VP4 Differentially Regulates TRAF2 Signaling, Disengaging JNK Activation while Directing NF-kappa B to Effect Rotavirus-specific Cellular Responses*

Rachel LaMonicaDagger §, Salih S. KocerDagger §, Jennet NazarovaDagger ||, William Dowling§**, Erika GeimonenDagger §, Robert D. ShawDagger , and Erich R. MackowDagger §||DaggerDagger

From the Dagger  Department of Medicine, § Department of Molecular Genetics and Microbiology, and || Molecular Cell Biology Program, State University of New York, Stony Brook, New York 11794,  Northport Veterans Affairs Medical Center, Northport, New York 11768, and ** Department of Retrovirology, Walter Reed Army Institute of Research, Rockville, Maryland 20850

Received for publication, January 18, 2001, and in revised form, March 21, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rotaviruses rapidly activate NF-kappa B and induce the secretion of selected chemokines after infection. The ability of rotavirus particles lacking genomic RNA to activate NF-kappa B suggested that rotavirus proteins direct cell signaling responses. We identified conserved TNFR-associated factor (TRAF) binding motifs within the rotavirus capsid protein VP4 and its N-terminal VP8* cleavage product. TRAFs (-1, -2, and -3) are bound by the rhesus rotavirus VP8* protein through three discrete TRAF binding domains. Expression of VP4 or VP8* from rhesus or human rotaviruses induced a 5-7-fold increase in NF-kappa B activity and synergistically enhanced TRAF2-mediated NF-kappa B activation. Mutagenesis of VP8* TRAF binding motifs abolished VP8* binding to TRAFs and the ability of the protein to activate NF-kappa B. Expression of pathway-specific dominant negative (DN) inhibitors DN-TRAF2 or DN-NF-kappa B-inducing kinase also abolished VP8*-, VP4-, or rotavirus-mediated NF-kappa B activation. These findings demonstrate that rotavirus primarily activates NF-kappa B through a TRAF2-NF-kappa B-inducing kinase signaling pathway and that VP4 and VP8* proteins direct pathway activation through interactions with cellular TRAFs. In contrast, transcriptional responses from AP-1 reporters were inhibited 5-fold by VP8* and were not activated by rotavirus infection, suggesting the differential regulation of TRAF2 signaling responses by VP8*. VP8* blocked JNK activation directed by TRAF2 or TRAF5 but had no effect on JNK activation directed by TRAF6 or MEKK1. This establishes that fully cytoplasmic rotaviruses selectively engage signaling pathways, which regulate cellular transcriptional responses. These findings also demonstrate that TRAF2 interactions can disengage JNK signaling from NF-kappa B activation and thereby provide a new means for TRAF2 interactions to determine pathway-specific responses.

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ABSTRACT
INTRODUCTION
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DISCUSSION
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NF-kappa B is a rapid transcriptional activator present in the cytoplasm of cells bound to the Ikappa B inhibitor protein (3, 4). Cytoplasmic signaling pathways which lead to the phosphorylation and proteosome degradation of Ikappa B, permit nuclear translocation and transcriptional activation of NF-kappa B (3, 4). Both NF-kappa B-inducing kinase (NIK)1 and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 (MEKK1) are discrete upstream effectors of the NF-kappa B activation pathway. NIK and MEKK1 phosphorylate Ikappa B kinases, which in turn phosphorylate Ikappa Bs (4, 5).

NIK-mediated NF-kappa B activation is stimulated by tumor necrosis factor receptor (TNFR) family-signaling pathways (4, 6, 7). TNFR-associated factors (TRAFs) are a family of adapter proteins that transduce TNFR ligand binding signals to cytoplasmic kinase cascades (6, 8, 9). The C-terminal domain of TRAF1, TRAF2, TRAF3, and TRAF5 proteins bind PXQX(T/S) sequences within the cytoplasmic tails of TNFRs, whereas TRAF6 binds to discrete TNFR sequences (9-17).

TRAF2, TRAF5, and TRAF6 are capable of activating NF-kappa B and Jun-N-terminal kinase (JNK) when overexpressed (6, 11, 18). TRAF2 binds mitogen-activated protein kinase kinase kinases, NIK, and apoptosis signal-regulating kinase (ASK1) (19-23). TRAF2 also interacts with germinal center kinase and activates germinal center kinase-related, which are upstream of MEKK1 in JNK activation pathways (24, 25). TRAF2 contains an N-terminal ring finger domain that is required to activate NIK, and as a result, TRAF2 plays a central role in receptor-mediated NF-kappa B and JNK activation (9). Deleting either the TRAF2 ring finger domain (DN-TRAF2) or functional kinase portions of NIK (DN-NIK) results in a dominant negative (DN) effect on TNFR-induced NF-kappa B activation (6, 26).

Rotaviruses are the single most important cause of severe dehydrating diarrhea in children under 2 years of age. Rotavirus infection causes the rapid activation of NF-kappa B and the induction of NF-kappa B-directed chemokines, including IL-8 (1, 2, 27). However, noninfectious, genetically inactivated rotavirus also activates NF-kappa B and induces IL-8 both transcriptionally and translationally when added to cells (1). Inoculation of cells with recombinant viral-like particles (VLPs), which lack viral RNA but contain rotavirus proteins VP2, VP4, VP6, and VP7, similarly activates NF-kappa B and directs IL-8 secretion (1, 2). However, rotavirus infection does not result in the production of TNFalpha or IL-1beta , which are themselves capable of activating NF-kappa B (1, 2). Collectively, these findings suggest that rotavirus proteins or the viral entry process itself activate NF-kappa B during rotavirus infection.

Rotaviruses are nonenveloped icosahedral viruses with 11-double-stranded (ds) RNA segments inside a 70-nm triple-layered particle (28). VP4 spikes project from the rotavirus surface, and trypsin cleavage of VP4 (86 kDa) into VP8* (28- kDa) and VP5* (60-kDa) fragments is required for viral infectivity (28). The VP5* fragment permeabilizes membranes, and proteolytically activated rotaviruses enter cells rapidly by a mechanism consistent with direct membrane penetration (29-33). Virion exposure to low intracellular calcium concentrations results in the uncoating of outer capsid proteins and leaves a transcriptionally active double-layered particle within the cytoplasm of the infected cell (34). Genomic dsRNAs remain within double-layered particles that contain a dsRNA-dependent RNA polymerase. Within the cell, double-layered particles synthesize and extrude viral mRNAs into the cytoplasm through pores in the double-layered particle capsid (35).

Rotaviruses infect intestinal epithelial cells (IECs) that line the lumen of the gut, have absorptive and barrier functions, and serve an immunologic role by eliciting primary cytokine and chemokine responses (36, 37). IEC barrier functions are regulated by TNF, and IECs secrete chemokines and respond to inflammatory cytokines by activating NF-kappa B (38-43). In the gut, TNFalpha activates NF-kappa B, whereas IL-10 suppresses TNFalpha -directed responses, and transcription of both TNFalpha and IL-10 are regulated by NF-kappa B and JNK activation (44-48). Although both TNFalpha and dsRNA can activate NF-kappa B, these elements do not appear to play a role in rotavirus-induced NF-kappa B activation, and the means by which rotavirus particles activate NF-kappa B but fail to induce TNFalpha has not been explained.

Here we report that the rotavirus capsid protein, VP4, and its VP8* cleavage product contain conserved PXQXT sequences, which are required for binding to cellular TRAFs and for NF-kappa B activation. Both rotavirus and VP8* selectively direct transcription from kappa B but not other (activator protein-1 [AP-1], cyclic AMP response element [CRE], serum response element [SRE]) DNA enhancer elements via a TRAF2-NIK-signaling pathway. Consistent with the ability of VP8* to block AP-1 transcription, VP8* inhibited JNK activation directed by TRAF2 and TRAF5 but not other JNK activators, suggesting that VP8* selectively regulates PXQXT-directed TRAF-signaling responses to activate NF-kappa B and inhibit JNK activation. These findings indicate that rotavirus proteins engage a central cytoplasmic-signaling pathway to selectively direct cellular transcriptional responses and suggest a means for the selective regulation of TRAF2-directed signaling.

    MATERIALS AND METHODS
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INTRODUCTION
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Reagents-- Isopropyl beta -D-thiogalactopyranoside and o-nitrophenyl-beta -D-galactopyranoside were from Lab Scientific Inc. (Livingston, NJ). Rabbit anti-TRAF1 (S19), anti-TRAF2 (C20), and anti-TRAF3 (C20) were from Santa Cruz Biotechnology. Rabbit anti-rhesus rotavirus serum was previously described (30, 49, 50). Glutathione-agarose and monoclonal anti-FLAG were from Sigma, and anti-Myc antibodies were from Upstate Biotechnology Inc. Protein G-agarose and Dulbecco's modified Eagle's medium were from Life Technologies, Inc. Luciferase reporter assay reagents were obtained from Promega.

Cells and Virus-- Rhesus rotavirus (RRV) was cultivated and titered in MA104 cells as previously described (55). Human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. HT-29 cells were grown as previously described on 50% Dulbecco's modified Eagle's medium and 50% Ham's F-12 medium (Life Technologies, Inc.) in 10% fetal calf serum (2). Media were endotoxin-free (<0.01 enzyme units/ml) as measured by the limulus amebocyte lysate test (Sigma). 293 cells were infected or mock-infected 24 h post-transfection with reporter and expression plasmids. Virus was adsorbed to cells at a multiplicity of infection of 5 for 1 h, and monolayers were washed with media and incubated at 37 °C in 5% CO2. Lysates were prepared as described below and at indicated times were assayed for luciferase and beta -galactosidase activity or used for co-immunoprecipitations.

Plasmids-- Expression plasmids for FLAG-TRAF1, FLAG-TRAF2, FLAG-TRAF3, DN-TRAF2 (87), and DN-NIK (624) in pRKF were previously described and provided by D. V. Goeddel (Tularik Inc.) (20). TRAF5 and TRAF6 in pcDNA3 were provided by J. C. Reed (Burnham Institute) (17). Luciferase reporter plasmids containing the indicated number of repeated enhancer sequences upstream of a luciferase gene and positive control MEKK1 and AP1 expression plasmids were obtained from Stratagene: pNF-kappa B-Luc (TGGGGACTTTCCGC)5, pAP1-Luc (TGACTAA)7, pCRE-Luc (AGCCTGACGTCA GAG)4, pSRE-Luc (AGGATGT CCATATTAGGACCTCT)5, pFC-MEKK, and pFC-AP1. pSV-beta gal expression plasmid was from Promega. pBIND, pACT, pBIND-Id, and pACT-MyoD mammalian 2-hybrid plasmids were from CLONTECH. Expression vectors for RRV VP8* and VP4 were constructed by polymerase chain reaction amplification using oligonucleotide pairs below and inserted directionally into pBIND or pEGFP-N (CLONTECH) at the BglII and EcoRI or BamHI and XbaI sites to generate Gal4- or FLAG-tagged proteins. Individual TRAF binding motif sequences within VP8* were amplified using oligos below and inserted into pGEX-KG (BamHI-EcoRI) to generate GST fusion proteins.

Specific primers for amplifying coding sequences of VP8* and VP4 were synthesized with 5'-BamHI, EcoRI, or XhoI sites and directionally cloned and sequenced. Mutations were made in VP8* by site-directed mutagenesis as previously described using complimentary primers to each PXQXT site (29). Oligonucleotide pairs are denoted by their nucleotide position in the RRV gene 4 sequence (30). VP8 contained amino acids 1-231 and was amplified by oligos containing bases 10-31 (5") and 682-702 (3'). RRV VP4 contained amino acids 1-776 and was amplified by oligos containing bases 10-31 (5') and 2315-2340 (3'). VP8*-TRAF binding domain 1 contained amino acids 1-83 and was amplified by oligos containing bases 10-31 (5') and 241-258 (3'). VP8*-TRAF binding domain 2 contained amino acids 146-184 and was amplified by oligos containing ATG and bases 458-477 (5') and 541-570 (3'). VP8*-TRAF binding domain 3 contained amino acids 203-231 and was amplified by oligos containing ATG and bases 619-639 (5') and 682-702 (3').

Cell Transfection-- Plasmid DNA was extracted by alkaline lysis and purified by CsCl density gradient centrifugation before transfection. The HEK 293 cell line was used for transfection experiments since intestinal cells are poorly transfected. Transient transfections for co-immunoprecipitation experiments, luciferase reporter assays, and Western blotting were performed using FuGene6 (Roche Molecular Biochemicals) or an enhanced Ca3(PO4)2 method as previously described (51). 293 cells (5 × 105) were plated in 6-well plates and at 80% confluence transfected for 4 h with a constant amount of total plasmid DNA in control and experimental wells. Media was replaced, and 24-36 h post-transfection, cells were washed with phosphate-buffered saline and lysed or infected as described above. Cells were lysed on ice for 30 min in luciferase reporter lysis buffer (Promega) or 0.1% Nonidet P-40 immunoprecipitation lysis buffer (52).

Immunoprecipitation and Immunoblots-- Cells were lysed as above, clarified by centrifugation, precleared with protein A-, protein G-, or glutathione-agarose and incubated with appropriate antibodies for 2 h or overnight at 4 °C. Protein A- or protein G-agarose was added and incubated for 1 h at 4 °C. Alternatively, lysates were incubated with GST fusion proteins bound to glutathione-agarose. Beads were washed 5 times with 0.1% Nonidet P-40 lysis buffer, and bound proteins were separated by SDS-polyacrylamide gel electrophoresis (10%) and transferred to nitrocellulose for Western blotting. Western blot analysis was performed as previously described (52, 53) with 1 µg/ml primary antibody and species-specific horseradish peroxidase-conjugated sera (Amersham Pharmacia Biotech) (1:2000 dilution in 1% bovine serum albumin, TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20). Reactive proteins were detected by chemiluminescence using the ECL reagent (Amersham Pharmacia Biotech).

GST Fusion Protein Expression and Binding Assays-- GST or GST fusion proteins were expressed in bacteria (BL21) from pGEX plasmids. Bacteria were grown to an A600 of 0.6 in LB and induced with 1 mM isopropyl-beta -D-galactopyranoside for 2 h at 30 °C. GST fusion proteins were purified on glutathione-agarose resin (Sigma) as previously described (31). Bound GST proteins were washed extensively in phosphate-buffered saline and eluted with 10 mM glutathione, and protein was quantitated by Bio-Rad protein assay. GST or GST fusion proteins (25 µg) bound to glutathione-agarose were used to precipitate proteins from cell lysates.

Reporter Assays-- Cells were transfected in duplicate or triplicate as above with constant amounts of plasmid DNAs including luciferase reporter plasmids, a beta -galactosidase expression plasmid (pSV-beta gal), and experimental and reporter plasmids. After lysis, luciferase reporter assays were performed in duplicate and read in a Turner Designs luminometer (TD-20/20). 10 µl of lysate was added to 50 µl of assay buffer (Promega) and quantitated for 15 s. Luciferase activity was normalized to relative amounts of beta -galactosidase activity present within each lysate as previously described (53). All experiments were repeated multiple times. Error bars represent the range of normalized luciferase responses among replicate samples, and representative experiments are presented.

Mammalian 2-Hybrid Analysis-- VP8* was cloned into pBIND, and TRAF2 was cloned into pACT mammalian 2-hybrid vectors fused to GAL4 or VP16 proteins, respectively. These plasmids as well as control pBIND and pACT or known interactive positive controls pBIND-Id and pACT-MyoD were transfected into 293 cells along with the GAL4-luciferase reporter plasmid, pG5luc, as indicated. pBIND-Id contains the inhibitor of DNA-binding protein (Id), and pACT-MyoD contains the muscle determination factor MyoD fused to binding and activation domain elements, respectively (54). Identical amounts of DNA were transfected into each well. Lysates were harvested 36 h post-transfection and quantitated as above.

JNK Activation Assays-- Two assays were used to monitor JNK activation. A phospho-JNK antibody Western blot assay was used to detect phosphorylated JNK from cells transfected with TRAF and VP8* plasmids. Briefly, an identical amount of plasmid DNA was transfected into 293 cells using a pNGFP plasmid control. Cell lysates were prepared, standardized by beta -galactosidase activity within lysates, and immunoprecipitated with anti-JNK1 antibody and subsequently assayed for JNK1 activation by Western blotting with an antibody specific for phosphorylated JNK1 protein. A radiolabeled c-Jun phosphorylation assay was used to assay the ability of VP8* to inhibit TRAF5-, TRAF6-, and MEKK1- directed JNK activation. 293 cells were transfected, and 36 h post-transfection lysates were standardized by beta -galactosidase activity and assayed for their ability to phosphorylate GST-c-Jun-(1-79) as previously described (Stratagene) (55).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VP8* Binding to Cellular TRAFs-- We previously demonstrated that RRV infection, inoculation of cells with genetically inactivated RRV or inoculation of VLPs that lack viral nucleic acids, induced IL-8 and activated NF-kappa B in HT-29 or 293 cells (1, 2). These responses required trypsin activation of rotavirus particles, suggesting that cell entry is required for virus- or VLP-mediated NF-kappa B activation. However, rotaviruses do not induce TNFalpha or IL-1beta responses, which are capable of activating NF-kappa B. The ability of inactivated virus or VLPs to activate NF-kappa B further suggested that viral proteins may mediate NF-kappa B activation after entry (1).

During an analysis of rotavirus capsid proteins we identified TRAF binding motifs within VP4 and its cleavage product VP8* that could specify NF-kappa B activation (Fig. 1). PXQX(T/S) sequences are present in the cytoplasmic tail of TNFRs and mediate binding to selective TRAFs (-1, -2, and -3). The PXQX(T/S) sequence within the TNFR2 cytoplasmic tail is required for TNF-directed NF-kappa B activation (9, 12, 13, 53, 56). PXQXT sequences are conserved in the VP8* proteins of 117 different Group A rotaviruses available within GenBankTM and absent from all other rotavirus proteins. Most rotaviruses (95) and nearly all human strains (65/66) contain 2 PXQX(T/S) sequences that are positionally conserved in VP8* at residues 69-73 and 225-229, respectively, of RRV (Fig. 1). RRV contains a third PXQX(T/S) TRAF binding motif within a hypervariable region of VP8*.


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Fig. 1.   Conservation of TRAF binding elements within rotavirus VP4 proteins. Rotavirus proteins within GenBankTM were analyzed for the presence of PXQX(T/S) TRAF binding elements. The rotavirus VP4 protein and its VP8* cleavage products were found to contain PXQXT motifs at conserved positions within the VP8* portion of VP4. 117 different Group A rotaviruses were found to contain PXQXT sequences within VP8*, but not other rotavirus proteins. All human rotaviruses contained at least one PXQXT, whereas nearly all (65/66) human rotaviruses contained two motifs coinciding with the first and third PXQXT sites within the RRV VP8* protein.

VP4 TRAF Binding Motifs Bind Expressed TRAFs-- PXQXT sequences in the cytoplasmic tails of TNFRs mediate interactions with selected TRAFs, and TRAFs engage signaling pathways that activate NF-kappa B. Biochemical and mammalian 2-hybrid analyses were used to define VP8* TRAF binding interactions. Cells were co-transfected with plasmids expressing Gal4-VP8* fusion proteins and TRAFs (-1, -2, or -3). VP8* was immunoprecipitated with anti-Gal4 antibody, complexes were separated by polyacrylamide gel electrophoresis, and co-precipitated TRAFs were detected by Western blot using TRAF-specific sera. Fig. 2A demonstrates that the rotavirus VP8* protein binds TRAF1, -2, and -3 from cell lysates. A mammalian 2-hybrid analysis was also used to assess VP8* binding to TRAF2 (Fig. 2B). When VP8*-pBIND and TRAF2-pACT were co-transfected into cells, we observed a specific 6-10-fold activation of the Gal4-luciferase reporter compared with individually transfected TRAF2-pACT or VP8*-pBIND or empty pBIND or pACT vectors. These findings indicate that VP8* specifically interacts with TRAF2 within cells (Fig. 2B).


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Fig. 2.   Expressed VP8* and GST-VP8* TRAF domains bind TRAFs. A, the pBIND-VP8* expression plasmid (4 µg, GAL4-VP8* fusion) was co-transfected into cells along with TRAF1, -2, or -3 (2 µg) (51). After anti-GAL4 immunoprecipitation of cell lysates, the binding of VP8* to TRAF1, -2, or -3 was detected by Western blot using TRAF-specific antibodies and compared with the total TRAF content of lysates (52, 53). B, mammalian 2-hybrid analysis. VP8*-pBIND, TRAF2-pACT, empty vectors, or a paired positive control pBIND-Id and pACT-MyoD (54) were transfected into 293 cells along with the GAL4-luciferase reporter plasmid, pG5luc, as indicated. Identical amounts of DNA were transfected into each well, and lysates were harvested 36 h post-transfection. Luciferase activity was quantitated relative to empty pBIND and pACT co-transfected cells. Experiments were performed in triplicate, and the results are representative of three separate experiments.

To determine if specific PXQXTs mediate VP8*-TRAF interactions, we investigated the ability of TRAFs to bind GST fusion proteins containing potential RRV VP8*-TRAF binding domains (PXQXTs; RRV BD1, -2, -3). Cells transfected with FLAG-tagged TRAF1, TRAF2, or TRAF3 proteins were lysed and reacted with GST or GST-VP8*-TRAF binding domain proteins. VP8* BD complexes were co-precipitated with glutathione-agarose, and bound TRAFs were detected by Western blotting. In contrast to GST controls, each GST-VP8* BD fusion protein was capable of binding expressed TRAFs (Fig. 3A).


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Fig. 3.   PXQXT motifs direct VP8* binding to TRAFs. A, GST fusion proteins containing each TRAF binding domain (PXQXT) of the RRV VP8* protein were used to precipitate expressed TRAFs. Residues contained within each binding domain of the RRV VP4 protein are as follows: BD-1, amino acids 1-83; BD-2, amino acids 146-184; BD-3, amino acids 203-231. Cells were transfected with 2 µg of TRAF1, -2, or -3 plasmids, and TRAFs bound by GST (25 µg) or equivalent amounts of GST binding domain fusion proteins were evaluated after Western blot analysis of FLAG-tagged TRAFs (53). B, the PXQXT motifs within each binding domain GST fusion protein was mutated (mut) and evaluated for its ability to co-precipitate TRAF3. The Pro and Gln residues of BD1 and BD3 were mutated to alanines, whereas Gln and Thr residues were mutated within BD2. TRAF3 binding was assessed as in A using identical amounts of GST BD fusion proteins.

To determine whether PXQXT motifs directed VP8* binding to TRAFs, each binding domain was mutagenized, and binding to TRAFs was reassessed. Fig. 3B demonstrates that mutagenized VP8* BDs were incapable of binding TRAFs and that PXQXT elements were required for VP8*-TRAF interactions.

VP8*/VP4-TRAF Interactions Mediate NF-kappa B Activation-- To determine if expressed VP8* and VP4 activate NF-kappa B, we co-transfected 293 cells with an equivalent amount of empty control plasmid or plasmids expressing VP8* or VP4. An NF-kappa B-luciferase reporter plasmid and a beta -galactosidase expression plasmid (SV40 promoter) were co-transfected into cells to monitor and standardize NF-kappa B activation. Transfecting increasing amounts of VP8* plasmid resulted in an increase in NF-kappa B activation with a 6-7-fold increase in NF-kappa B activation (2 µg) compared with control plasmid-transfected cells (Fig. 4A). Similarly, NF-kappa B activity was increased 5-6-fold in VP4-transfected cells and by the human KU rotavirus VP8* protein (5-fold induction; not shown). These findings demonstrate that rotavirus VP8* and VP4 proteins are capable of activating NF-kappa B in the absence of other viral proteins or RNA and in the presence of endogenous levels of cellular TRAFs.


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Fig. 4.   VP8* and VP4 activate NF-kappa B and synergizes TRAF2-mediated NF-kappa B activation. 293 cells were transfected with the indicated amounts of pEGFPVP4 or pEGFPVP8* expression vectors, an NF-kappa B-luciferase reporter plasmid (0.5 µg), and an SV40 promoter-driven beta -galactosidase expression plasmid (0.5 µg) in the absence (A) or presence (B) of co-transfected TRAF2 (51). NF-kappa B activation from the luciferase reporter was measured in triplicate and standardized to beta -galactosidase activity of lysates (53). Error bars represent the range of beta -galactosidase-adjusted luciferase reporter responses. Fold activation was determined relative to a reporter construct co-transfected with an identical amount of an pEGFP-N plasmid control. All wells were transfected with identical amounts of plasmid DNA. Findings were reproduced in at least eight separate experiments, and a representative experiment is presented.

Synergistic Enhancement of TRAF2-mediated NF-kappa B Activation by VP8*-- TRAF2 itself activates NF-kappa B when overexpressed. To determine if VP4 synergizes NF-kappa B activation directed by TRAF2, we co-transfected a constant amount of TRAF2 into cells along with increasing amounts of VP8*, VP4, or control plasmid. When TRAF2 and VP8* were co-expressed in cells we observed a synergistic 4-fold activation of NF-kappa B over cells transfected with TRAF2 and a control plasmid (Fig. 4B). Similar results were obtained with VP4. These findings indicate that VP8* enhances TRAF2-directed NF-kappa B activation and functionally links VP8*-TRAF2 interactions to cellular transcriptional responses.

NF-kappa B Activation by VP8*-TRAF Binding Domain Mutants-- The RRV VP8* protein contains 3 PXQXT motifs that could mediate TRAF binding and direct NF-kappa B activation. We mutated each binding domain sequentially within the full-length VP8* protein by site-directed mutagenesis and evaluated the ability of the triple mutant or VP8*s with only one PXQXT binding domain to activate NF-kappa B. Fig. 5 shows that mutating all three PXQXT motifs within VP8* abolishes the ability of the protein to activate NF-kappa B. Similar results were obtained with VP8* constructs containing only binding domains 2 (BD2) or 3 (BD3). In contrast, VP8* containing only binding domain 1 (BD1) retained its ability to activate an NF-kappa B-luciferase reporter, and NF-kappa B activity was close to that of the intact VP8*. These findings suggest that the first binding domain within the RRV VP8* protein is primarily responsible for NF-kappa B activation.


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Fig. 5.   TRAF binding domain 1 of the RRV VP8* is required for NF-kappa B activation. TRAF binding domains within the full-length RRV VP8* protein were selectively mutagenized to generate double and triple PXQXT mutants. The ability of full-length VP8* constructs containing only one TRAF binding domain (BD1, BD2, or BD3) or VP8* in which all three TRAF BDs have been mutagenized (BD-neg) were evaluated for their ability to activate NF-kappa B. BD1, BD2, and BD3 constructs contain full-length VP8* proteins with only the first, second, or third TRAF binding domains, respectively. Cells were transfected and evaluated as in Fig. 4 and standardized to beta -galactosidase activity within each lysate. Findings were reproduced in at least two separate experiments, and a representative experiment is presented.

VP8* and Rotavirus Selectively Direct Transcription from kappa B Sites-- TRAF2 also directs transcription from AP-1 enhancer binding sites. To determine if VP8* and rotavirus direct additional cellular transcriptional responses, we transfected cells with a series of enhancer-directed luciferase reporters to assay cellular responses to expressed VP8* or RRV infection. Both RRV infection and VP8* expression directed transcription from kappa B reporters but not from reporters directed by AP1, CRE, or SRE enhancer elements (Fig. 6). VP8* expression reproducibly inhibited AP-1 transcriptional responses ~5-fold below basal levels. Although RRV infection did not reduce AP-1 transcription in this assay, infection did not activate the AP-1 reporter. These findings indicate that both rotavirus and the VP8* protein direct pathway-specific transcriptional responses. Even though TRAF2 normally activates both NF-kappa B and JNK-directed AP-1 transcription, VP8*-TRAF2 interactions did not direct AP-1 transcription.


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Fig. 6.   VP8* and rotavirus selectively activates NF-kappa B transcription. We expressed VP8 in the presence of pAP1-Luc, pCRE-Luc, or pSRE-Luc reporter plasmids, and assayed luciferase activity as above. VP8* (1 µg) was co-transfected into 293 cells along with AP-1, CRE, SRE, or NF-kappa B luciferase reporter plasmids (0.5 µg) and a constant amount of plasmid DNA (Fig. 6). Fold activation was determined relative to reporter construct alone and equivalent µg amounts of co-transfected pEGFP-N control plasmid in triplicate. MEKK1 (1 µg) activated the AP-1 luciferase reporter 27-fold over controls in this assay. For RRV activation assays, cells were infected or mock-infected with RRV 36 h post-transfection at a multiplicity of infection of 10, and lysates were assayed for luciferase activity 20 h post-infection. MEKK1 activated luciferase reporter activity 15-27-fold with each reporter construct. These experiments were repeated three times with similar results.

DN-TRAF2 and DN-NIK Block Rotavirus and VP8*-mediated NF-kappa B Activation-- To determine whether VP8 specifically engages a TRAF2-NIK-signaling pathway of NF-kappa B activation, we tested the ability of pathway-specific dominant negative inhibitors DN-TRAF2 and DN-NIK to block VP8-induced NF-kappa B activation. The expression of DN-TRAF2 or DN-NIK dramatically reduced or abolished VP8*- or VP4-mediated NF-kappa B activation (Fig. 7A). This demonstrates that VP8* and VP4 proteins direct NF-kappa B activation by engaging a TRAF2-NIK-signaling pathway.


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Fig. 7.   DN-TRAF2 and DN-NIK block VP8*- and rotavirus-directed NF-kappa B activation. A, indicated amounts (µg) of VP8* and dominant negative TRAF2 (DN-TRAF2) or NIK (DN-NIK) expression plasmids were co-transfected into 293 cells along with an NF-kappa B-luciferase reporter and pSVbeta -gal plasmids (0.5 µg) as in Fig. 3. An identical amount of plasmid DNA was transfected into each well using an pEGFP-N plasmid control. Fold activation was determined relative to reporter construct alone and equivalent µg amounts of co-transfected pEGFP-N control plasmid with or without DN-TRAF2 or DN-NIK. This experiment was repeated more than six times with similar results. B, 293 cells were transfected with the indicated amounts of expression vectors and an NF-kappa B-luciferase reporter plasmid (0.5 µg) as in Fig. 3. 24 h post-transfection, cells were infected or mock-infected with RRV at a multiplicity of infection of 10, and lysates were assayed for luciferase activity at 2 and 20 h post-infection. Fold activation was determined relative to mock-infected cells transfected with the luciferase reporter with or without DN-TRAF2 or DN-NIK and equivalent µg amounts of co-transfected pEGFP-N control plasmid. No detectable differences in beta -galactosidase activity were observed between infected or uninfected cells. Experiments were performed in triplicate, and the results are representative of two separate experiments.

We have previously demonstrated that rotavirus infection causes the rapid nuclear translocation of NF-kappa B in cells by EMSA assay and that inactivated rotavirus or rotavirus VLPs activate NF-kappa B (1). To determine whether rotavirus also activates NF-kappa B through the TRAF2-NIK pathway engaged by VP8*, we tested whether DN-TRAF2 or DN-NIK proteins are capable of blocking rotavirus-induced NF-kappa B activation. Cells were co-transfected as above with the pNF-kappa B-Luc reporter in addition to DN-TRAF2 or DN-NIK plasmids. Twenty-four hours post-transfection, cells were infected at a multiplicity of infection of 10 with RRV, and NF-kappa B-directed luciferase activity was assayed either 2 or 20 h post-infection. Rotavirus infection activated NF-kappa B (2.5-5-fold); however, cells transfected with DN-TRAF2 or DN-NIK abolished RRV-mediated NF-kappa B activation at either time point (Fig. 7B). Similar to VP8*, these findings indicate that rotavirus also engages a TRAF2-NIK pathway of NF-kappa B activation and that additional NF-kappa B activation pathways do not appear to be activated during rotavirus infection.

VP8* Expression Selectively Inhibits TRAF2- and TRAF5-directed JNK Activation-- In addition to NF-kappa B activation, TRAF2 also activates JNK and downstream AP-1 transcription. However, transcriptional activation of AP-1 was reproducibly depressed 5-fold by co-expression of VP8*. To determine if VP8* blocked JNK activation, we assessed the ability of cotransfected VP8* to block JNK activation directed by TRAF2, TRAF5, TRAF6, and MEKK1. Using a phospho-JNK-specific antibody, Fig. 8A demonstrates that increasing amounts of expressed VP8* blocked TRAF2-directed JNK activation. Fig. 8B demonstrates that transfection of increasing amounts of VP8* plasmid resulted in increased VP8* expression. Using a c-Jun phosphorylation assay, TRAF5-directed JNK activation was also inhibited by VP8* co-expression (Fig. 8C). In contrast, TRAF6- and MEKK1-directed JNK activation were unaffected by VP8* co-expression. Similar to TRAF2, TRAF5 binds PXQXT motifs in CD40, which are present in VP8*, whereas MEKK1 and TRAF6 activate JNK independent of PXQXT binding interactions. These findings demonstrate the selective inhibition of TRAF2- and TRAF5-directed JNK activation by VP8* and are consistent with a requirement for VP8* PXQXT-TRAF binding interactions in selectively directing NF-kappa B activation and blocking JNK activation.


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Fig. 8.   VP8* expression selectively inhibits JNK activation. A, cells were transfected with TRAF2, control, or VP8* plasmids as indicated. An identical amount of plasmid DNA was transfected into each well using a pNGFP plasmid control. Cell lysates were prepared, standardized by beta -galalactosidase activity, and immunoprecipitated with anti-JNK1 antibody. Blots were assayed for JNK activation with an antibody specific for phosphorylated JNK1 protein. Densitometry of bands and comparison relative to pNGFP-transfected controls are graphically presented above the corresponding phospho-JNK-1 blot. This experiment was repeated twice with similar results. B, to demonstrate that VP8* expression increased with increasing plasmid transfection, 293 or HT-29 cells were transfected with the indicated amounts of VP8* expression vector. VP8* was immunoprecipitated (IP) using a rabbit anti-RRV hyperimmune sera and Western-blotted with anti-FLAG (M2) monoclonal antibody. C, TRAF5, TRAF6, and MEKK1 (4 µg) were transfected into cells with or without co-transfected VP8* (4 µg) and, 36 h post-transfection, assayed for JNK activity using a c-Jun phosphorylation assay as previously described (55). The ability of lysates (standardized by equivalent amounts of beta -galactosidase) to radiolabel c-Jun-1-79 was analyzed by SDS-polyacrylamide gel electrophoresis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rotaviruses infect IECs of the proximal small intestine and effect diarrheal responses in infected individuals. IECs perform both barrier and immune functions and have the ability to respond to and secrete chemokines and cytokines (36, 39, 41, 42, 57). Although rotaviruses acutely infect IECs, infected cells are still viable for 36-48 h post-infection, providing a broad time frame for rotaviruses to regulate and direct cellular responses (58, 59). As a result, the ability of rotaviruses to direct cellular responses is central to our understanding of viral pathogenesis as well as to our understanding of host immunity to natural and vaccine-derived rotavirus infections. Studies presented here provide the first demonstration that rotavirus proteins directly engage intracellular-signaling pathways to effect cellular transcriptional responses.

Human intestinal cells reportedly specify chemokine responses through cytoplasmic-signaling pathways and require TRAF2-directed NF-kappa B activation to elicit IL-8 responses (36, 41, 42, 57). Rotavirus activation of NF-kappa B and induction of IL-8 from IECs requires proteolytic cleavage of VP4 into VP8* and VP5*, consistent with requirements for viral entry into cells (1, 2, 28, 32). Here we report that signaling elements within the rotavirus VP4 protein bind cellular TRAFs and activate NF-kappa B. The ability of the KU human rotavirus VP8* protein to activate NF-kappa B further demonstrates that VP4 functions similarly in both human and animal rotaviruses. DN-TRAF2 or DN-NIK proteins abrogated VP8*-, VP4-, and rotavirus infection-directed NF-kappa B activation, demonstrating that the rotavirus protein activates NF-kappa B by engaging the TRAF2-NIK-signaling pathway and that VP4-signaling interactions may be the primary means for rotavirus-mediated NF-kappa B activation. These findings suggest that virally introduced VP8* as well as newly synthesized VP4 proteins are capable of engaging intracellular-signaling pathways and directing specific transcriptional responses after rotavirus infection.

The TNF family of receptors (TNFR2, TNFR1, LTB-R, CD30, LMP1, CD40) activate NF-kappa B by engaging the TRAF2-NIK-signaling pathway through direct or indirect interactions of their cytoplasmic tails (6, 8, 53, 60). The rotavirus VP8* protein mimics TNFR2 interactions with TRAF1, 2-, and -3 and, similarly, effects NF-kappa B activation. Individual PXQXT motifs within VP8* specify TRAF binding interactions, and since most rotaviruses contain two conserved PXQXT motifs within VP8* (the first and third of RRV), multiple TRAF BDs may be functionally important to the rotavirus VP4 protein. The ability of VP8* to bind multiple TRAFs further suggests that VP8* and VP4 may serve as scaffolding proteins for the recruitment and assembly of rotavirus-specific signaling complexes. However, NF-kappa B activation studies indicate that the first RRV VP8* binding domain appears to be primarily responsible for TRAF2-directed NF-kappa B activation. Although this may not be the case for other VP4s, it is interesting to note that all human rotaviruses contain the first PXQXT site in VP8*, and 65/66 contain the third site, suggesting the functional conservation of both binding domains.

The rotavirus VP4 protein is present on the surface of virions as a dimer (61, 62). It is possible that VP4 dimers contribute to the oligomerization of TRAFs required for NIK recruitment and NF-kappa B activation. Overexpression of VP8* has also been shown to result in the functional oligomerization of the protein (63). The three TRAF binding domains present within VP4 or the ability of TRAFs to recruit additional TRAFs could also facilitate or enhance the formation of signaling complexes, which permit VP4 to direct NF-kappa B activation (19, 64, 65). Since the first TRAF BD contributes most to VP4-directed NF-kappa B activation, VP4 dimers may be the primary means for signaling complex activation, and additional TRAF BDs may only slightly enhance VP4 complex formation and NF-kappa B activation.

The results presented suggest that rotaviruses do not activate NF-kappa B via dsRNA-dependent mechanisms, which direct NF-kappa B activation downstream of TRAF2 (66). A dsRNA interactive protein, which blocks protein kinase R (PKR) activation, has been identified in group C rotaviruses (67); however, this domain is not present in group A rotaviruses, and group A rotavirus proteins that perform this function have not been identified. The ability of DN-TRAF2 to inhibit rotavirus-directed NF-kappa B activation as well as studies that demonstrate the continued expression of viral and cellular proteins during rotavirus infection suggest that PKR inhibitory proteins are also likely to exist for group A rotaviruses.

Currently it is not possible to introduce mutated recombinant rotavirus genes back into rotaviruses (reverse genetics). However, it is clear that establishing reverse genetics for rotaviruses would dramatically enhance our ability to investigate the role of these and other rotavirus proteins in effecting specific cellular responses. Rotaviruses elicit cytokine and chemokine responses, and IECs play central roles in immunity, secretion, and barrier functions (1, 2, 38, 39, 41, 42, 68). From the conservation of rotavirus-signaling elements within VP4 and the pathway that it engages, at this point we can only surmise that VP4 signaling is an important element of rotavirus-induced cellular responses (3, 19, 42, 43, 60, 69-73). However, NF-kappa B activation by pathogenic Escherichia coli has recently been shown to induce galanin receptors on IECs that direct galanin-dependent increases in intestinal chloride secretion, and these findings suggest the possibility that VP4-signaling elements could contribute to rotavirus disease (38, 74).

TRAF2 is a central activator of both kappa B and AP-1 transcriptional responses, and TNFalpha transcription is regulated by both kappa B and AP-1 enhancer elements (19, 20). Rotaviruses do not induce TNFalpha from cells, and this is consistent with the demonstration that rotavirus or VP4 activate NF-kappa B, but not AP-1, transcriptional responses (1, 2, 46, 75-77). TNFalpha plays a central role in intestinal inflammatory responses and reportedly affects IEC barrier functions (44, 45, 47, 48, 78, 79). It will be interesting to investigate whether VP8*-TRAF2 interactions regulate the induction of TNFalpha , alter tight junctional proteins, or induce galanin receptors (38, 39, 58, 68, 74). As a result, the ability of rotaviruses to selectively activate NF-kappa B provides a potential role for signaling pathway activation in both host immune response and viral pathogenesis.

The hepatitis C virus core protein has been shown to effect opposite responses to those shown for VP4, suppressing NF-kappa B activity and activating AP-1, although it is not known whether TRAFs play a role in directing these responses (80). These studies have demonstrated that a rotavirus protein blocks TRAF2- and TRAF5-directed JNK activation and thereby differentially regulates TRAF2-specific signaling responses. Our findings indicate that protein interactions with TRAF2 are capable of disengaging TRAF2 from JNK while directing NF-kappa B activation and thereby suggest that cellular correlates, which similarly regulate TRAF2 function, may be identified. Since TRAF2 interacts with germinal center kinase, germinal center kinase-reduced, and receptor-interacting protein (RIP) to direct JNK activation, it is possible that VP4 disengages TRAF2 interactions with these JNK-signaling effectors (24, 25). These results suggest a new selective mechanism for regulating TRAF2-signaling responses that are central to cytokine induction and inflammation.

    ACKNOWLEDGEMENTS

We thank Dave Goeddel for providing TRAF1, -2, -3, and NIK expression plasmids, John C. Reed for TRAF5 and 6 plasmids, Yasutaka Hoshino for providing KU VP4 plasmids, and Imelyn Fernandez and Joanne Mackow for technical assistance.

    FOOTNOTES

* This work was supported by Veterans Administration Merit and Department of Defense/Veterans Affairs awards and National Institutes of Health Grants AI31016, AI42150, and AI44917 (to E. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Depts. of Medicine and Molecular Genetics and Microbiology, HSC T17, Rm. 60, SUNY, Stony Brook, NY 11794. Tel.: 631-444-2120; Fax: 631-444-8886; E-mail: EMackow@mail.som.sunysb.edu

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M100499200

    ABBREVIATIONS

The abbreviations used are: NIK, NF-kappa B-inducing kinase; MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; JNK, Jun-N-terminal kinase; DN, dominant negative; IL, interleukin; VLP, viral-like-particle; ds, double-stranded; IEC, intestinal epithelial cell; AP-1 activator protein-1, CRE, cyclic AMP response element; SRE, serum response element; RRV, rhesus rotavirus; GST, glutathione S-transfersase; Id, inhibitor of DNA-binding protein; BD, binding domain; PKR, protein kinase R.

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RESULTS
DISCUSSION
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