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INTRODUCTION |
NF-
B is a rapid transcriptional activator present in the
cytoplasm of cells bound to the I
B inhibitor protein (3, 4). Cytoplasmic signaling pathways which lead to the phosphorylation and
proteosome degradation of I
B, permit nuclear translocation and
transcriptional activation of NF-
B (3, 4). Both NF-
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-
B
activation pathway. NIK and MEKK1 phosphorylate I
B kinases, which in
turn phosphorylate I
Bs (4, 5).
NIK-mediated NF-
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-
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-
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-
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-
B and the induction of NF-
B-directed
chemokines, including IL-8 (1, 2, 27). However, noninfectious,
genetically inactivated rotavirus also activates NF-
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-
B and directs IL-8 secretion (1, 2).
However, rotavirus infection does not result in the production of
TNF
or IL-1
, which are themselves capable of activating NF-
B
(1, 2). Collectively, these findings suggest that rotavirus proteins or the viral entry process itself activate NF-
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-
B
(38-43). In the gut, TNF
activates NF-
B, whereas IL-10
suppresses TNF
-directed responses, and transcription of both TNF
and IL-10 are regulated by NF-
B and JNK activation (44-48).
Although both TNF
and dsRNA can activate NF-
B, these elements do
not appear to play a role in rotavirus-induced NF-
B activation, and
the means by which rotavirus particles activate NF-
B but fail to
induce TNF
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-
B activation. Both rotavirus and VP8* selectively direct
transcription from
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-
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.
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MATERIALS AND METHODS |
Reagents--
Isopropyl
-D-thiogalactopyranoside
and o-nitrophenyl-
-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
-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-
B-Luc
(TGGGGACTTTCCGC)5, pAP1-Luc
(TGACTAA)7, pCRE-Luc (AGCCTGACGTCA
GAG)4, pSRE-Luc (AGGATGT
CCATATTAGGACCTCT)5, pFC-MEKK, and pFC-AP1. pSV-
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-
-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 
galactosidase expression plasmid
(pSV-
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
-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
-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
-galactosidase activity and assayed for their ability to phosphorylate
GST-c-Jun-(1-79) as previously described (Stratagene) (55).
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RESULTS |
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-
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-
B activation.
However, rotaviruses do not induce TNF
or IL-1
responses, which
are capable of activating NF-
B. The ability of inactivated virus or
VLPs to activate NF-
B further suggested that viral proteins may
mediate NF-
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-
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-
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.
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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-
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.
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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.
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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-
B Activation--
To
determine if expressed VP8* and VP4 activate NF-
B, we co-transfected
293 cells with an equivalent amount of empty control plasmid or
plasmids expressing VP8* or VP4. An NF-
B-luciferase reporter plasmid
and a
-galactosidase expression plasmid (SV40 promoter) were
co-transfected into cells to monitor and standardize NF-
B
activation. Transfecting increasing amounts of VP8* plasmid resulted in
an increase in NF-
B activation with a 6-7-fold increase in NF-
B
activation (2 µg) compared with control plasmid-transfected cells
(Fig. 4A). Similarly, NF-
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-
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- B and synergizes TRAF2-mediated
NF- B activation. 293 cells were
transfected with the indicated amounts of pEGFPVP4 or pEGFPVP8*
expression vectors, an NF- B-luciferase reporter plasmid (0.5 µg),
and an SV40 promoter-driven -galactosidase expression plasmid (0.5 µg) in the absence (A) or presence (B) of
co-transfected TRAF2 (51). NF- B activation from the luciferase
reporter was measured in triplicate and standardized to
-galactosidase activity of lysates (53). Error bars
represent the range of -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.
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Synergistic Enhancement of TRAF2-mediated NF-
B Activation by
VP8*--
TRAF2 itself activates NF-
B when overexpressed. To
determine if VP4 synergizes NF-
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-
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-
B
activation and functionally links VP8*-TRAF2 interactions to cellular
transcriptional responses.
NF-
B Activation by VP8*-TRAF Binding Domain Mutants--
The
RRV VP8* protein contains 3 PXQXT motifs that
could mediate TRAF binding and direct NF-
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-
B. Fig. 5 shows
that mutating all three PXQXT motifs within VP8*
abolishes the ability of the protein to activate NF-
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-
B-luciferase reporter, and NF-
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-
B activation.

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Fig. 5.
TRAF binding domain 1 of the RRV VP8* is
required for NF- 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- 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 -galactosidase activity within each lysate. Findings
were reproduced in at least two separate experiments, and a
representative experiment is presented.
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VP8* and Rotavirus Selectively Direct Transcription from
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
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-
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- 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- 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.
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DN-TRAF2 and DN-NIK Block Rotavirus and VP8*-mediated NF-
B
Activation--
To determine whether VP8 specifically engages a
TRAF2-NIK-signaling pathway of NF-
B activation, we tested the
ability of pathway-specific dominant negative inhibitors DN-TRAF2 and
DN-NIK to block VP8-induced NF-
B activation. The expression of
DN-TRAF2 or DN-NIK dramatically reduced or abolished VP8*- or
VP4-mediated NF-
B activation (Fig.
7A). This demonstrates that
VP8* and VP4 proteins direct NF-
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- 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- B-luciferase reporter
and pSV -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- 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 -galactosidase activity were observed between
infected or uninfected cells. Experiments were performed in triplicate,
and the results are representative of two separate experiments.
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We have previously demonstrated that rotavirus infection causes the
rapid nuclear translocation of NF-
B in cells by EMSA assay and that
inactivated rotavirus or rotavirus VLPs activate NF-
B (1). To
determine whether rotavirus also activates NF-
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-
B activation.
Cells were co-transfected as above with the pNF-
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-
B-directed luciferase activity was assayed
either 2 or 20 h post-infection. Rotavirus infection activated
NF-
B (2.5-5-fold); however, cells transfected with DN-TRAF2 or
DN-NIK abolished RRV-mediated NF-
B activation at either time point
(Fig. 7B). Similar to VP8*, these findings indicate that
rotavirus also engages a TRAF2-NIK pathway of NF-
B activation and
that additional NF-
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-
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-
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 -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
-galactosidase) to radiolabel c-Jun-1-79 was analyzed by
SDS-polyacrylamide gel electrophoresis.
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DISCUSSION |
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-
B
activation to elicit IL-8 responses (36, 41, 42, 57). Rotavirus
activation of NF-
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-
B. The ability of the KU human rotavirus VP8*
protein to activate NF-
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-
B activation, demonstrating that the rotavirus protein activates
NF-
B by engaging the TRAF2-NIK-signaling pathway and that
VP4-signaling interactions may be the primary means for
rotavirus-mediated NF-
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-
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-
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-
B activation studies indicate that the first RRV VP8*
binding domain appears to be primarily responsible for TRAF2-directed NF-
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-
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-
B activation (19,
64, 65). Since the first TRAF BD contributes most to VP4-directed
NF-
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-
B activation.
The results presented suggest that rotaviruses do not activate NF-
B
via dsRNA-dependent mechanisms, which direct NF-
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-
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-
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
B and AP-1 transcriptional
responses, and TNF
transcription is regulated by both
B and AP-1
enhancer elements (19, 20). Rotaviruses do not induce TNF
from
cells, and this is consistent with the demonstration that rotavirus or
VP4 activate NF-
B, but not AP-1, transcriptional responses (1, 2,
46, 75-77). TNF
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 TNF
, alter tight
junctional proteins, or induce galanin receptors (38, 39, 58, 68, 74).
As a result, the ability of rotaviruses to selectively activate NF-
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-
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-
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.