From the Nef is a membrane-associated cytoplasmic
phosphoprotein that is well conserved among the different human (HIV-1
and HIV-2) and simian immunodeficiency viruses and has important roles
in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In
order to identify Nef binding proteins and map their site(s) of
interaction, we targeted a highly conserved acidic sequence at the
carboxyl-terminal region of Nef sharing striking similarity with an
acidic sequence at the c-Raf1-binding site within the Ras effector
region. Here, we used deletion and site-specific mutagenesis to
generate mutant Nef proteins fused to bacterial glutathione
S-transferase in in vitro precipitation assays
and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence
motif containing the core sequence Asp-Asp-X-X-X-Glu
(position 174-179). Significantly, we demonstrate that
substitution of the nonpolar glycine residue for either or both of the
conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background
completely abrogated binding of c-Raf1 in vitro. In
addition, lysates from a permanent CEM T-cell line constitutively
expressing the native HIV-1 Nef protein was used to coimmunoprecipitate
a stable Nef-c-Raf1 complex, suggesting that molecular interactions
between Nef and c-Raf1, an important downstream transducer of cell
signaling through the c-Raf1-MAP kinase pathway, occur in
vivo. This interaction may account for the Nef-induced
perturbations of T-cell signaling and activation pathways in
vitro and in vivo.
The Nef protein of the human and simian immunodeficiency viruses
(HIV-1,1 HIV-2, and SIV) is
predominantly a membrane-associated cytoplasmic phosphoprotein (1-3)
synthesized from multiply spliced transcripts (4) early during the
viral infection process and is also packaged into mature virions (5,
6). By using in vitro cell culture assay systems, Nef has
been shown to be dispensable for virus replication (7-9) but is
necessary for enhancing virus production and infection of
recipient cells in vitro (5, 6, 9-11). In the
SIVmac239 model for nef gene function in
vivo, Nef has been shown to be necessary for maintaining high
viral loads and for induction of an AIDS-like disease in rhesus monkeys
infected with recombinant SIVmac239 with an intact
nef gene (12). In the transgenic mouse model for Nef
function in vivo, it has also been found that the HIV-1 Nef
alters normal T-cell activation responses in thymocytes (13),
suggesting that the function of Nef in vivo is tied to its
ability to perturb T-cell signaling pathways. In addition, evidence
from several in vitro studies have also supported a link
between Nef expression and defective T-cell signaling and activation.
Notably, Nef expression has been tied to its ability to interfere with
signaling and cellular activation in T-lymphocytes (14-18),
phosphatidylinositol 3-kinase signaling in NIH/3T3 cells (19), and
tumor necrosis factor One of the best characterized signaling defects induced by Nef
expression is the down-regulation of the cell-surface CD4 viral receptor molecule (2, 13, 21) by a mechanism that involves physical
interaction with p56Lck kinase (22) and requires a common
di-leucine containing sequence element at the cytoplasmic tail of the
CD4 receptor molecule that also overlaps with the
p56Lck-binding site (23, 24). The molecular interactions
between Nef and p56Lck kinase (22) and between Nef and
other members of the Src family of protein tyrosine kinases (25) occur
through the highly conserved proline (PXXP)3
repeat motif in Nef and the conserved Src homology 2 (SH2) and/or SH3
domains of the protein kinases (22, 25). Other classes of cellular
proteins have been reported to associate with the Nef protein in
vitro or in vivo, including the p21-activated serine/threonine protein kinase (26-28), the MAP/ERK-1 protein kinase
(17), p53 (17), protein kinase C (29, 30), a 46-kDa cellular
phosphoprotein (31, 32), numerous T-cell-derived cytosolic and
membrane-associated proteins (3, 17, 32, 33), and The ability of Nef to alter T-cell signaling and activation pathways
in vitro and in vivo suggests a mechanism that
may involve specific molecular interactions between Nef and a limited
number of cellular signaling proteins. In order to define this
mechanism, we set out to identify and map the T-cell signaling proteins
and their specific protein-protein interaction domains that are targets of the HIV-1 Nef protein in vitro and in vivo. In
the present report, we describe the identification of a relatively well
conserved acidic sequence motif, consensus
Asp/Glu-Asp-X-X-X-Glu (where X represents any
other amino acid), at the carboxyl-terminal region of the primate
lentiviral Nef proteins (35), that is structurally quite similar to the
corresponding acidic sequence, Asp-Pro-Thr-Ile-Glu-Asp (amino acids
33-38), at the c-Raf1-binding site within the conserved core effector
loop of the Ras oncoprotein (36). Furthermore, we describe that the
c-Raf1 kinase physically interacts with the HIV-1LAI Nef
protein at the acidic sequence motif which can be effectively blocked
by single point mutations that substituted a nonpolar glycine residue
for either of the conserved aspartic acid residues at positions 174 (Asp-174 Mutagenesis and Construction of nef Expression Plasmids
Site-specific Mutagenesis--
Oligonucleotide-directed
mutagenesis (37) was performed on plasmid pSG5- Deletion Mutagenesis--
A 360-bp
EcoRV-Ecl136II nef gene fragment
spanning nucleotides 8555-8929 and encoding amino acid residues
136-206 at the carboxyl-terminal region of the
HIV-1LAI(HTLV-111B) Nef protein was subcloned into the
SmaI site of pBluescript plasmid (Stratagene, CA), then
released with BamHI and EcoRI enzymes, and cloned
into the BamHI and EcoRI sites of the pGEX-2T
plasmid (39) to generate the pGEX-Nef1C expression plasmid. The 360-bp
BamHI-EcoRI fragment was used to generate a panel
of carboxyl-terminal nef deletion mutants by digestion with
unique restriction enzymes, followed by repair of sticky ends with
Klenow enzyme. The blunt-ended fragments were then cloned into the
pGEX-2T or pGEX-3X plasmids (39) to generate the following expression
plasmids: pGEX- c-Raf1 Expression Plasmids--
The pGEX-Raf-(50-133)
expression plasmid was created by polymerase chain reaction cloning in
which the primer pairs, 5'-GTAGGATCCGATCCTTCTAAGACAAGC-3' and
5'-CTAGAATTCATCCAGGAAATCTACTTG-3' containing a 5'-BamHI and 3'-EcoRI restriction site, were used to release a 245-bp
EcoRI-BamHI fragment from the pKS+cRaf-1 plasmid
(a gift from Dr. Debra Morrison, FCRDC, Frederick, MD) which encodes
amino acids 50-133 of the human c-Raf1 protein. The fragment was then
cloned into the BamHI-EcoRI sites of pGEX-2T
vector. The correct clone was authenticated by DNA sequence analysis.
The expression vectors were then used to transform competent
Escherichia coli XL-1 or JM109 cells (Stratagene, La Jolla,
CA) for expression of fusion proteins. The full-length human c-Raf1
protein (residues 1-648) was expressed as a GST-Raf-(1-648) fusion
protein in the Sf9 insect (Spodeptera frugiperda)
cell line with the pVL1393 baculovirus expression vector.
Protein Expression and Purification
Fresh overnight cultures of E. coli XL-1 or JM109
cells (Stratagene, La Jolla, CA) harboring the various pGEX expression
plasmids were diluted 1:10 in Luria-Bertani (LB) broth supplemented
with 100 µg/ml ampicillin, and the cultures were grown and induced with 1 mM isopropyl- Preparation of Soluble Recombinant Nef Proteins
Full-length wild-type and mutant recombinant GST-Nef fusion
proteins bound to glutathione-agarose beads were digested with thrombin
protease enzyme according to the recommendations of the manufacturer
(Boehringer Mannheim) to remove the 26-kDa GST fusion tag, and the
thrombin activity was inactivated with the protease inhibitor mixture
(Boehringer Mannheim). The resulting protease-cleaved soluble Nef
proteins were recovered by centrifugation of the glutathione-agarose beads to which the GST tag was still bound, followed by buffer exchange
through a small Sephadex G-25 column (Amersham Pharmacia Biotech). The
recovered recombinant Nef proteins were analyzed by SDS-PAGE and
quantitated by staining with Coomassie Brilliant Blue R-250. The
samples were adjusted to contain the same amount of protein.
Preparation of Soluble CEM Cell Lysate
The CEM T-lymphoblastoid cell line was maintained at 37 °C in
a humidified 5% CO2 incubator in RPMI 1640 media
supplemented with 4 mM glutamine, 15% heat-inactivated
fetal bovine serum, and 100 units each of streptomycin and penicillin
(Atlanta Biologicals, Norcross, GA). Cells in exponential growth phase
were collected at 500 × g for 5 min at 4 °C and
washed once with ice-cold PBS. Whole cell lysates were prepared in cell
lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.5% Triton X-100) supplemented with 0.1 mM sodium
orthovanadate, 5 mM sodium fluoride, 1 mM
phenylmethylsulfonyl fluoride, 100 µg/ml
L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone, 100 µg/ml L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone,
and 10 µg/ml each of leupeptin and pepstatin A (Boehringer Mannheim) at 4 °C, then cleared by centrifugation at 100,000 × g for 30 min at 4 °C. Cleared cell lysates containing
5 × 107 CEM cell eq/ml were used immediately or
frozen at Coprecipitation Assay
Soluble CEM whole cell lysates containing 5 × 107 CEM cell eq/ml were incubated with 50 µl (1 µg of
protein) of GST beads or GST-Nef affinity beads with gentle mixing for
2 h at 4 °C, and the protein-bound beads were extensively
washed with an excess of ice-cold cell lysis buffer containing protease
inhibitors and processed for SDS-PAGE and Western blot analysis as
described above.
Direct Protein Binding Assay
For the direct protein binding assay, aliquots containing 1 µg
of thrombin-cleaved soluble recombinant Nef proteins were incubated with 50 µl (1 µg of protein) of glutathione-agarose affinity beads containing either the bound recombinant GST-Raf-(50-133) or
GST-Raf-(1-648) fusion proteins for 2 h at 4 °C as before. The
protein-bound beads were then extensively washed with cell lysis buffer
and processed for SDS-PAGE and Western blot analysis as before.
SDS-PAGE and Immunoblot Analysis
Cellular proteins bound to the GST beads and GST fusion protein
affinity beads were solubilized by boiling with 2× SDS-PAGE sample
buffer and resolved by fractionation on reducing SDS-12% polyacrylamide gels (41). The fractionated proteins were then electrophoretically transferred (42) to Hybond-polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Burkinghamshire, UK),
and the membranes were blocked for 2 h at 23 °C in TBS (10 mM Tris, pH 7.5, 100 mM NaCl) buffer containing
0.1% Tween 20 and 5% BSA (fraction V) (Sigma). The membranes were
then incubated with a 1:1000 dilution of mouse anti-human c-Raf1
monoclonal antibody (mAb) that was raised against amino acids 162-378
of the human c-Raf1 kinase (Transduction Laboratories) or with a 1:500
dilution of the 55S mAb against the HIV-1LAI(HTLV-111B) Nef
protein (43), in TBS-T (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 1% BSA for 12 h
at 4 °C. Following extensive washes for 10 min each in TBS-T, the
membranes were incubated with a 1:5000 dilution of a sheep anti-mouse
IgG:horseradish peroxidase conjugate in TBS-T with 1% BSA at 23 °C
for 60 min. The membranes were then extensively washed as before, and
the complexes were visualized with the enhanced chemiluminescence
detection system (Amersham Pharmacia Biotech, Burkinghamshire, UK).
Coimmunoprecipitation Assay
Whole cell lysates containing 5 × 108 CEM cell
eq/ml were prepared from permanent CEM T-cell lines transduced with the
HIV-1LAI(HTLV-111B) wild-type nef allele in the
forward (NEF) and reverse (FEN) directions with the pLXSN retroviral
expression vector (CLONTECH, Inc., La Jolla, CA)
and were incubated either with the 55S anti-Nef (43) or the anti-c-Raf1
mAb for 12 h at 4 °C in cell lysis buffer containing protease
and phosphatase inhibitors, followed by incubation with 100 µl of
33% (v/v) protein G-Sepharose beads at 4 °C for an additional 2 h. The immunoprecipitates were washed extensively with cell lysis buffer, then solubilized by boiling in SDS-PAGE sample buffer, and resolved by fractionation on SDS-PAGE as described before. The
fractionated proteins were transferred to Hybond-polyvinylidene difluoride membrane as before; the membrane was then incubated either
with the anti-Raf1 or anti-Nef mAbs, and specifically bound antigen-antibody complexes were visualized with the enhanced
chemiluminescence system as before.
Recombinant Nef Fusion Proteins--
Recombinant GST- Laboratory of Leukocyte Biology,
NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, § Laboratory for Genetic
Disease Research, National Human Genome Research Laboratory,
Bethesda, Maryland 20892, ¶ Laboratory of Genomic Diversity,
SAIC,
Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, and ** Department of Biology, Morgan State
University, Baltimore, Maryland 21239
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-activated sphingomyelin signaling in human
glial cells (20).
-cop protein (34),
an important component of non-clathrin-coated pit vesicles involved in
cellular membrane trafficking.
Gly) or 175 (Asp-175
Gly) and by combined Asp-174
Gly and Asp-175
Gly mutations at both positions. Our results define
the direct binding of c-Raf1 kinase to a conserved acidic sequence in
the carboxyl-terminal region of the HIV-1 Nef protein in
vitro and suggest a possible molecular model for investigating the
Nef-induced T-cell signaling defects involving the c-Raf1-MAP kinase
signaling pathway.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
9Nef which contains
the full-length HIV-1Lai (HTLV-111B) nef gene
(GenBankTM M15654, nucleotides 8152-8773) (38), utilizing
the Muta-Gene kit (Bio-Rad). The mutagenic oligonucleotide,
5'-CTGCATGGAATGGGTGACCCGGAGAGA-3' (mutated
nucleotides are in boldface and codon changes are underlined), which
substituted a glycine for the aspartic acid at position 174 (D174G
change), spans nucleotide positions 8659-8685 of the HIV-1Lai nef gene (38); the oligonucleotide,
5'-CTGCATGGAATGGATGGCCCGGAGAGAGA-3' (nucleotide
positions 8659-8685) substituted a glycine for the aspartic acid at
position 175 (D175G change); oligonucleotide 5'-CTGCATGGAATGGGTGGCCCGGAGAGAGAA-3'
spans nucleotides 8659-8688 and introduced double aspartic acid
to glycine substitutions at positions 174 and 175, respectively
(D174G/D175G change); and oligonucleotide
5'-CTGCATGGAATGGGTGGCCCGGAGAGAGGAGTGTTAGAGTGG-3', spans nucleotides 8659-8700 and introduced triple point mutations in
which aspartic acids 174 and 175 and glutamic acid 179, respectively, were changed to glycine (D174G/D175G/E179G change). The resulting wild-type and mutated full-length nef genes were cloned into
the BamHI and EcoRI sites of the pGEX-2T plasmid
(Amersham Pharmacia Biotech) (39), generating the pGEX-wtNef and
pGEX-Nef174D
, pGEX-Nef175D
,
pGEX-Nef174:175DD
, and
pGEX-Nef174:175:179DDE
nef expression plasmids,
respectively. The correct clones were verified by DNA sequence
analysis.
12nef, which contains a 180-bp
BamHI-HaeI11 nef gene fragment (codons
136-194); pGEX-
31nef containing codons 136-177 encoded
by a 123-bp BamHI-Nci1 fragment; pGEX-
41nef containing an 88-bp
BamHI-Mae111 fragment encoding codons 136-165;
and pGEX-
29nef containing a 290-bp
MaeIII-Ecl136II fragment encoding codons
165-206.
-D-thiogalactoside
(Sigma) as described previously (39). Cell pellets were collected by
centrifugation and washed in ice-cold PBS (pH 7.4, Mg2+/Ca2+-free) supplemented with 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml
L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone, 100 µg/ml L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone,
10 µg/ml pepstatin A, 20 µg/ml aprotinin, and 10 µg/ml leupeptin,
and Triton X-100-soluble extracts containing the recombinant proteins
were prepared essentially as described before (40). The
detergent-soluble protein extracts were dialyzed against PBS, pH 7.4, containing 0.5% Triton X-100 and protease inhibitors (Boehringer
Mannheim), and the GST and recombinant GST fusion proteins were
purified by coupling to glutathione-agarose beads (Sigma) by gentle
mixing at 4 °C for 1 h as described (39, 40). The coupled
affinity beads were sequentially washed with excess PBS, pH 7.4, containing 0.5% Triton X-100, PBS containing 0.5% Triton X-100 and
300 mM NaCl, and finally with PBS. The protein-bound affinity beads were analyzed and quantitated by Coomassie Blue R-250
staining following SDS-PAGE analysis (41). The coupled affinity beads
were then adjusted to approximately 1 µg of protein per 50-µl beads
and stored as a 33% (v/v) slurry at 4 °C in PBS supplemented with
protease inhibitors.
70 °C until use.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Nef fusion
proteins containing overlapping amino acid deletions between residues
136 and 206 at the carboxyl-terminal region of the HIV-1LAI
Nef protein were expressed in an insoluble form in E. coli
bacteria and then solubilized by extraction with the Sarkosyl-Triton
X-100 procedure (40). The detergent-soluble fusion proteins were
finally purified by directly coupling to glutathione-agarose beads as
described previously (39, 40). Aliquots of affinity beads containing
1-3 µg of the purified GST fusion proteins were resolved by
fractionation on SDS-PAGE and quantitated by staining the gel with
Coomassie Brilliant Blue R-250 (Fig. 1,
lanes 1-6). Three of the resulting Nef fusion proteins carrying carboxyl- or amino-terminal deletions, GST-
12Nef
(lane 3), GST-
31Nef (lane 5), and GST-
41Nef
(lane 6), respectively, show anomalous mobilities on the SDS
gel. In addition, samples of the glutathione-agarose beads containing 1 µg of the GST-bound wild-type full-length Nef (Fig. 3B, lane
2) and mutant full-length Nef proteins containing individual or
combined point mutations within the conserved acidic sequence motif
(Fig. 3B, lanes 3-6) were also analyzed by SDS-PAGE and
quantitated by Coomassie Blue staining.
View larger version (79K):
[in a new window]
Fig. 1.
Purification of recombinant Nef deletion
proteins. Bacterially expressed GST- Nef deletion mutants were
affinity purified by coupling to glutathione-agarose beads, and samples
containing 1-3 µg of protein were separated by electrophoresis on an
SDS-12% polyacrylamide gel and stained with Coomassie Brilliant Blue
R-250 as described under "Experimental Procedures." The fusion
protein sample beads are GST (lane 1), GST-Nef1C (lane
2), GST-
12Nef (lane 3), GST-
29Nef (lane
4), GST-
31Nef (lane 5), and GST-
41Nef (lane
6), respectively. Molecular mass markers are shown by
numbers in the margin.
Similarity between the Nef Acidic Sequence and a Corresponding c-Raf1-binding Site Sequence in Ras-- During structure-function analysis on the predicted Nef protein sequences of HIV-1, HIV-2, and SIV, we observed a well conserved acidic hexapeptide sequence with the general consensus, Asp/Glu-Asp-X-X-X-Glu, located at the carboxyl-terminal region of the primate lentiviral proteins (35), which suggests that this sequence may have an important role in the function of Nef. Interestingly, this sequence has a number of characteristic features in common with a corresponding acidic sequence, Asp-Pro-Thr-Ile-Glu-Asp (amino acids 33-38), at the c-Raf1-binding site sequence within the highly conserved core effector region of the mammalian Ras oncoprotein (36). In particular, both the Nef and Ras acidic sequence motifs share a similar pair of conserved acidic amino acids located at one end of the sequence and a single glutamic or aspartic acid residue positioned at the opposite end of the sequence (see Table I). Similarly, the relative spacing of three residues between the conserved terminal acidic amino acids is also well conserved within the Nef and Ras sequence motifs. However, these oppositely positioned single and double acidic amino acids are oriented differently in the two proteins, such that the two aspartic acid residues at positions 174 and 175 in the HIV-1LAI, positions 204 and 205 of HIV-2ISY, and positions 203 and 204 in SIVMM239 Nef proteins, respectively, are equivalent to the aspartic acid at position 38 and the glutamic acid at position 37 of Ras. Similarly, the highly invariant glutamic acid at positions 179, 209, and 208, respectively, in the HIV-1, HIV-2, and SIV Nef protein sequence, corresponds to the aspartic acid at position 33 within the conserved Ras effector sequence (36). Furthermore, whereas the lone proline residue (position 34) within the Ras sequence is tightly conserved in the subfamily of GTP-binding proteins, the analogous proline residue is conserved only in the predicted Nef protein sequence of the various SIV isolates. In the HIV-1 and HIV-2 Nef protein sequence, this proline residue exhibits a much greater degree of genetic drift.
|
Interaction between c-Raf1 and the Carboxyl-terminal Region of
Nef--
To determine whether the conserved carboxyl-terminal acidic
sequence, Asp-Asp-Pro-Glu-Arg-Glu, spanning amino acids 174-179 within
the HIV-1LAI Nef protein is important for mediating
specific interaction with c-Raf1 in vitro, we utilized the
GST-Nef affinity beads containing different carboxyl-terminal deletions
within amino acids 136-206 (see Fig. 1) in precipitation assays to
pull down the c-Raf1 protein from detergent lysates of the CEM
T-lymphoblastoid cell line. The results, shown in Fig.
2A, demonstrate that the soluble c-Raf1 protein was specifically precipitated from CEM cell
lysates with the affinity beads containing amino acid residues 136-206
of Nef (lane 3, GST-Nef1C), residues 136-194 (lane
4, GST-12Nef), residues 136-177 (lane 5,
GST-
31Nef), and residues 165-206 (lane 7, GST-
29Nef),
respectively. As expected, the empty GST-agarose beads did not
precipitate c-Raf1 (lane 2). However, the affinity beads
containing amino acids 136-165 of HIV-1 Nef (fusion protein
GST-
41Nef) also did not precipitate the c-Raf1 protein from the CEM
extract (Fig. 2A, lane 6). The missing sequence from
GST-
41Nef encompasses amino acids 165-177 and includes the conserved acidic sequence, Asp-Asp-Pro-Glu (positions 174-177) within
the predicted c-Raf1-binding site (Table I). The results of the
experiments presented in Fig. 2A and summarized in Fig. 2B indicate that binding of the CEM cell-derived c-Raf1
protein to the carboxyl terminus of Nef requires a minimal sequence
located between amino acids 165 and 177. The results also implicate the importance of the acidic sequence motif at positions 174-179 in the
binding.
|
A Di-aspartic Acid Motif Is Critical for C-Raf1 Binding to
Nef--
Since the carboxyl-terminal end of the Nef sequence deleted
from the GST-41Nef fusion protein above,
Leu-His-Pro-Val-Ser-Leu-His-Gly-Met-Asp-Asp-Pro-Glu (amino acids
165-177), also contains the highly conserved pair of aspartic acid
residues (positions 174 and 175), we investigated the contributions of
this pair of acidic residues to binding of the c-Raf1 protein in
vitro. We introduced single, double, and triple point amino acid
substitutions (37) within the conserved acidic Nef sequence such that
the conserved aspartic acid residues at positions 174 and 175, and the
glutamic acid residue at position 179 within the full-length HIV-1 Nef
genetic background (38), were mutated to a glycine residue. Both the
full-length wild-type and mutant Nef were expressed in E. coli as GST-Nef fusion proteins and were purified by coupling to
glutathione-agarose beads and used in the precipitation assays to pull
down the c-Raf1 protein.
|
HIV-1 Nef Protein Directly Interacts with c-Raf1 in Vitro-- The possibility that interaction between matrix-bound GST-Nef fusion proteins and soluble c-Raf1 protein in the CEM T-cell extracts required an additional cellular factor(s) has not been ruled out in the above experiments. Thus, to demonstrate that binding of c-Raf1 to Nef in vitro involves a direct mechanism, which is direct physical interaction between the two proteins, we utilized a direct binding assay in which soluble recombinant Nef proteins free of the GST tag were reacted with matrix-bound full-length GST-Raf1-(1-648) and a truncated GST-Raf1-(50-133) fusion protein (36, 46-49) containing the Ras-binding domain (RBD) within a minimal peptide (36, 46, 48).
As shown in the results of Fig. 4A, affinity beads containing both the truncated GST-Raf-(50-133) with the known RBD (lane 2) and full-length GST-Raf-(1-648) fusion protein (lane 7) effectively pulled down the soluble recombinant wild-type Nef protein but did not precipitate the soluble recombinant Nef proteins containing the single D174G (lanes 3 and 8), double D174G/D175G (lanes 4 and 9), and triple D174G/D175G/E179G (lanes 5 and 10) substitution mutations, respectively, within the conserved acidic Nef sequence motif. As expected, soluble matrix-free bacterial GST protein was not precipitated with the GST-Raf-(50-133) (Fig. 4A, lane 1) nor the GST-Raf-(1-648) (lane 6) affinity beads. The soluble wild-type Nef protein in lane 12 was used as a positive control in the Western blot assay. The integrity of the purified soluble recombinant Nef proteins used in the above direct binding assay was assessed by SDS-PAGE analysis and Coomassie Blue staining (Fig. 4B, lanes 1-5). The results of the direct binding assay demonstrate that Nef and c-Raf1 are sufficient and necessary for direct physical interaction in vitro. In addition, the results confirm the previous findings (Fig. 3A) showing that aspartic acid 174 within the acidic sequence motif is critical for physical interaction with c-Raf1, as the single D174G substitution completely abrogated the binding (Fig. 4A, lanes 3 and 8). Although the single D175G at position 175 was not analyzed in the direct binding assay, we predict that the results will be similar to that with the D174G mutant. Taken together, the results of Fig. 4A demonstrate that the minimal sequence for binding of c-Raf1 to the HIV-1 Nef encompasses the conserved acidic sequence motif, Asp-Asp-X-X-X-Glu (positions 174-175) within the carboxyl-terminal region of Nef, and also indicate that the primary Nef-binding site in the c-Raf1 protein maps within a minimal peptide (residues 50-133) previously shown to be critical for binding to Ras (36, 48-50).
|
Coimmunoprecipitation of a Nef-c-Raf1 Complex from a Stable Nef Expressing Cell Line-- We next determined whether the interaction we have observed in vitro between the recombinant Nef and c-Raf1 proteins also occurs in vivo. We reasoned that if a stable Nef-c-Raf1 complex exists in vivo, then it can be pulled down with an antibody specific for either the HIV-1 Nef or c-Raf1 protein by coimmunoprecipitation assay on soluble lysates of permanent T-cell lines constitutively expressing the native Nef protein. Thus, using lysates of CEM T-cell lines permanently infected with pLXSN-nef expression vectors carrying the HIV-1LAI (HTLV-111B) wild-type nef allele in either the forward (NEF) or reverse (FEN) orientations, both the 72-kDa c-Raf1 and 27-kDa Nef protein were coimmunoprecipitated as a complex with the 55 S anti-Nef mAb from the NEF cell line (Fig. 5, lane 1) but not from the FEN cell line which does not express the Nef protein (Fig. 5, lane 2).
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DISCUSSION |
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Several studies suggest that the Nef proteins of HIV-1, HIV-2, and
SIV have a critical role in modulating transduction of T-cell
activation signals as well as in T-cell activation (2, 7, 13-18, 50).
It is possible that Nef exerts its effects on T-cell signaling and
activation by its ability to associate with specific factors involved
in signaling pathways in vivo (17, 22, 23, 25-28, 30, 33).
Thus, we investigated interaction between HIV-1 Nef and the c-Raf1
protein in vitro and identified a well conserved acidic
sequence motif with consensus,
Asp/Glu-Asp-X-X-X-Glu (where
X represents any other amino acids), found at the
carboxyl-terminal regions of the HIV-1, HIV-2, and SIV Nef proteins,
that is critical for interaction with c-Raf1 in vitro. We
have shown that the Nef acidic sequence is structurally quite similar
to the corresponding sequence, Asp-Pro-Thr-Ile-Glu-Asp, that spans
amino acids 33-38 within the c-Raf1-binding site in the conserved core
effector loop of the Ras GTP-binding protein (36, 46). However, this sequence is oriented in the reverse position relative to the Nef sequence (Table I). Analysis of GST-Nef truncations has allowed us
to define the minimal region for binding c-Raf1 in vitro in precipitation pull-down assays. This region was mapped to a sequence spanning amino acids 165-177 within the carboxyl-terminal region of
the HIV-1 Nef protein, which is critical for the Nef-c-Raf1 interaction. Additionally, we showed that c-Raf1 residues spanning the
region between amino acids 50 and 133, which contains a minimal domain
for binding Ras (46, 48, 50), are also sufficient for binding the
full-length recombinant HIV-1 Nef, since single substitution mutations
involving either Asp-174
Gly or Asp-175
Gly changes are
sufficient to completely abrogate c-Raf1 binding in vitro.
Significantly, we demonstrated that the Asp-174
Gly mutation was
sufficient to block direct binding of the soluble Nef mutant to both
the full-length GST-Raf-(1-648) protein and a truncated
GST-Raf-(50-133) containing the RBD. Although the Asp-175
Gly
point mutation was not analyzed in the direct binding assay, we expect
that it will also effectively block direct binding with c-Raf1 in
vitro. In addition, we also showed that a pairwise Asp-174
Gly
and Asp-175
Gly and triple Asp-174
Gly, Asp-175
Gly, and
Glu-179
Gly substitution mutation within the conserved acidic Nef
sequence completely blocked direct interaction with c-Raf1 in
vitro.
We have also demonstrated the in vivo association between the native HIV-1 Nef protein and c-Raf1 proteins in a stable complex that was coimmunoprecipitated from lysates of the NEF cell line expressing both the Nef and c-Raf1 proteins but not from the FEN cell line that expressed the c-Raf1 protein but did not express Nef.
We occasionally were able to precipitate a tripartite Nef-c-Raf1-MEK-1 complex in our in vitro precipitation assay, but we did not observe a similar triple complex in lysates of the NEF expressing CEM T-cell (data not shown). Since a highly stable c-Raf1-MEK-1 complex is known to exist in vivo and is important for signaling through the c-Raf1-MAP kinase pathway (51), we cannot totally rule out the possibility that Nef may interact with such a complex in vivo. It has previously been shown that the HIV-1 Nef exists in a complex with mitogen-activated protein kinase in vitro (21), but no evidence was presented to show whether Nef and MEK-1 associate with each other. Presumably, MEK-1 forms a specific complex with c-Raf1 (51) without interference by the complex formed between Nef and c-Raf1 in vitro. Although we do not yet know the reason for the discrepancy between our in vitro and in vivo results with regard to the Nef-c-Raf1-MEK-1 tripartite complex, it may be that such a triple complex is not sufficiently stable in vivo to allow detection with the coimmunoprecipitation assay used in the present study.
While the present work was in progress, we were excited to learn of the
findings of Aiken et al. (52) and LaFrate et al. (53) showing that introduction of a single Asp-174 Lys or pairwise
Asp-174
Lys and Asp-175
Lys substitutions of aspartic acids 174 and 175 for lysine effectively blocked the ability of both the HIV-1
and SIV Nef proteins to down-regulate the CD4 receptor (52, 53). These
results parallel our present findings showing that a single Asp-174
Gly or Asp-175
Gly substitution and a double Asp-174
Gly and
Asp-175
Gly substitution introduced within the HIV-1 Nef acidic
sequence, Asp-Asp-Pro-Glu-Arg-Glu (residues 174-179), can effectively
abrogate interaction between Nef and c-Raf1 proteins in
vitro. The ability of the same pairwise mutations at the highly
conserved pair of acidic amino acids at positions 174 and 175 to
simultaneously block the Nef-c-Raf1 binding in vitro and the
Nef-induced CD4 down-regulation function in vivo strongly
suggests that c-Raf1 may have a role in Nef-induced CD4 modulation.
In studies with long term survivors with non-progressive HIV-1
infection, it has been shown that these individuals are infected with
an HIV-1 strain that has a high frequency of defective nef alleles (54). It is of interest to also note that a significant number
of these defective nef alleles also harbor the aspartic acid
to lysine (Asp-174 Lys) substitution at position 174 in the Nef
protein that are also defective in the ability to down-regulate the CD4
receptor (55). Whether or not the Nef-c-Raf1 interaction we described
in the present study is also linked to other Nef functions such as CD4
down-regulation (2, 13, 21) and modulation of T-cell signaling pathways
(2, 13-18) remains to be determined. However, the ability of Nef to
interfere with transduction of signals suggests that the viral protein
may directly interact with specific proteins of T-cell signaling
pathways.
Activated Ras, a target of the c-Raf1 protein, has been shown to recruit c-Raf1 to the plasma membrane where it becomes activated by tyrosine phosphorylation (56, 57). Whether Nef functions by disrupting interaction between Ras and c-Raf1 and therefore interfering with the Ras-Raf-MAP kinase signaling cascade remains to be determined. However, our present findings provide the first demonstration of the direct interaction between Nef and the c-Raf1 kinase in vitro. Although we have no data to suggest that Nef and Ras compete for binding c-Raf1, it is of interest to note the close similarity in amino acid sequence conservation between the c-Raf1-binding sites in Nef (residues 174-179) and Ras (residues 33-38) (36). Both sequences are acidic with similar orientation and spacing of critical acidic amino acids, and they are similarly located within conserved flexible or disordered secondary loop structures (44, 45).
Abrogation of the Nef-c-Raf1 interaction in vitro (this study) and the Nef-induced CD4 down-regulation in vivo (52, 53) by single or double mutations that substituted an uncharged nonpolar (aliphatic) or positively charged (basic) amino acid for the negatively charged (acidic) amino acid residues at positions 174 and 175 in the Nef sequence suggests that strong charge-charge interactions may be involved in both functions. A mechanism was previously suggested for involvement of conserved charge-charge interactions between the acidic side chain groups of Asp-33, Glu-37, and Asp-38 of Ras with the corresponding basic side chain groups of Lys-84, Lys-87, and Arg-89, respectively, within the RBD of c-Raf1 (36, 49). Therefore, it is tempting to speculate that Nef may function by presenting itself as a "false" G-protein (2, 58), and it may also participate as a downstream mediator or interceptor of signals passing through the Ras-Raf-MAP kinase signaling pathway. Like Ras (56, 57), Nef is associated with the inner face of the plasma membrane (1-3) and may physically interact with c-Raf1 upon recruitment to the membrane by Ras (56, 57). Therefore, direct physical interaction between Nef and the c-Raf1 kinase within the described acidic sequence motif could have important significance for the mechanism(s) of the Nef-induced T-cell signaling defects and CD4 down-regulation functions in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. Frank Ruscetti and Dr. David Derse for their critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 2P20RR011606-02.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.
To whom correspondence should be addressed: Dept. of Biology,
Spencer Hall, Rm. 110, Morgan State University, 1700 Cold Spring Ln.,
Baltimore, MD 21251. Tel.: 410-319-4005; Fax: 410-426-4732; E-mail:
ksamuel{at}moac.morgan.edu.
1 The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; bp, base pair; MAP, mitogen-activated protein; RBD, Ras-binding domain.
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REFERENCES |
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