From the
Among the primate lentiviruses (human immunodeficiency virus
(HIV) -1, HIV-2, and simian immunodeficiency virus (SIV)), the nef gene is highly conserved and encodes a myristylated protein of
The nef gene was first identified as an open reading
frame that overlaps with the 3`-long terminal repeat of the human
immunodeficiency virus type 1
(HIV(
Nef encodes a myristylated, phosphorylated protein of approximately 27
kDa that forms homomeric oligomers and intramolecular disulfide
bonds(1, 8, 9, 10, 11, 12, 13, 14) .
Myristylation has been reported to be important for its
function(13, 14, 15, 16, 17) .
In infected cells, Nef primarily localizes to the cytoplasm and
intracellular membranes (18) and preferentially associates with
the cytoskeleton(15) . However, it has also been reported to be
present in the nucleus(19, 20) .
Initially, Nef was
reported to have a negative effect on virus replication and
transcription(12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29) .
However, later studies demonstrated that Nef expression has no effect
on viral replication in T-cell lines but may have a positive effect on
virus replication in peripheral blood mononuclear cells (30, 31, 32) or fetal thymic and liver implants
in mice having severe combined immunodeficiency(33) . Thus, the
role of this protein on virus replication is controversial. This may be
due, in part, to the use of various alleles of Nef and different cell
culture systems. Nevertheless, studies in SIV-infected macaques have
revealed that the preservation of a full-length Nef is necessary for
the maintenance of high viral loads and for the progression of
disease(34) . Although the basis for this requirement is still
not known, it may be related to a role of Nef in T-cell
activation(28, 29, 35, 36, 37, 38, 39, 40) .
Indeed, several studies indicate that the expression of Nef affects
a signal transduction pathway. Nef has been shown to down-regulate the
expression of CD4 on T-lymphocytes in
vitro(41, 42, 43, 44) . This effect
of Nef is mediated by an endocytotic mechanism (17, 39, 45) that involves the targeting of CD4
for lysosomal degradation(17, 39, 45) .
Furthermore, we recently reported that the expression of a CD8-Nef
fusion protein in T-cells leads to inhibition or activation of early T
cell signaling events depending on its localization within the cell
(40). When expressed at the inner surface of the plasma membrane, the
activation markers CD69 and CD25 were induced, and the cells died by
apoptosis. Cells that survived contained truncated Nefs.
To
elucidate the pathway by which Nef functions, attempts have been made
to identify cellular proteins that complex with Nef. Harris and Coates (46) reported that baculovirus-expressed glutathione S-transferase-Nef fusion proteins associate with cellular
proteins of various sizes which depend on their subcellular location.
We demonstrated that Nef expressed either as a CD8-Nef fusion protein
or native Nef itself specifically interacted with a serine kinase in
human T-lymphocytes(47) . This kinase was found to phosphorylate
proteins of 62 and 72 kDa that coimmunoprecipitated with Nef.
In the
present study, the regions of Nef that are important for these
interactions with the kinase activity were investigated. Using a
transient expression assay, we determined that various alleles of Nef
are capable of associating with the kinase, and the kinase is present
in non-lymphoid cells from several different species. We found that
stability of the molecule affects the association of Nef with the
cellular kinase activity. Moreover, two different regions of Nef are
critical for the association with this activity; the first has been
mapped to a domain that overlaps a centrally located, highly conserved
portion of the molecule, and the second represents a membrane targeting
signal.
Site-specific mutations, carboxyl-terminal truncations, and
amino-terminal deletions in Nef were introduced into the
pCMV/CD8-SF2Nef, pCMV/CD8-SIV
Chimeric CD8-Nef plasmids
that reciprocally exchanged the centrally located conserved region of
HIV-1
Plasmid DNA
(15 µg) was transfected into
Results from studies with CD8-SF2Nef 1-94 and
CD8-SF2Nef 1-127 suggest that the highly conserved central region
of Nef (HIV-1
Since the arginines at positions 109 and 110 of SF2Nef are
also present in SIV
Because Nef has been reported to be
phosphorylated by protein kinase C (8, 9) and phosphorylation of Nef
may be important for function(12) , potential phosphorylation
sites were also selected for mutation. Serine 14, threonine 84, and
serine 107 of Nef were mutated to alanine residues. In each case, these
amino acid substitutions did not affect the associated kinase activity (Fig. 4A, lanes 15 and 16 and Fig. 6). In addition, the conserved cysteine at position 146 also
was converted to an alanine. This residue has been implicated as being
important for intrachain disulfide bond formation and
function(11) . A change in this residue also did not affect the
associated kinase activity (Fig. 6). These findings suggest that
neither protein kinase C phosphorylation of Nef nor the formation of
intrachain disulfide bonds are critical for this association.
Finally, we found that the reciprocal exchange of the centrally
located conserved domain between CD8-SF2Nef and
CD8-SIV
We previously reported the association of a cellular serine
kinase with HIV
Using amino-terminal and carboxyl-terminal deletion mutants
of Nef, we localized the region of Nef that is important for the
association with the kinase activity to amino acids 45-127. This
region overlaps the centrally located, highly conserved domain (amino
acids 74-152, Fig. 3) of HIV-1 and SIV Nef proteins. While
the basic region and the putative amino-terminal phosphorylation site
(Thr
Using point
mutations in HIV-1
Although we have mapped the region of Nef critical for the
association with the kinase activity, we cannot differentiate between
the possibilities that this region functions as a binding domain for
the serine kinase and/or, one or both of the phosphorylated substrates
(62- or 72-kDa proteins). It remains possible that one or both of the
phosphorylated proteins may be the kinase(s) in lymphoid cells. The
lack of phosphorylation of the 72-kDa protein in non-lymphoid cells
indicates that this protein is not the kinase in these cells.
Identification and characterization of the 62- and 72-kDa proteins
should resolve this question.
Our finding with the myristylation
mutant indicates that Nef interacts directly with a membrane-associated
kinase activity and supports the idea that membrane targeting is likely
to be important for Nef function. In this cellular compartment, Nef may
affect an intracellular signaling pathway(s).
In this regard, we
observed differential effects of Nef on early signaling events in
T-cells depending on its subcellular expression (40). When Nef was
expressed on the inner surface of the plasma membrane, an activated,
apoptotic phenotype was observed. Only cells expressing truncated forms
of Nef survived this event. Because membrane targeted Nef associates
with the kinase activity and truncated forms of Nef do not, these
results suggest that Nef may influence intracellular activation through
its interaction with the cellular serine kinase. Whether the
Nef-associated kinase activity is linked to other functions of Nef (e.g. CD4 down-regulation and enhancement of viral
infectivity) remains to be determined.
Nef has been shown to be
important for the maintenance of high viral loads and the progression
of SIV-infected macaques to disease(34) . Our observations that
Nef of the pathogenic strain SIV
In summary, our results define two
regions of Nef that regulate one of its biochemical properties: the
central homology region and the myristylation signal sequence.
Moreover, this study demonstrates that the Nef-associated kinase
activity represents a conserved function of primate lentiviral Nef
proteins. It will be important to determine whether this association is
necessary for viral replication and/or disease progression in
vivo.
We thank Greg Harrowe for his expert technical
assistance. The full-length nef gene of SIV
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
27 kDa (HIV-1) or
34 kDa (HIV-2, SIV). Previously, we found
Nef expressed either as a CD8-Nef fusion protein or as a native protein
in virally infected T cell lines associates with a cellular serine
kinase. This kinase activity phosphorylated two proteins of 62 and 72
kDa that coimmunoprecipitate with Nef in in vitro kinase
assays. Using transient expression, various Nef alleles and mutants
have been analyzed for association with the cellular kinase activity.
The ability of Nef to associate with the kinase activity is conserved
among several alleles of HIV-1 as well as SIV
and is
observed in non-lymphoid cell lines of simian and murine origins. Two
separate regions of HIV-1
Nef are critical for the
associated kinase activity. One domain overlaps with a central highly
conserved region found in all primate lentivirus nef genes and
has been provisionally mapped to amino acids 45-127. Because
membrane localization of Nef is important for the associated cellular
kinase activity, the second domain represents a membrane targeting
signal. Moreover, point mutations within the central region that
abrogate the Nef-associated kinase activity in HIV-1
Nef
have the same effect when introduced into SIV
Nef.
)-1)
(1) . It is conserved among all
primate lentiviruses, i.e. HIV-1, HIV-2, and simian
immunodeficiency virus (SIV)(2, 3) . The viral gene
product is translated from multiply spliced transcripts and is
expressed together with the regulatory proteins Tat and Rev early in
the viral replicative
cycle(4, 5, 6, 7) .
Cells and Antibodies
The Jurkat T-cell lines
that constitutively express the CD8/HIV-1 Nef fusion
protein (J.CN) and HUT 78 cells chronically infected with HIV-1
(E-line) were cultured as described previously(47) . COS-7
cells were grown in Dulbecco's minimal essential medium
containing 10% fetal bovine serum, 1% glutamine, and 1%
penicillin-streptomycin. The hybridomas, 51.1 and MH-SVM26, which
produce monoclonal antibodies directed against the human CD8 molecule
or HIV-1 gp41, respectively, were obtained from the American Type
Culture Collection and cultured in HY media containing 20% fetal bovine
serum. Hybridoma supernatants containing CD8-specific monoclonal
antibody (
-CD8) or gp41-specific monoclonal antibody (
-gp41)
were collected and either partially purified by ammonium sulfate
precipitation or used directly for immunoprecipitation analyses. The
rabbit anti-HIV-1
Nef antibody (
-Nef) was provided by
Chiron Corporation (Emeryville, CA).
Plasmid Construction and Transfection
To express
native Nef in COS-7 cells, the complete nef gene of
HIV-1 was inserted into the pRc/CMV expression vector
(Invitrogen) generating the plasmid pCMV/SF2Nef. In addition, the
complete nef gene of HIV-1
and the pathogenic
SIV
strain were fused to the extracellular and
transmembrane domains of the human CD8 molecule thereby generating a
hybrid CD8-SF13Nef or CD8-SIV
Nef fusion that was
subsequently cloned into the pRc/CMV plasmid as described
previously(40) . These plasmids were designated pCMV/CD8-SF13Nef
and pCMV/CD8-SIV
Nef, respectively.
Nef, or the
pCMV/SF2Nef expression vectors using the single-stranded
oligonucleotide-directed mutagenesis strategy (Bio-Rad). The
amino-terminal deletion mutants of Nef were fused in-frame to the
extracellular and transmembrane domains of CD8. All of the mutations
were confirmed by DNA sequencing and by the presence of introduced
restriction endonuclease cleavage sites.
Nef for the homologous region of SIV
Nef were constructed as follows. Unique KpnI and EcoRV sites, which did not alter the amino acid sequence of
the Nef protein, were engineered into the SIV
plasmid by site-directed mutagenesis. The KpnI-EcoRV fragments from the HIV-1
and
SIV
Nefs were subsequently exchanged. The domain
substitution results in the replacement of amino acids 78-139 of
HIV-1
Nef for amino acids 107-167 of
SIV
Nef and vice versa.
5
10
COS-7
cells by calcium-phosphate precipitation. Twenty-four h after
transfection, the cultures were split in half and propagated for an
additional 24 h before harvesting for protein labeling and kinase
analyses.
Metabolic Labeling, Immunoprecipitation, in Vitro Kinase
Assay, and Pulse-chase Analysis
The procedures for metabolic
labeling, cellular extraction, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, and the in vitro kinase assay were
performed as described previously(47) . For pulse-chase
analyses, transfected COS-7 cells were starved with methionine- and
cysteine-free RPMI 1640 media for 30 min and pulse labeled for 15 min
with labeling medium containing 500 µCi/ml S-Translabel (ICN) at 37 °C. After washing the
monolayers twice with Dulbecco's phosphate-buffered saline, 10 ml
of Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum was added to the cells, except for those representing the
zero time point which was extracted immediately. After chase times of
30 min, 1, 2 and 4 h, the respective cells were washed twice with
Dulbecco's phosphate-buffered saline and extracted.
Immunoprecipitations were done as described above.
Kinase Association Is a Highly Conserved Property of
Nef and Can Be Detected in Several Cell Types
Previously, using
a CD8-Nef fusion protein or native Nef, we identified a serine kinase
activity that interacts with HIV-1 Nef expressed in human
T-cell lines (47). This kinase was responsible for the phosphorylation
of two proteins of 62 and 72 kDa that specifically coimmunoprecipitate
with native Nef and CD8-Nef fusion proteins. We also reported that
HIV-1
Nef truncated at amino acid 95 (CD8-SF2Nef
(1-94)) no longer associated with the kinase activity, as
indicated by the absence of the 62 or 72 kDa phosphorylated proteins in
Nef immunoprecipitates. In order to develop a rapid assay for screening
different alleles and mutants of Nef for the associated kinase
activity, transient expression of Nef in simian COS-7 cells was
conducted (Fig. 1A). By metabolic labeling, substantial
expression of CD8-SF2Nef, CD8-antisense Nef, CD8-SF2Nef (1-94),
CD8-SIV
Nef, and CD8-SF13 Nef could be detected in
transfected COS-7 cells (Fig. 1A, lanes
2-6).
Figure 1:
The Nef-associated kinase activity is
found in simian fibroblast cells expressing different Nef alleles. A, COS-7 cells transfected with CD8-antisense SF2Nef (Anti, lanes 1, 2, 7, and 8), CD8-SF2Nef (SF2, lanes 3 and 9), CD8-SF2(1-94) (SF2(1-94), lanes 4 and 10), CD8-SF13 Nef (SF13, lanes 5 and 11), and CD8-SIV Nef (SIV, lanes 6 and 12) were extracted with (lanes 1-6) or without (lanes 7-12)
metabolic labeling. Immunoprecipitations were done by using a control
anti-gp41 monoclonal antibody (lanes 1 and 7) or an anti-CD8
monoclonal antibody (lanes 2-6 and 8-12).
An in vitro kinase assay was performed on the unlabeled
immunoprecipitates (lanes 7-12). B, kinase
assays were performed on immunoprecipitates from either COS-7 (lanes 1 and 2) or Jurkat cells (J.CN,lanes 3
and 4) expressing CD8-SF2Nef. A control anti-gp41 monoclonal
antibody (
-gp41,lanes 1 and 4) or an
anti-CD8 monoclonal antibody (
-CD8,lanes 2 and 3) were used. Molecular size standards are marked on the right
and the positions of the phosphorylated 62- (p62) and
72-kDa proteins are indicated on the
left.
In vitro kinase assays performed on
immunoprecipitates from unlabeled, transfected cell extracts
demonstrated that besides CD8-SF2Nef, CD8-SF13 Nef, and
CD8-SIV Nef also interacted with this kinase (Fig. 1A, lanes 9, 11, and 12). The 62-kDa protein was specifically phosphorylated in
these immunoprecipitates but not in immunoprecipitates containing
CD8-antisense Nef (anti) or a truncated Nef
(SF2Nef(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94) ) (Fig. 1A, lanes 8 and 10). Several
other phosphorylated bands are observed in lanes 10 and 11. These were not observed reproducibly and, thus, were
considered to be nonspecific (for example compare Fig. 2A, lane 10 and Fig. 1A, lane 10). The phosphorylated 62-kDa protein from T-cell lines
and COS-7 cells was similar by partial V8-protease mapping (data not
shown). Since three different alleles of Nef were capable of
interacting with the kinase, it is likely that this property of Nef is
conserved. Moreover, because the kinase activity was found in cultures
of human T-cell lines (Jurkat, HUT-78)(47), simian fibroblasts (COS-7),
and murine fibroblasts (NIH3T3; data not shown) that express Nef, the
kinase itself is conserved. However, the phosphorylation of the 72-kDa
substrate was not observed in the latter two cell types (COS-7 and
NIH3T3) (Fig. 1B). Thus, phosphorylation of the 72-kDa
protein may be T-cell-specific.
Figure 2:
The
central conserved region of Nef is important for association with the
cellular kinase activity. A, COS-7 cells transiently
expressing CD8-SF2 Nef (SF2,lanes 1, 2, 8, and 9), CD8-SF2(1-94) (1-94,lanes 3 and 10), CD8-SF2(1-127) (1-127,lanes 4 and 11),
CD8-SF2(45-210) (45-210,lanes 5 and 12), and CD8-SF2(70-210) (70-210,lanes 6 and 13) were extracted with (lanes
1-6) or without (lanes 8-13) metabolic
labeling as in Fig. 1. Immunoprecipitations were performed either with
an anti-gp41 monoclonal antibody (lanes 1 and 8) or a CD8-specific monoclonal antibody (lanes 2-6 and 9-13). An in vitro kinase assay was
performed on the immunoprecipitates from unlabeled extracts (lanes
8-13). B, truncation of Nef at amino acid 127
results in an unstable protein. Pulse-chase analysis was performed
using cells transiently expressing either CD8-SF2 Nef (lanes
5-8) or CD8-SF2(1-127) (lanes 1-4).
Metabolically labeled cells were extracted at 0, 0.5, 1.0, and 2 h
after the initial 15-min pulse. Immunoprecipitations were
performed using a monoclonal antibody to CD8. The increased mobilities
of CD8-SF2 Nef and CD8-SF2(1-127) during the chase period are
probably due to the glycosylation of the CD8 portion of the molecule.
The positions of CD8-SF2 Nef and CD8-SF2(1-127) are indicated on
the right.
The Central Domain of Nef Is Critical for Association
with the Kinase Activity
To determine the region of Nef
important for the observed kinase activity, amino-terminal and
carboxyl-terminal truncations of Nef in the CD8-SF2Nef plasmid were
constructed, and the deletion mutants were analyzed for their ability
to complex with the kinase activity in COS-7 cells (Fig. 2A). Deletion of amino acids 95-210 resulted
in the loss of the associated kinase activity, whereas introduction of
a stop codon immediately after amino acid 127 resulted in a greatly
reduced, but detectable phosphorylation of the 62-kDa protein (Fig. 2A, lanes 10 and 11). This
mutant is truncated at the same position as Nef from the
HIV-1 strain(48) . Unlike the CD8-SF2Nef
1-94 protein that is detectable by metabolic labeling, the
CD8-SF2Nef 1-127 protein is expressed weakly and appears to
undergo proteolytic degradation. Since the stability of Nef may affect
kinase association, pulse-chase analyses were performed to compare the
metabolic half-life of CD8-SF2Nef 1-127 with wild type CD8-SF2
Nef (Fig. 2B). Previously, we found that the metabolic
half-life of CD8-SF2Nef was about 8 h(47) . In contrast, the
metabolic half-life of the CD8-SF2Nef 1-127 protein was less than
1 h. Other CD8-SF2Nef truncated proteins encoded by amino acids
1-145 and 1-167 were even less stable than the SF2 Nef
1-127, and therefore had none or barely detectable levels of
associated kinase activity (summarized in Fig. 6). From these
results, we conclude that carboxyl-terminal truncations of Nef beyond
amino acid 94 resulted in the expression of unstable fusion proteins.
Figure 6:
Summary of in vitro kinase assay
results. A schematic representation of HIV-1 Nef is
indicated at the top (white bar). The central domain
homologous to HIV-2 and SIV Nefs (SIV Homology) is flanked by
acidic regions depicted as partially hatched boxes. The
amino-terminal basic domain is represented by a black box. Numbers indicate amino acid position. The conserved cysteines
Cys
, Cys
, and Cys
of HIV-1 Nef
are indicated. Nef mutants used in this study are indicated below, and numbers indicate the amino acids encoded by each mutant. The
results of the in vitro kinase assay are shown on the right.
Wild-type (++), reduced (+), weak (±), and
undetectable (-) levels of kinase activity in COS-7 cells (as
determined by phosphorylation of the 62-kDa protein) are indicated on
the right. The SC mutation, which utilizes an alternative open reading
frame as described in the text, was introduced into SF2 Nef (SF2(SC)) and is depicted as a box filled with wavy
lines. HIV-1
and SIV
Nefs are
shown as boxes filled with diagonal lines. The chimeric Nef
proteins are indicated as SF2(SIV) and SIV(SF2).
Amino-terminal deletions of CD8-SF2Nef that resulted in truncated
proteins encoded by amino acids 45-210, 70-210, and
89-210 of Nef (Fig. 6) were also analyzed for the ability
to associate with the kinase activity. The deletion of amino acids
1-44 affected neither the stability of the protein nor the
ability of the protein to associate with the kinase activity (Fig. 2A, lanes 5 and 12). The
deletion of amino acids 1-69 was stable yet did not exhibit
associated kinase activity (Fig. 2A, lanes 6 and 13). However, the deletion of amino acids 1-88
adversely affected protein stability, and consequently no kinase
activity was detected (Fig. 6). From these amino-terminal and
carboxyl-terminal deletions, we have provisionally mapped the domain of
Nef important for association with kinase activity to amino acids
45-127.
Nef amino acids 74-152, Fig. 3)
contains an important domain for associated kinase activity. To further
examine this possibility, we constructed a CD8-SF2Nef mutant that
resembles the HIV-1
allele(49) . In nef of
HIV-1
, a single nucleotide deletion at position 9117
results in the utilization of an alternative open reading frame that
continues for 34 amino acids until another single nucleotide insertion
at position 9219 restores the original nef open reading frame.
These mutations were placed into the CD8-SF2nef gene so that
the alternate open reading frame was utilized between amino acids 105
and 138. The mutant protein was metabolically stable (data not shown),
yet no associated kinase activity was detected (summarized in Fig. 6). These findings further indicate that the central region
between amino acids 94 and 127 of Nef is important for the association
with the kinase activity.
Figure 3:
Alignment of HIV-1, HIV-2, and SIV Nefs in
the central, conserved region. Sequence comparisons were done using the
protein sequence alignment program, BLAST (50). All the sequences (51)
were aligned and compared to the Nefs of HIV-1 and
HIV-1
. HXB2 Nef is prematurely truncated at amino acid
123 as indicated by a stop codon (asterisk). Dashes indicate positions where the amino acids are identical to the HXB2
and BRU sequences. Non-identical amino acids are indicated in single-letter amino acid code. The coordinate of the last
amino acid residue in each of the aligned sequences is indicated on the
right. A gap (indicated by a period) was introduced in the
HIV-1
sequence to facilitate the alignment. A consensus
sequence (Consensus) of amino acid residues that are identical
among all of the aligned sequences (with the exception of
HIV-1
; see text for a description of the SC allele) are
shown in capital letters, and their positions (#) are
indicated; other positions of non-identity are shown (
). Dots are placed above the Nef residues that were mutagenized
in the SF2 allele for this study. The position of the stop codon for
the SF2(1-94) truncated Nef is indicated (+). The
truncation of SF2 Nef at amino acid 127 (CD8-SF2(1-127)) directly corresponds to the
site of truncation in HXB2 Nef (amino acid 123). Potential protein
kinase C (PKC) phosphorylation sites, and the
conserved acidic region (Acidic) are indicated on the bottom
by bars.
Specific Amino Acid Substitutions in the Conserved Region
of Nef Can Affect Association with the Kinase Activity
Alignment
of HIV-1, HIV-2, and SIV Nef sequences indicated that several amino
acids in the region between 94-127 were identical among all
primate lentiviral Nef proteins (Fig. 3, note consensus sequence
between 94 and 127). Of the conserved amino acids, 2 adjacent arginine
residues at positions 109 and 110 of SF2 Nef represented a charged
region. Because hydrophobic moment analyses (52) performed on
the entire SF2 Nef molecule indicated that this region had a high
degree of hydrophilic character, both of these amino acids were mutated
to leucine residues. The mutant protein (CD8-SF2Nef
RR
-LL) was stable but lost the ability to
associate with the kinase activity (Fig. 4A, lanes 3 and 12). Several other phosphorylated bands are observed
in various lanes (lanes 11-18). These were not observed
reproducibly and were considered to be nonspecific (for example compare Fig. 4A, lane 12 and Fig. 4B, lane 7). Mutation of the individual arginines resulted in the
loss of associated kinase activity only when arginine 110, but not when
arginine 109, was changed to leucine (Fig. 4A, lanes
13 and 14).
Figure 4:
Specific, highly conserved amino acids in
the central homology domain of Nef are important for the association
with the cellular kinase activity. A, COS-7 cells transiently
expressing CD8-SF2Nef (SF2,lanes 1, 2, 10, and 11),
CD8-SF2(RR
-LL) (R
-R
-LL,lanes 3 and 12), CD8-SF2(R
-L) (R
-L,lanes 4 and 13), CD8-SF2(R
-L) (R
-L,lanes 5 and 14), CD8-SF2(T
-A) (T
-A,lanes 6 and 15), CD8-SF2(S
-A) (S
-A,lanes 7 and 16), CD8-SIV
Nef (SIV,lanes
8 and 17), and CD8-SIV(R
R
-LL) (SIV
),lanes 9 and 18) were extracted with (lanes 1-9) or without (lanes 9-18) metabolic labeling. Immunoprecipitations
were performed with monoclonal antibodies against gp41 (lanes 1 and 15) or CD8 (lanes 2-9, and 11-18). An in vitro kinase assay (lanes
10-18) was done as described in Fig. 1. The positions of p62
and CD8-Nef are marked on the right. B, a point mutation in
CD8-SF2Nef eliminates the ability of Nef to associate with the cellular
kinase activity in T-cell lines. Jurkat cells that
constitutively express CD8-SF2Nef (lanes 1, 2, 4, and 5) and
CD8-SF2Nef(R
-R
-LL) (lanes 3, 6, and 7) were extracted with (lanes
1-3) or without (lanes 4-7) metabolic
labeling. Immunoprecipitations were performed with monoclonal
antibodies against gp41 (lanes 1, 4, and 6)
or CD8 (lanes 2, 3, 5, and 7). An in vitro kinase assay (lanes 11-20) was
performed as described in Fig. 1. A 34 kDa band representing a
proteolytic degradation product of
CD8-SF2Nef(R
-R
-LL) is observed in lane
3. The positions of CD8-Nef, p62, and p72 are
marked.
To determine whether the CD8-Nef
RR
-LL fusion protein associates with the
cellular kinase activity in T-cell lines, we have stabily expressed
this mutant in Jurkat T-cells and have found that the protein is
metabolically stable, yet does not associate with the cellular kinase
activity (Fig. 4B). These results confirm those obtained
in COS cells with this mutant (CD8-Nef
R
R
-LL, Fig. 4A, lane
12).
Nef, the arginine to leucine
substitutions were introduced into CD8-SIV
Nef at
positions 137 and 138. This SIV
Nef mutant,
CD8-SIV
Nef R
R
-LL, like its
HIV-1
counterpart CD8-SF2Nef
R
R
-LL, was metabolically stable but
defective for the associated kinase activity (Fig. 4A,
compare lanes 11 and 12 with lanes 17 and 18). These results indicate that the homologous arginine
residues in SIV
are also critical for the association
with the kinase activity.
Nef gave rise to stable chimeric
proteins that did not associate with the kinase activity despite
retaining the conserved arginine at residue 110 (SF2) or 138
(SIV
) (SF2(SIV) and SIV(SF2), Fig. 5). These results suggest that protein
conformation may be important for association of Nef with the kinase
activity.
Figure 5:
Analysis of HIV/SIV Nef chimeras in the
kinase assay. COS-7 cells transiently expressing CD8-SF2Nef (SF2,lanes 1, 5, 9, and 12),
CD8-SIV (SIV,lanes 2 and 6), CD8-SIV(SF2KpnI-EcoRV) (SIV(SF2),
lanes 3, 4, 7, 8, 11, and 14), and CD8-SF2(SIVKpnI-EcoRV) (SF2(SIV), lanes 10 and 13) were extracted
with (lanes 1-4 and 9-11) and without (lanes 5-8 and 12-14) metabolic labeling.
Immunoprecipitations were done using a monoclonal antibody to CD8. The
positions of p62 and CD8-Nef are indicated on the
right.
Myristylation of Native Nef Is Critical for the
Associated Kinase Activity
Several reports have indicated that
myristylation is important for Nef
function(13, 15, 16, 17, 44) .
To determine whether myristylation of native Nef was important for the
Nef-associated kinase activity, a myristylation defective mutant, that
converts glycine at position 2 to alanine, was constructed. The ability
of the mutant Nef to associate with the kinase activity in transient
assays was tested. Whereas native SF2 Nef was observed to associate
with the kinase activity ( Fig. 6and Fig. 7, lanes 6 and 8), the myristylation negative mutant
(G-A) and the control antisense Nef (fen) did not associate
with the kinase activity (Fig. 7, lanes 5 and 7). These results indicate that the myristylation of Nef is
also a critical determinant for the association with the kinase
activity. Because both the CD8-Nef fusion protein and the native Nef
associated with the kinase activity, and the myristylation-defective
Nef did not, we conclude that the targeting of Nef to intracellular
membranes is important for association with a cellular kinase activity.
Figure 7:
The myristylation of Nef is critical for
the association with the cellular serine kinase activity. COS-7 cells
transiently expressing HIV-1 antisense Nef (fen, lanes 1 and 5), HIV-1
native Nef (Nef, lanes 2 and 6), a myristylation
defective HIV-1
Nef (myr
, lanes 3 and 7), together with HUT 78 cells
chronically infected with HIV-1
(E-line, lanes 4 and 8) were extracted with (lanes
1-4) or without (lanes 5-8) metabolic
labeling. Immunoprecipitations were done with a polyclonal rabbit
anti-Nef serum. An in vitro kinase assay was performed as
described in Fig. 1 (lanes 5-8). The positions of the
72-kDa phosphorylated protein (p72), p62, and Nef are
indicated on the right.
Nef in T-cell lines(47) . In the
present study, we demonstrate that the association with this kinase is
a highly conserved property of Nef and is found in lymphoid and
non-lymphoid cells from different species. We have also found that Nef
from HIV-1
-infected peripheral blood mononuclear cells is
capable of associating with the cellular serine kinase activity (data
not shown). Since phosphorylation of the 72-kDa protein in COS-7 and
NIH3T3 cells was not detectable, the association of this protein with
Nef and/or its phosphorylation by the serine kinase may be specific for
T-cells.
) of Nef are dispensable for association with the
kinase activity (45-210, Fig. 6), further deletion of the
region between amino acids 45 and 70, which includes the amino-terminal
acidic domain (amino acids 63-69), eliminates the Nef-associated
kinase activity (70-210, Fig. 6). These findings indicate
that the retention of only the conserved domain of Nef is not
sufficient for its ability to associate with the kinase activity and
suggest that multiple sites along this protein interact with the kinase
or its substrates. Alternatively, the amino-terminal region (amino
acids 45-70) might be required to maintain a proper conformation
of Nef. The latter possibility is consistent with the prediction from
NMR spectrographic studies of proteolytic peptide fragments that amino
acids 66-206 of HIV-1
Nef may represent a
separable functional domain (53). Furthermore, we observe that
carboxyl-terminal truncations of Nef are relatively unstable. This
effect may be due to the presence of internal PEST-like sequences (54) in Nef which, as a result of truncations, are now present
near the carboxyl-terminus, and signal the targeting of newly
synthesized proteins for premature degradation.
Nef, we have determined that several
conserved amino acids that are located in different regions of the
central Nef homology domain are important for the association with the
kinase activity. While mutation of potential protein kinase C
phosphorylation sites did not affect the ability of Nef to complex with
the cellular kinase activity, mutation of the double arginines at
positions 109 and 110 of HIV-1
Nef and positions 137 and
138 of SIV
Nef did. One of these
residues, arginine 110 of HIV-1
Nef, was critical since
conversion of this residue to leucine abrogates the associated kinase
activity. Because reciprocal substitution of the conserved domains of
HIV-1
Nef and SIV
Nef did
not result in kinase association, it appears that the conserved central
domain of Nef interacts with its flanking regions in conferring a
conformation that is important for association with the kinase
activity.
also associates with
the kinase activity and that the region of Nef important for this
association maps to the central homology domain raise the possibility
that this association with the kinase may be important for viral
pathogenesis in vivo.
was constructed and provided by Paul Luciw (University of
California, Davis, CA).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.