig. (Fig.2).2). In HT-29 cells, p38 was not activated by either RRV or I-RRV (Fig. (Fig.2A)2A) (data not shown). However, in Caco-2 cells, p38 was activated by RRV from 8 h PI and remained so until at least 24 h PI (Fig. (Fig.2B).2B). In MA104 cells, p38 was activated from 6 h PI by RRV, with levels peaking at 8 h PI and remaining high until at least 16 h PI (Fig. (Fig.2C).2C). In contrast to JNK, p38 showed little if any activation by nonreplicating I-RRV in Caco-2 cells (Fig. (Fig.2B)2B) and was not activated by I-RRV in MA104 cells (Fig. (Fig.2C).2C). Compared to mock-infected cells, no activation of p38 was observed between 5 and 60 min PI with RRV in each cell type (Fig. 2A, B, and C).

FIG. 2.
Kinetics of p38 activation following RRV infection. Lysates prepared from HT-29 (A), Caco-2 (B), and MA104 (C) cells that had been mock infected or infected with RRV or I-RRV at an MOI of 10 for the indicated times were analyzed by Western blotting using ...

Dependence of rotavirus-induced JNK and p38 activation on viral MOI.

The level of RRV required to induce activation of JNK and p38 in intestinal cell lines was examined (Fig. (Fig.3).3). In HT-29 cells, an MOI of 0.05 was insufficient to induce detectable JNK activation as assessed by Western blotting for p-JNK (Fig. (Fig.3A).3A). At an MOI of 0.5, a low level of JNK activation was observed. Strong activation of JNK was seen at an MOI of 5, with no further increase at an MOI of 20 or 50. In Caco-2 cells, a low level of JNK activation was observed at an MOI of 0.05, whereas an MOI of 0.5 to 50 led to induction of similar high levels of activated JNK (Fig. (Fig.3B).3B). In MA104 cells, JNK activation was not detectable at an RRV MOI of 0.05 or 0.5 (Fig. (Fig.3C).3C). However, a moderate level of JNK activation was observed in MA104 cells at an MOI of 5, and activation was further increased to a plateau at MOI of 20 and 50. In Caco-2 and MA104 cells, RRV MOI of 0.05 and 0.5, respectively, were required to induce detectable levels of p38 activation (Fig. 3B and C). Higher MOI of up to 50 produced a consistently high level of p38 activation in these two cell lines.

FIG. 3.
Effect of RRV MOI on JNK and p38 activation. Lysates prepared from HT-29 (A), Caco-2 (B), and MA104 (C) cells infected with RRV at the indicated MOI were analyzed at 8 h PI (HT-29 and MA104) and 16 h PI (Caco-2) by Western blotting using antibodies against ...

JNK and p38 activation by various rotavirus strains.

To determine if JNK activation and p38 activation are general properties of rotaviruses, Caco-2 and HT-29 cells were infected with rotaviruses isolated from several different species (Fig. (Fig.4).4). Monkey strains RRV and SA11 and porcine rotavirus CRW-8 induced strong activation of JNK at 8 h PI in HT-29 cells when compared with mock-infected cells, whereas no JNK activation was seen for human rotavirus Wa and the UK bovine rotavirus at this time (Fig. (Fig.4A).4A). At 16 h PI in HT-29 cells, Wa and UK rotavirus produced a low level of activation of lower-molecular-weight isoforms (p46) of JNK (Fig. (Fig.4B).4B). RRV, SA11, and CRW-8 also strongly induced JNK and p38 activation in Caco-2 cells at 16 h PI, as compared with mock-infected cells (Fig. (Fig.4A).4A). At this time, the UK strain induced moderate levels of p38 and JNK activation, while Wa induced a low level of activation of p38 and the lower-molecular-weight JNK isoforms. By 24 h PI in Caco-2 cells, both Wa and UK had induced higher levels of p38 and JNK activation (Fig. (Fig.4B4B).

FIG. 4.
JNK and p38 activation by multiple rotavirus strains. HT-29 and Caco-2 cells were infected with rotavirus strains RRV, SA11, CRW-8, Wa, and UK at an MOI of 10 or mock infected (M). (A) Cell lysates were prepared at 8 h PI (HT-29) or 16 h PI (Caco-2) and ...

Effect of JNK and p38 inhibition on rotavirus replication.

To assess if the activities of JNK and p38 are required for efficient virus propagation in cell culture, the effects of JNKi and p38i at nontoxic levels on RRV replication in HT-29, Caco-2, and MA104 cells were determined. As we found no evidence of activation of the MEK-ERK1/2 pathway during rotavirus infection, as assessed by ERK1/2 phosphorylation (data not shown), the effect of MEKi was also assessed as a negative control. First, the selected concentrations of JNKi were tested to show their effectiveness in inhibiting virus-induced JNK activity (Fig. (Fig.5A).5A). Western blotting for p-c-Jun demonstrated clearly that JNKi reduced c-Jun phosphorylation in RRV-infected HT-29, Caco-2, and MA104 cells. In infected HT-29 cells treated with 10 μM JNKi, a low level of p-c-Jun remained above that seen in mock-infected cells. In RRV-infected MA104 cells, JNKi partially (5 μM) and completely (20 μM) reduced p-c-Jun to levels seen in mock-infected cells. In RRV-infected Caco-2 cells, JNKi at 5 μM reduced p-c-Jun to a level similar to that seen in mock-infected cells, while JNKi at 20 μM reduced p-c-Jun to below background levels.

FIG. 5.
Effects of JNK and p38 inhibition on RRV replication. (A) HT-29, Caco-2, and MA104 cells were mock infected (M) or infected with RRV at an MOI of 10 in the presence of DMSO alone (−) or the JNK inhibitor SP600125 (JNKi) at the indicated concentrations. ...

RRV replication in HT-29 cells was not significantly altered by the presence of JNKi or p38i at 5 μM or 10 μM (Fig. (Fig.5B).5B). However, JNKi at 5 μM inhibited RRV production in Caco-2 and MA104 cells by 33% and 60%, respectively, compared with controls (Fig. 5C and D). At 20 μM, JNKi reduced RRV growth in Caco-2 and MA104 cells by 76% and 75%, respectively, showing a clear dose-dependent effect of JNK inhibition on RRV replication in these cell lines. Treatment with p38i at 20 μM and 50 μM also reduced RRV titers in Caco-2 cells by 51% and 79%, respectively (Fig. (Fig.5C).5C). At 5 μM and 20 μM, p38i did not affect RRV production in MA104 cells (Fig. (Fig.5D).5D). MEKi had no effect on RRV replication in any of the cell lines tested (Fig. 5B, C, and D). In summary, JNKi treatment produced a dose-dependent inhibition of RRV replication in Caco-2 and MA104 cells, while p38i treatment produced a dose-dependent inhibition in Caco-2 cells.

Effect of JNK and p38 inhibition on expression of RRV structural proteins and cell-associated RRV titers.

To help delineate the effect of JNK and p38 inhibition on virus replication, the expression of antigenic RRV structural proteins in Caco-2 cells infected with RRV in the presence or absence of inhibitors was analyzed by flow cytometry (Fig. (Fig.6).6). At an MOI of 0.1 a large shift in fluorescence intensity was observed in the untreated infected cell population, creating a second peak compared to uninfected cells (Fig. (Fig.6A).6A). The histogram plots from cells infected at a MOI of 0.1 in the presence of inhibitors were indistinguishable from those from untreated cells, demonstrating no change in RRV structural antigen expression. At an MOI of 0.1 and in the absence of inhibitors, 63.1% ± 0.1% of the cell population was positive for viral antigen. In the presence of JNKi and p38i, the proportions of infected cells remained unchanged, at 67.7% ± 3.7% and 62.3% ± 4.1%, respectively. Similarly, at an MOI of 1, no difference was observed between the histogram plots for inhibitor-treated and untreated cells (Fig. (Fig.6B).6B). At this MOI, the proportion of the cell population positive for viral antigen remained at 85%, regardless of inhibitor treatment. Similar results were obtained for RRV infection of MA104 cells (data not shown). The cell-associated virus fraction also was analyzed to determine whether titers of infectious RRV in Caco-2 cells were affected by inhibitors. In the presence of 20 μM JNKi and 50 μM p38i, the infectious virus titer in the cell-associated fraction was reduced by a mean ± standard deviation of 70% ± 1.9% and 58% ± 1.5%, respectively. These results indicate that RRV cell entry and production of structural viral proteins in Caco-2 and MA104 cells were not affected by inhibition of JNK and p38, despite reduced titers of cell-associated infectious virus.

FIG. 6.
Effects of JNK and p38 inhibition on RRV structural antigen expression. Caco-2 cells were mock infected or infected with RRV at an MOI of 0.1 (A) or 1.0 (B) in the presence of DMSO, the JNK inhibitor SP600125 (JNKi; 20 μM) or the p38 inhibitor ...

Effect of JNK and p38 inhibition on IL-8 and c-jun mRNA induction by RRV.

JNK and p38 inhibitors at nontoxic concentrations did not inhibit RRV replication in HT-29 cells. Therefore, the effects of these inhibitors on the RRV-induced transcription of genes that are influenced by AP-1 could be determined in HT-29 cells. Quantitation of IL-8 and c-jun mRNA expression was performed by real-time PCR on mRNA extracted from RRV-, I-RRV-, or mock-infected HT-29 cells that had been treated with JNKi, p38i, or, as a control, DMSO alone (Fig. (Fig.7).7). An increase in IL-8 mRNA expression of 120-fold following RRV infection was observed at 8 h PI, compared with that in mock-infected cells (Fig. (Fig.7A).7A). In the presence of JNKi, IL-8 mRNA levels following RRV infection were reduced by 43%, demonstrating a significant role for JNK activation in RRV-induced IL-8 mRNA expression. Interestingly, although p38 was not activated during RRV infection of HT-29 cells, p38i reduced IL-8 mRNA expression by 86% compared to that in the control. This suggests that basal p38 activity may be important for optimal IL-8 mRNA induction by RRV. Nonreplicating I-RRV exposure increased IL-8 gene transcription by 23-fold over that in mock-treated cells (Fig. (Fig.7B).7B). Thus, I-RRV increased IL-8 transcription to a lesser extent than did replicating virus. IL-8 mRNA induction by I-RRV was reduced in the presence of JNKi or p38i by 38% and 72%, respectively. As shown in Fig. Fig.7C,7C, RRV infection also resulted in up-regulation of c-jun mRNA levels by 3.3-fold over mock-infected cells. This increase in c-jun mRNA transcription was reduced by 92% by JNKi and 93% by p38i. Thus, as found for IL-8 mRNA induction, basal levels of p38 activity may be required for maximal induction of c-jun mRNA. IL-8 and c-jun mRNA were also induced by RRV infection of Caco-2 cells. Their levels increased by 9- and 10-fold, respectively (data not shown). This is consistent with previous studies showing much lower induction of IL-8 in Caco-2 cells than in HT-29 cells (5, 46). JNKi and p38i were not used to define the role of JNK and p38 in Caco-2 cell gene expression during RRV infection as their inhibition of RRV replication in this cell line would confound this analysis. Overall, these studies show the importance of RRV-induced JNK activation and basal levels of p38 activity for IL-8 and c-jun gene regulation in HT-29 cells.

FIG. 7.
Role of JNK and p38 in RRV-induced gene expression. HT-29 cells were mock infected or infected with RRV or I-RRV at an MOI of 10 for 8 h in the presence of DMSO alone (−), SP600125 (JNKi), or SB203580 (p38i). Cellular RNA was extracted and analyzed ...


The transcription factor AP-1 plays an important role in cellular gene expression induced by rotavirus. AP-1 binding to the IL-8 promoter is increased following rotavirus infection, and AP-1 binding sites within the IL-8 promoter are required for optimal expression of this gene (6). Additionally c-Jun, an AP-1 component that is also transcriptionally regulated by AP-1, is induced in Caco-2 cells as a result of rotavirus infection (6, 12). We therefore investigated if cellular kinases known to be involved in AP-1 activation were themselves activated during rotavirus infection. Our data clearly indicate that JNK and p38 are activated by infection with multiple rotavirus strains and can influence both RRV-induced mRNA expression and RRV replication.

Several viruses and bacteria are known to activate JNK and p38, and the subsequent effects on cellular processes have been investigated. Reovirus activation of JNK leads to apoptosis in infected cells through a c-Jun-independent mitochondrial pathway (8). As it has been reported that intestinal cell lines show evidence of apoptosis following rotavirus infection, the role of JNK activation in this process may warrant further investigation (7, 48). Activation of JNK and p38 by influenza virus infection of lung epithelial cells has been linked to induction of the proinflammatory cytokines TNF-α and RANTES (30, 35). JNK and p38 activation by bacterial pathogens has also been linked to intestinal inflammation. Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli O157:H7 can cause acute intestinal inflammation and activate multiple MAPK, including JNK and p38, which play a role in IL-8 induction by these pathogens (13, 23). These studies highlight the potential importance of JNK and p38 activation in inflammatory responses to epithelial cell infection. It is therefore possible that JNK and p38 activation may play a role in rotavirus-induced diarrhea, either by directly affecting intestinal cell function or by promoting an inflammatory response through cytokine induction.

As JNK activation was induced by inactivated virus, and activation was not seen earlier than 2 h PI, the mechanism leading to JNK activation does not appear to involve viral replication or engagement of cellular receptors. One potential explanation is that JNK activation is stimulated by viral dsRNA or virion proteins released within the cell following entry. The mechanism of activation of p38 may differ from that of JNK, as only replicating virus could activate p38, and this activation was seen later than that of JNK. Under many conditions, both JNK and p38 are activated simultaneously as they share common upstream activators. However, there are numerous activators with various specificities for JNK and p38 that can cause differential activation of these proteins (33). For example, p38 but not JNK is activated during ischemia of rat heart, whereas during subsequent reperfusion JNK becomes activated while p38 remains active (4).

JNK and p38 activation was observed following infection with each of the rotavirus strains tested in our study. This suggests that the ability to activate JNK and p38 is a property common to most, if not all, rotaviruses. However, human rotavirus strain Wa and bovine strain UK induced delayed activation of JNK and p38, implying some differences exist between strains in their ability to induce activation of these kinases. Our study also shows that the levels of rotavirus-induced activation of JNK and p38 plateau above a certain MOI. This probably relates to the observation that the number of cells infected also plateaus with increasing MOI (P. Halasz and B. S. Coulson, unpublished data). It is likely that JNK and p38 activation becomes saturated in each cell and cannot be increased by exposure to more virus. We showed that a level of 0.05 to 5 infectious particles per cell was required to induce activation of JNK and p38. This suggests that once an individual cell is infected, these kinases are activated within that cell.

Cellular expression of rotavirus spike protein VP4 or VP4 subunit VP8* by cDNA transfection leads to NF-κB activation. TRAF binding domains within VP8* are required for this effect, and dominant-negative TRAF expression can reduce rotavirus-induced NF-κB activation (34). As TRAF signaling can also cause JNK activation, an effect not observed following VP8* expression, it has been suggested that VP8* selectively bypasses or down-regulates TRAF-directed JNK activation (34). Based on these findings, the JNK activation observed during rotavirus infection in our study is unlikely to involve TRAF protein signaling.

A substantial decrease in viral replication was evident in Caco-2 cells following inhibition of JNK or p38. This effect was not observed in HT-29 cells in which inhibition, at least of JNK activity, was incomplete due to the use of lower concentrations of inhibitors in order to avoid cellular toxicity. A marked decrease in RRV replication in MA104 cells was also observed following inhibition of JNK but not p38. These data show that optimal RRV replication requires JNK and p38 activity, at least in certain cell types, and raise the possibility that rotaviruses have acquired the ability to activate these kinases to aid their replication. A decrease in rotavirus replication following p38 inhibition has been reported previously (42). JNK and p38 activities have been shown to be required for the optimal replication of other viruses. For example, JNK and p38 activation by herpes simplex virus requires expression of particular viral proteins, and blockade of JNK translocation to the nucleus resulted in decreased virus production (14, 19, 38). Coxsackievirus B3 also activates both JNK and p38, which appears to aid virion release (47). Encephalomyocarditis virus activates p38 and requires p38 activity for efficient translation of viral proteins (22). It is unclear if JNK and p38 play a direct role in rotavirus replication or whether downstream targets or gene products under the control of AP-1 promoters are involved.

In RRV-infected Caco-2 cells, JNK and p38 inhibition did not cause a decrease in structural viral antigen expression or the proportion of infected cells, despite reduced yields of infectious virus. As reduced titers of infectious virus were produced within cells following inhibitor treatment, our results suggest a block to late stages of RRV replication and not a generalized block to virus transcription, translation, or virion release. Stages of replication that might be affected include viroplasm formation, assembly of viral cores, and budding into the endoplasmic reticulum. However, we cannot formally rule out a block to production of viral nonstructural proteins, a less abundant viral structural protein, or dsRNA. Further experimental work is required to elucidate the specific mechanism of the negative effect of JNK and p38 inhibition on RRV replication.

The induction of IL-8 expression by rotavirus is a well-established phenomenon. Our data show for the first time, in a quantitative assay, the extent of IL-8 mRNA induction by RRV in HT-29 cells. The 120-fold increase in IL-8 mRNA we demonstrated is consistent with observations of a 12-fold to 114-fold increase in IL-8 protein secreted from rotavirus-infected cells in previous studies (5, 41, 46). The IL-8 gene promoter contains binding sequences for NF-κB and AP-1, and mutation or deletion of either of these sites affects levels of transcription induced by rotavirus in HT-29 cells (6). Other studies have shown that both JNK and p38 are involved in the regulation of the IL-8 gene in response to various stimuli (reviewed in reference 23). Our novel demonstration that inhibition of p38 and JNK can reduce IL-8 mRNA induction following RRV infection is consistent with these previous findings. In our assays, IL-8 mRNA induction was reduced by 43% after inhibition of JNK. Due to toxicity, the levels of JNK inhibitor used did not completely block JNK activity, so a further decrease in IL-8 transcription might be achieved with increased JNK inhibition. It is likely that JNK activation plays a partial role in IL-8 transcription in response to rotavirus infection and that the IL-8 gene is regulated during infection by the combined effects of AP-1 and NF-κB activation (6, 24, 34, 41). The major AP-1 components involved in IL-8 promoter binding following rotavirus infection appear to be JunD and c-Fos, with c-Jun and the Fos protein subfamily member Fra-2 playing lesser roles (6). Therefore, the role of JNK in rotavirus-induced IL-8 transcription is likely to be mediated by the direct phosphorylation of JunD and c-Jun. The mechanism of activation of c-Fos and Fra-2 following rotavirus infection is as yet unknown.

As p38 is not activated by rotavirus in HT-29 cells, the increase in IL-8 and c-jun mRNA expression induced by rotavirus cannot be attributed to AP-1 activation by p38. This is plausible as AP-1 represents multiple different homodimeric and heterodimeric complexes, some of which may not be required for increased mRNA expression from a particular gene. Indeed, our findings suggest that activation of JNK is sufficient for IL-8 and c-jun promoter-specific AP-1 activation in HT-29 cells. However, p38 inhibition led to a large decrease in the levels of IL-8 mRNA seen during RRV infection in HT-29 cells. This suggests either a substantial role for basal levels of p38 activity in IL-8 transcription or that transcriptional enhancement by JNK and NF-κB is somewhat dependent upon p38 activity. Interestingly, p38 is known to stabilize IL-8 mRNA. This represents a possible posttranscriptional mechanism by which IL-8 mRNA levels may be elevated during rotavirus infection (25, 50). The p38 activation that we observed in rotavirus-infected Caco-2 and MA104 cells suggests that p38 may more strongly influence AP-1-directed transcription, through ATF2 activation, in these cell types as compared to HT-29 cells.

We observed a 3.3-fold increase in c-jun mRNA levels following RRV infection of HT-29 cells. This is in agreement with Cuadras et al., who judged a 2-fold increase in mRNA as significant in their microarray analysis and found a 4.9-fold increase in c-jun mRNA levels at 12 h PI of Caco-2 cells (12). The c-jun promoter includes two binding sequences for AP-1. Previous studies suggest that the upstream site binds heterodimers of c-Jun and ATF2, whereas the other site preferentially binds heterodimers of Fos and Jun families (44, 49). In our experiments, inhibition of JNK or p38 reduced RRV-induced c-jun expression by greater than 90%. This suggests that JNK and p38 act in a mutually dependent manner to achieve optimal RRV-induced expression of c-jun, and it appears likely that both AP-1 sites are involved. As c-jun is transcriptionally up-regulated during RRV infection, at least part of the increased phosphorylation of c-Jun seen in the present study is probably due to increased levels of cellular c-Jun. Given our data, it seems reasonable to propose that a number of the many cellular genes regulated in response to rotavirus infection are influenced by the activation of JNK and p38 and subsequent activation of AP-1 (12).

The activation of JNK and p38 during rotavirus infection and their involvement in AP-1-directed transcription are important new findings that advance our understanding of the cellular processes induced by rotavirus. The potential importance of JNK and p38 activation in rotavirus pathogenesis and their role in replication make the JNK and p38 signaling pathways possible new targets for antirotaviral therapies.


We are grateful to C. Kirkwood for HT-29 cells. We thank Peter Halasz, Kate Graham, Fiona Fleming, Jennifer Koplin, and Natalie Sanders for technical advice and reagents.

This work was supported by fellowship grants 299862 and 350253 and project grants 208900 and 350252 from the National Health and Medical Research Council of Australia (NHMRC). B.S.C. is a Senior Research Fellow of the NHMRC.


Published ahead of print on 23 August 2006.


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