Previous Article
Journal of Virology, June 2000, p. 5388-5394, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Probing the Structure of Rotavirus NSP4: a Short
Sequence at the Extreme C Terminus Mediates Binding to the
Inner Capsid Particle
Judith A.
O'Brien,*
John A.
Taylor, and
A.
R.
Bellamy
Microbiology and Virology Research Group,
School of Biological Sciences, University of Auckland, Auckland,
New Zealand
Received 9 November 1999/Accepted 15 March 2000
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ABSTRACT |
The rotavirus nonstructural glycoprotein NSP4 functions as the
receptor for the inner capsid particle (ICP) which buds into the lumen
of the endoplasmic reticulum during virus maturation. The structure of
the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been
investigated by using limited proteolysis and mass spectrometry.
Digestion with trypsin and V8 protease reveals a C-terminal
protease-sensitive region that is 28 amino acids long. The minimal
sequence requirements for receptor function have been defined by
constructing fusions with glutathione S-transferase and
assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and
sufficient for ICP binding and that this binding is cooperative. These
observations are consistent with a model for the structure of the NSP4
cytoplasmic region in which four flexible regions of 28 amino acids are
presented by a protease-resistant coiled-coil tetramerization domain,
with only the last ~20 amino acids of each peptide interacting with
the surface binding sites on the ICP.
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TEXT |
Rotavirus is the single most
important cause of severe, dehydrating diarrhea in young children
worldwide and accounts for as many as 125 pediatric deaths per year in
the United States and 873,000 childhood deaths each year in developing
countries (10). The mechanism by which rotavirus induces
intestinal secretion of fluid and electrolytes is unknown, but
proposals include destruction of enterocytes in the small intestine
(11), alterations in transepithelial fluid balance
(5), a toxin-like effect by a viral nonstructural protein,
NSP4 (3), or activation of the enteric nervous system (15). The virus particle consists of a three-layer protein
capsid that contains a segmented, double-stranded RNA genome. The
assembly of mature rotavirus involves a unique budding process in which cytoplasmically assembled immature virus particles bud into the lumen
of the rough endoplasmic reticulum (ER). The transfer is mediated by an
interaction between the double-layered rotavirus inner capsid particle
(ICP; approximate molecular mass, 5 × 107 Da) and the
cytoplasmic tail of NSP4 (1, 2, 19, 24). This region of NSP4
contains a putative coiled-coil stem structure and a C-terminal
receptor domain that is sensitive to proteolysis (26), but
the precise size of the domain that interacts with the ICP and the
number of interactions involved remain to be established.
Here, we have used a combination of limited proteolysis and mass
spectrometry to probe the structure of the C-terminal region of NSP4.
The minimal receptor domain and characteristics of the NSP4-ICP
interaction have been defined by using site-directed mutagenesis and
quantitative analysis of binding kinetics. The results demonstrate that
28 residues form an exposed C-terminal domain, of which the last 17 to
20 (amino acids 156 to 175) are required for binding of the ICP to the
receptor. In solid-phase binding assays that reflect some aspects of
the in vivo interaction, multiple receptors appear to interact with the
ICP surface.
Protease digestion of the cytoplasmic domain of NSP4.
Previous
biophysical studies of the cytoplasmic domain of NSP4 showed that the
protein is a tetramer in which a putative coiled-coil domain forms the
subunit interface. This region was found to be resistant to limited
trypsin digestion while, under identical conditions, the C terminus was
removed (26). We refined this analysis to probe the
structure of NSP4 with emphasis on the size of the protease-sensitive
C-terminal region while monitoring the integrity of the coiled-coil domain.
This was achieved by expressing cDNA encoding the C-terminal 90 amino
acids of the cytoplasmic domain as a glutathione
S-transferase (GST) fusion protein (GSTC90) in
Escherichia coli DH5
cells (23, 25) by using
the pGex-2TK vector (Pharmacia), which incorporates a kinase site
adjacent to the thrombin cleavage site. Fusion protein adsorbed to 500 µl of glutathione-agarose beads (Sigma) was washed with protein
kinase buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 12 mM
MgCl2) and radiolabelled by addition of 150 µl of protein kinase reaction mixture (protein kinase buffer containing 50 U of
bovine heart kinase [Sigma] in 40 mM dithiothreitol and 50 µCi of
[
-32P]ATP). The mixture was vortexed and incubated for
30 min at 4°C. The reaction was terminated by the addition of 5 ml of
stop solution (10 mM sodium phosphate [pH 8.0], 10 mM sodium
pyrophosphate, 10 mM EDTA, 1 mg of bovine serum albumin per ml). The
beads were centrifuged at 500 × g for 2 min and washed
extensively with 0.1 M Tris-HCl (pH 8.0). The radioactively labelled
fusion protein was then washed with thrombin cleavage buffer (50 mM
Tris-HCl [pH 8.0], 150 mM NaCl, 2.5 mM CaCl2) and
incubated with 2 U of bovine thrombin (Sigma) per mg of fusion protein
for 90 min at 37°C with shaking. After centrifugation, the
supernatant was collected, concentrated by using a 3.5-ml Microsep
concentrator (10K cutoff; Filtron Technology Corporation), and
incubated with glutathione-agarose beads on ice for 10 min to remove
contaminating GST. Thrombin was removed by incubating the preparation
with para-aminobenzamidine-agarose beads (Sigma) on ice for 10 min.
This procedure yielded the cytoplasmic domain of NSP4 (C90) with a
32P label at the N terminus.
Purified receptor protein (10 µg) in thrombin cleavage buffer was
incubated with protease (0.1 µg) in a total volume of 20
µl at
temperatures ranging from 0 to 32°C. Proteolysis was terminated
by
precipitation of digested protein with cold 10% trichloroacetic
acid
(TCA) followed by centrifugation for 5 min. The precipitated
protein
was dissolved in Laemmli loading buffer, neutralized with
1 M Tris (pH
11), and boiled for 3 min prior to analysis by gel
electrophoresis as
described by Schägger and von Jagow (
22).
Limited
digestion of radiolabelled C90 with either trypsin or
V8 protease
produced smaller fragments (~8 kDa) that retained
the radioactive
label, indicating that the most accessible cleavage
sites are those
located in the C-terminal region of the molecule
(Fig.
1).
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FIG. 1.
Limited proteolysis of 32P-labelled C90 by
using trypsin or V8 protease. 32P-labelled C90 (10 µg) in
thrombin cleavage buffer was digested with 0.1 µg of trypsin at 0°C
(A) or V8 protease at 32°C (B) for 0 to 60 min. Reactions were
terminated by precipitation of the protein with ice-cold 10% TCA
followed by centrifugation for 5 min, and the digestion products were
resolved by SDS-PAGE. Gels were dried and exposed to X-ray film for
1 h to produce autoradiographs. Lane 1, nonradioactive protein
size markers (Sigma); lane 2, undigested C90; remaining lanes, C90
digested for the time indicated.
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Mass spectrometry of C90 and its digestion products.
Mass spectra for undigested and digested C90 (Fig.
2) were obtained by using the
matrix-assisted laser desorption/ionization (MALDI) process
(13). Preparations of the C90 variant or C90 limited
digestion products (25 µg) were purified by reversed-phase high-performance liquid chromatography (HPLC) using a C8
Brownlee cartridge column (Applied Biosystems) and elution in a 10 to
70% acetonitrile gradient containing 0.08% trifluoroacetic acid.
Samples were mixed in a 1:1 ratio with sinapinic acid
(3,5-dimethoxy-4-hydroxycinnamic acid; Hewlett-Packard Corp.), vortexed
briefly, and loaded onto individual mesas on the tip of an inert probe.
Peptide standards with known molecular masses (goosefish angiotensin I,
1,281.49 kDa; somatostatin, 1,637.90 kDa; human insulin, 5,807.7 kDa;
equine cardiac cytochrome c, 12,359 kDa; Hewlett-Packard
Corp.) were analyzed in the same fashion. Molecular masses were
determined in a Hewlett-Packard G2025A time-of-flight mass spectrometer
by comparison of the spectra for unknown samples with those obtained for standards (G2025A Software A.01.00; Hewlett-Packard Corp.). Undigested C90 yielded a molecular mass of 11,649 Da (cf. predicted monomeric value of 11,657 Da), whereas trypsin-digested C90 contained four distinct fragments (unresolved in Fig. 1) ranging in size from
8,372 to 8,903 Da. The spectrum for the major V8 protease-resistant product indicated a molecular mass of 8,347 Da.
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FIG. 2.
Mass spectrometry of proteolytic fragments of NSP4.
Purified C90 (50 µg) was digested with trypsin (0.5 µg) for 2 h at 0°C or V8 protease (0.5 µg) for 60 min at 32°C. The
reactions were terminated by the addition of protease inhibitors (0.5 mM Pefabloc for trypsin and 5 µM dichloroisocumarin for V8 protease).
Mass spectra for digested and undigested samples were obtained by
MALDITOF mass spectrometry. Shown are undigested C90 (A),
trypsin-digested C90 (B), and V8 protease-digested C90 (C).
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The origins of the protease-resistant NSP4 fragments generated by
trypsin and V8 protease digestion were then deduced by inspection
of
the amino acid sequence of C90 (Fig.
3).
The N-terminal serine
residue bearing the radioactive phosphate label
(bold type in
Fig.
3) clearly was retained because all the major
proteolytic
fragments were detectable by autoradiography. Trypsin
cleavage
could occur at the arginine residues in the kinase labelling
site
as well as at the C-terminal cleavage sites. Potential V8 protease
cleavage sites are confined to the C terminus.
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FIG. 3.
Origins of protease-resistant NSP4 fragments. The
origins of protease-resistant fragments of C90 generated by limited
digestion with trypsin or V8 protease were deduced from the amino acid
sequence of NSP4 (4). The sizes determined for these
fragments by mass spectrometry (Fig. 2) were compared with the sizes of
C90 peptides terminated at known trypsin (solid arrows) and V8 protease
(open arrows) cleavage sites and calculated by using GPMAW software
(Hewlett-Packard Corp.). The top bar represents the cytoplasmic domain
of NSP4, and the numbers refer to amino acid positions in the
full-length NSP4 sequence of rotavirus strain SA11 (shown in part).
Additional residues derived from the pGex-2TK vector are shown at the N
terminus, and residues inferred to be sensitive to limited proteolysis
are indicated (*). Boundaries of the -helical region are those
identified by Taylor et al. (26). The small bars represent
the protease-resistant fragments, with their predicted molecular
masses, which agreed (± 0.5%) with those measured by mass
spectrometry.
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The predicted sizes of four trypsin-resistant fragments terminating at
either Lys 151 or Arg 154 agreed closely (±0.5%) with
the molecular
masses of the proteolytic fragments obtained by
mass spectrometry.
Small peptides corresponding to C-terminal
tryptic fragments also were
found (data not shown), indicating
that the C-terminal region that
spans residues 151 to 175 is sensitive
to trypsin under conditions of
limited digestion. The size of
the major V8 protease-resistant product
(8,347 Da) is consistent
with the molecular mass expected for a
fragment cleaved at Glu
147 (8,384 Da). Other possible cleavages yield
fragments with
molecular masses of 7,895 Da (cleavage at Asp 143) or
9,580 Da
(cleavage at Glu 157), but fragments of these sizes were not
detected.
N-terminal sequencing (
18) and mass spectrometry
of the products
released from C90 by V8 protease digestion revealed the
presence
of C-terminal fragments corresponding precisely to amino acids
148 to 175, 148 to 160, 148 to 157, and 161 to 175 (Table
1).
Thus, it is clear from the results of
limited digestion with V8
protease that residues 147 to 175 are
exposed.
When the results of limited proteolysis with both enzymes are
considered, it is clear that Lys 146 represents the C-terminal
boundary
of the protease-resistant region of the cytoplasmic domain,
because
this residue is resistant to trypsin digestion, whereas
Glu 147 is
sensitive to cleavage with V8 protease. Overall, this
result suggests
that the C-terminal 28 amino acids of NSP4 adopt
a less ordered
conformation that renders this region of the protein
more accessible to
proteolytic
enzymes.
ICP-binding activity of truncated variants of NSP4.
Previous
studies of the binding of NSP4 and rotavirus ICPs revealed the
importance of the extreme C terminus for receptor activity (2,
24) and demonstrated that GST fusion proteins containing as
little as the C-terminal 20 amino acids retain the ability to bind ICPs
when immobilized on the surface of glutathione-agarose beads (25,
26). To assign a minimal ICP-binding domain more confidently,
further variants retaining fewer C-terminal amino acids were
constructed. A C16 variant was constructed by using the protocol
described previously for the C90 to C20 variants (26). The
C10, C12, and C14 variants were constructed by annealing complementary
oligonucleotides encoding the corresponding amino acids of NSP4 flanked
by 5' BamHI and 3' EcoRI sites. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA
sequencing using an Applied Biosystems automated sequencer.
GST fusion proteins were purified from cultures of
E. coli
DH5
cells (50 ml) expressing the receptor variants as follows.
Cultures were centrifuged (3,500 rpm, 10 min, 4°C, Sorvall RT7
benchtop centrifuge), and cell pellets were resuspended in buffer
(2 ml) containing 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 50
mM glucose.
The cell suspension was sonicated (10 s, Branson sonicator,
level 6)
after the addition of DNase (50 µg/ml), RNase (50 µg/ml),
lysozyme
(100 µg/ml), and Pefabloc [4(2-aminoethyl)-benzenesulfonyl
fluoride
hydrochloride; Boehringer Mannheim; 0.5 mM]. Cell debris
was pelleted
by microcentrifugation for 1 min. Cell lysates were
then incubated with
glutathione-agarose beads (100 µl) on ice
for 30 min, after which
fusion proteins were eluted with 10 mM
glutathione (200 µl) in 100 mM
Tris-HCl (pH 8.0) for 30 min at
room temperature. Dilutions (1/50) of
the eluted fusion protein
preparations in 100 mM Tris-HCl (pH 8.0) were
adsorbed to the
wells of a microtiter plate, and the receptor activity
of the
adsorbed protein was measured by enzyme-linked immunosorbent
assay
as described previously (
25). The relative levels of
adsorbed
receptor were compared by detecting adsorbed fusion protein by
using a rabbit polyclonal anti-GST antiserum. As anticipated,
fusions
incorporating 90, 44, or 20 amino acids exhibited strong
binding
activity (Fig.
4). In contrast, no
detectable binding
activity was found for the C10 to C16 fusions even
though the
levels of adsorbed receptor varied by no more than ±11% of
the
mean level for all variants (data not shown). This result indicates
that the minimal receptor domain consists of 17 to 20 amino acids
at
the extreme C terminus. Truncated variants in which methionine
175 was
mutated to isoleucine were also receptor negative, confirming
the
result demonstrated earlier that, in the full-length protein,
the
C-terminal methionine residue is required for ICP-binding
activity
(
24).
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FIG. 4.
Receptor activity of GST-NSP4 fusions. GST fusion
proteins were purified from cultures of cells (50 ml) expressing
N-terminally truncated variants of the cytoplasmic domain of NSP4. The
receptor activity for each variant was measured by enzyme-linked
immunosorbent assay. *, variants in which the C-terminal methionine
was mutated to isoleucine. The results represent the average of
duplicate measurements, and error bars indicate the range measured for
duplicate samples. OD, optical density.
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The dissociation constant for NSP4-ICP binding was measured for mutants
C90 to C20 by Scatchard analysis of the ICP-binding
activity of
GST-NSP4 fusions immobilized on the surface of glutathione-agarose
beads. Receptor assays used
125I-labelled ICPs and
glutathione-agarose-bound fusion protein (
25).
Bound-to-free
ratios were calculated from the ratio of bound counts
to input counts,
and the concentration of bound ICPs was calculated
from the counts
bound and the specific radioactivity of the labelled
ICPs. A value of
5 × 10
7 Da was used for the molecular mass of the ICP
that was based
on the molecular mass of the reovirus core particle
(
6). All
the Scatchard plots were concave downwards rather
than linear
(Fig.
5, left panels),
indicating that the binding event involved
positive cooperativity
(
20). The binding data was further analyzed
by using Hill
plots (Fig.
5, right panels). The fraction of binding
sites filled (Y)
was calculated as the ratio of counts bound to
the maximum counts bound
in the assay. The dissociation constant
(
Kd) was
obtained from the intercept of the
x axis {log
[Y/(1
Y)]
= 0}, and the Hill coefficient
n was obtained
from the slope of
the plot. The parameters of the linear fit confirmed
the cooperative
nature of the binding of ICPs to bead-immobilized
receptor (
7).
Derived values for
Kd
and the Hill coefficient
n are summarized
in Table
2.
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FIG. 5.
Scatchard and Hill plot analysis of ICP-binding activity
of bead-immobilized GST-NSP4. (Left) ICP-binding activity was measured
for each variant of NSP4, but only results for C90 and C20 are shown.
Assays contained 10 µl of bead-bound receptor and 0.3 to 15 µg of
125I-labelled ICPs (0.3 µg/µl, 1.5 × 104 to 2.1 × 104 cpm/µg). For Scatchard
analysis, bound-to-free ICP ratios were calculated from the ratio of
bound counts to input counts for the assay. The concentration of bound
ICPs was calculated from the counts bound and the specific
radioactivity of the labelled ICPs. (Right) Data for Scatchard analysis
of ICP-binding activity (left) were further analyzed to generate Hill
plots. Fractional saturation (Y) for each point was estimated from the
ratio of counts bound to the maximum counts bound in the assay. For all
plots, lines of best fit were applied to the data points.
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The
Kd results confirm that each variant
displayed comparable ICP-binding ability. Hill coefficients of >1
imply that binding
of one ligand molecule facilitates binding of others
to the same
receptor. For the NSP4-ICP interaction, the values derived
for
the Hill coefficients indicate that when ICPs bind to
bead-immobilized
receptor, the binding of the first virus particle
positively influences
the next binding event. A likely mechanism is
that the binding
of the immobilized receptor to the first site on the
surface of
the ICP increases the effective ICP concentration for
subsequent
interactions.
Isolation of NSP4-ICP complexes.
NSP4-ICP complexes were
obtained by incubating ICPs with bead-immobilized
33P-labelled GSTC90 fusion protein and then releasing
receptor-ICP complexes from beads by thrombin cleavage. Briefly,
glutathione-agarose beads (50-µl packed volume) were washed twice
with receptor assay buffer (10 mM Tris-HCl [pH 7.0], 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2) and incubated on ice for
45 min with 100 µg of 33P-labelled GSTC90 or its
Met175
Ile equivalent in the same buffer (radiolabelling was
performed as described above for 32P-labelled fusion
protein). The beads were pelleted by centrifugation for 5 s,
washed twice, and incubated with ICPs (100 µg) for 90 min at room
temperature in receptor assay buffer containing 0.1% octyl glucoside
(final volume, 400 µl). The samples were then centrifuged, the
supernatants were discarded, and the beads were washed three times with
thrombin cleavage buffer. NSP4-ICP complexes were released from the
beads by incubation with thrombin (2 U) in 85 µl of thrombin cleavage
buffer for 2 h at 37°C. The beads were removed by centrifugation
(700 × g, 10 s), and the supernatants were centrifuged
through MicroSpin S400 columns (Amersham Pharmacia Biotech) for 2 min
at ~700 × g to resolve the complexes from unbound NSP4.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
analysis of the complexes (Fig.
6) showed
that the GSTC90-ICP
sample (lane 2) contained Coomassie-stained bands
that comigrated
with either those of the ICP (lane 1) or C90 (lane 6).
The 14-kDa
band was identified as bound receptor by autoradiography
(Fig.
6, lower panel). The amount of NSP4 associated with the ICP was
quantified from the absorbency of the ICP (lanes 1 and 2) and
C90
(lanes 2 and 6) bands by using the NIH Image program. The
calculation
was based on the assumption that the molecular mass
of the rotavirus
ICP is close to that of the reovirus core particle
(5 × 10
7 Da [
6]). This analysis yielded a value
of approximately 40
C90 tetramers per particle. However, given the
likelihood of steric
hindrance of binding, this figure almost certainly
represents
only the level possible with bead-immobilized receptor
rather
than true saturation of all the binding sites on the surface of
the ICP. Decoration of ICPs with soluble rather than immobilized
NSP4
would eliminate this problem, but specific binding could
not be
demonstrated when soluble receptor was incubated with ICPs.
This
failure may reflect a lower affinity of binding for soluble
receptor
and is consistent with the inability of soluble variants
shorter than
C90 to block binding of ICPs in solid-phase assays
(data not shown) as
well as with the suggested mechanism of positive
cooperativity. The
isolation of complexes containing approximately
40 tetrameric C90
molecules per ICP does, however, have implications
for the nature of
potential receptor binding sites on the surface
of the ICP. The
icosahedral symmetry of rotavirus (
21,
27)
dictates that it
possesses 12 vertices, 20 faces, and 30 edges
(five-, three-, and
twofold axes of symmetry, respectively). If
40 or more molecules bind
to each particle, then NSP4 probably
does not bind to the 12 fivefold
vertex positions or to the 20
threefold faces of the particle. However,
if the receptor binding
sites are located on twofold symmetry axes (the
30 edges plus
390 local twofold axes between the VP6 trimers on each
face),
the decoration protocol using bead-immobilized C90 may have
achieved
an occupancy of only approximately 10% of the available
binding
sites.
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FIG. 6.
Analysis of NSP4-ICP complexes. ICPs were incubated with
bead-immobilized 33P-labelled GSTC90 or its Met175Ile
equivalent (GSTC90*). After centrifugation of the receptor-ICP
complexes through MicroSpin S400 columns, the eluates were incubated
with ice-cold 10% TCA. The precipitated proteins were pelleted by
centrifugation and analyzed by SDS-PAGE. The Coomassie-stained gel
(upper panel) was dried and exposed to X-ray film overnight (lower
panel). Lane 1, SA11 ICPs (10 µg); lane 2, C90-ICP eluate; lane 3, C90-no ICP eluate; lane 4, C90*-ICP eluate; lane 5, C90*-no ICP
eluate; lane 6, 33P-labelled C90 (4.4 µg). The arrowed
band corresponds to the anticipated position of C90 (lane 6).
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The results of this study confirm that the C terminus of NSP4 is more
sensitive to proteolysis than is the rest of the cytoplasmic
domain,
suggesting that this region of the protein may be disordered
and
flexible. Sequence alignments of NSP4 genes from human and
animal
rotavirus strains (
14) indicate that the C-terminal 20
amino
acids are reasonably well conserved, and circular dichroism
spectroscopy has demonstrated the presence of a high proportion
of
random conformation in this region of the protein (
26). A
number of potential trypsin and protease V8 cleavage sites are
contained within the upstream
-helical region (Fig.
3, residues
95 to 137) but presumably are protected by the high degree of
secondary
structure in this
region.
Our solid-phase binding assays indicate that the functional ICP binding
domain of NSP4 requires at least 17 of the C-terminal
20 amino acids
(156 to 175), including the final methionine residue.
Somewhat
conflicting results have been reported by Au et al. (
2),
who
measured rotavirus ICP binding by using membranes from
Spodoptera frugiperda cells containing NSP4 mutants. These workers found
that
loss of residues 161 to 175 abolished ICP binding activity,
while
deletion of the last three amino acids (173 to 175) diminished
but did
not abolish ligand binding. Since Met175
Ile versions
of each
N-terminal truncation consistently reinforced the requirement
for the
C-terminal methionine residue, the result obtained by
Au et al. is
difficult to reconcile with the results obtained
in this
study.
Quantitative analysis of the binding of ICPs to GST-NSP4 fusion
proteins immobilized on the surface of glutathione-agarose
beads showed
comparable levels of binding activity for all receptor-positive
variants. The
Kd for the interaction was
estimated to be on the
order of 7 × 10
11 M, which
agrees closely with the figure of 5 × 10
11 M
previously measured for membrane-anchored, full-length NSP4
expressed
in eukaryotic cells (
19). Although the solid-phase
binding
assays used here are unlikely to achieve saturation of
the ICP with
receptor, they nevertheless do reflect the situation
likely to occur in
vivo, in which multiple NSP4 molecules are
anchored in the ER membrane
with their C-terminal cytoplasmic
domains exposed and available for
interaction with the ICP. According
to the model first proposed for
animal enveloped viruses by Garoff
and Simons (
8), the
budding process is thought to be driven
by multiple interactions
between the receptor and the binding
sites on the surface of the
particle. Furthermore, lateral interactions
between peripheral or
integral membrane proteins seem now to be
a general feature that drives
the budding processes of different
viruses (
9). For
rotavirus, NSP4 has been shown to occur in
membranes associated with
two other viral proteins, VP7 and VP4
(
16). Thus, lateral
interactions between all three proteins
might supplement the positive
cooperativity demonstrated here
for the NSP4-ICP interaction and
together drive the transfer of
the immature virus particle across the
ER
membrane.
In summary, this work supports a model for NSP4 in which a disordered
C-terminal domain of 28 amino acids is presented to
the cytoplasm by
each subunit of the tetrameric receptor. Only
the last 17 to 20 residues of this region interact with the binding
sites on the ICP.
Future access to a high-resolution structure
of the ICP (B. McClain,
S. C. Harrison, and A. R. Bellamy, unpublished
observations)
should provide the means by which the precise molecular
details of this
unusual receptor interaction might be clarified.
Of particular interest
will be the reasons for the relatively
precise size limitation for the
ICP-binding domain and the requirement
for the C-terminal methionine
residue. Features of this interaction
may reflect those of others known
to involve large proteins and
short peptide domains
the presentation
of peptide antigens in
the cleft of the class I major
histocompatibility protein (
17)
and the binding of the
absolute C-terminal ends of target molecules
to the PDZ domains of
membrane proteins (
12) are two well-known
examples.
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ACKNOWLEDGMENTS |
We thank Christina Buchanan for advice on mass spectrometry.
Catriona Knight for assistance with reversed-phase HPLC, and David
Christie for N-terminal peptide sequencing. We are grateful to David
Christie, Vic Arcus, and Joerg Kistler for helpful discussions and
comments on the manuscript.
J.A.O. and J.A.T. were supported by grants from the Health Research
Council of New Zealand.
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FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Phone: 64 9 373 7599, ext. 8764. Fax: 64 9 373 7414. E-mail: j.obrien{at}auckland.ac.nz.
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Au, K.-S.,
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Journal of Virology, June 2000, p. 5388-5394, Vol. 74, No. 11
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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