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(Received for publication, October 10, 1995; and in revised form, February 21, 1996) From the
Vif is a 23-kDa protein encoded by human immunodeficiency virus,
type 1 (HIV-1) which is important for virion infectivity. Here, we
describe the phosphorylation of HIV-1 Vif and its role in HIV-1
replication. In vivo studies demonstrated that Vif is highly
phosphorylated on serine and threonine residues. To identify
phosphorylation sites and characterize the Vif kinase(s), Vif was
expressed in Escherichia coli and purified for use as a
substrate in in vitro kinase assays. The purified Vif protein
was phosphorylated in vitro on serine and threonine residues
by a kinase(s) present in both cytosol and membrane fractions.
Phosphorylation of Vif was stimulated by phorbol 12-myristate
13-acetate and inhibited by staurosporine and hypericin, a drug with
potent anti-HIV activity. The Vif kinase(s) was resistant to inhibitors
of protein kinase C, cAMP-dependent kinase, and cGMP-dependent kinase,
suggesting that it is distinct from these enzymes. To identify the
phosphorylation sites,
Vif, one of the human immunodeficiency virus type I
(HIV-1) ( Vif is a highly
basic protein consisting of 192 amino acids. Previous studies have not
demonstrated any post-translational modifications. In HIV-1-infected
cells, Vif is predominantly localized to the cytoplasm, where it exists
in both membrane-associated and cytosolic
forms(11, 12) . A small quantity of Vif (approximately
10 to 50 molecules) is associated with HIV-1 virus
particles(6, 13) , but whether virion incorporation is
important for Vif function is unknown. Membrane association of Vif is
important for its biological function(11, 14) .
Membrane localization requires C-terminal basic domains and an
interaction with a membrane-associated protein(s)(14) .
However, the putative protein required for membrane association of Vif
has not been identified. Little is known about the functional role
of phosphorylation of HIV-1 proteins. Several HIV-1 proteins, including
p24 In this report, we show that Vif is phosphorylated in
vitro and in vivo by a serine/threonine kinase(s). Three
phosphorylation sites are identified within the Vif C terminus. One
phosphorylation site, Ser
Figure 1:
Expression
and phosphorylation of Vif in vivo. A, HeLa cells
were infected with recombinant vaccinia virus VV-T7, and then
transfected with pTM-1 (lanes 1 and 3) or pTM-1/hVif (lanes 2 and 4). Cells were metabolically labeled
with [
Figure 2:
Purification of Vif from E. coli.
Vif was expressed in E. coli using the pD10Vif bacterial
expression plasmid. A, denaturing Ni
Figure 3:
Phosphorylation of Vif in vitro. In vitro kinase assays were performed using purified Vif
protein and [
Vif has been shown to exist in
both membrane-associated and soluble cytosolic forms(11) . To
determine whether the Vif kinase(s) colocalizes with Vif, the CEM
lysate was fractionated into an S200 soluble cytosolic fraction, P50
cellular membrane fraction, and P200 microsomal membrane fraction as
described (11) and the subcellular fractions were used for in vitro kinase assays. In a previous study(11) , we
demonstrated that these fractions contain approximately 40, 20, and
30%, respectively, of the total Vif in HIV-1-infected CEM cells. The
Vif kinase activity was present in all three fractions, although more
kinase activity was present in the membrane fractions (Fig. 3B), indicating that Vif and its kinases(s) show
a similar subcellular distribution. Phosphoamino acid analysis
demonstrated that approximately 60% of Vif phosphorylation in vitro occurred on threonine residues, and about 40% on serine residues (Fig. 3C, left), similar to the results obtained in
vivo. No tyrosine phosphorylation was detected. Similar results
were obtained when in vitro kinase assays were performed using
the S200, P50, and P200 subcellular fractions (Fig. 3C,
right). Thus, the Vif kinase(s) present in the cytosolic and
membrane fractions may be the same enzyme(s).
Figure 4:
Effects of kinase inhibitors and
activators on Vif phosphorylation in vitro. In vitro kinase assays were performed using purified Vif protein and
[
Figure 5:
Reverse-phase HPLC and phosphoamino acid
analysis of
Phosphoamino acid analysis of
the radioactive Vif peptides demonstrated that peptide I contains
phosphothreonine and peptide II contains both phosphoserine and
phosphothreonine (Fig. 5C). Inspection of the amino
acid sequences (Table 1) revealed that peptide I contains two
threonine residues, while peptide II contains two serine and three
threonine residues. To unequivocally identify the phosphorylated
residues, the peptides were subjected to radioactive sequencing.
Radioactive sequencing of peptide I showed that
Figure 6:
Identification of phosphorylation sites in
Vif. Radioactive sequencing of the
The preceding experiments
identify three phosphorylation sites in Vif, all within the C terminus,
Ser
Figure 7:
Tryptic phosphopeptide mapping of Vif
phosphorylated in vitro and in vivo.A, Vif
phosphorylated in vitro using
[
Figure 8:
Activity of Vif Ser
Many viral proteins are regulated by phosphorylation during
different stages of the virus life cycle. In this study, we show that
Vif, an essential accessory protein for HIV-1 replication, is
phosphorylated in vitro and in vivo by a cellular
kinase(s) and provide evidence that Vif phosphorylation is important
for HIV-1 replication in vivo. Phosphorylation of Vif in
vitro and in vivo occurred on serine and threonine
residues and generated similar patterns of two-dimensional tryptic
phosphopeptide mapping. Thus, phosphorylation in vitro and in vivo is likely to occur on the same sites, although the
relative amounts of phosphorylation at specific sites may differ. Three
phosphorylation sites (Ser Vif contains several potential phosphorylation sites,
including consensus sites for PKC, PKA, PKG, and casein kinase II.
Among these sites, Ser All three Vif phosphorylation sites identified in
this study are localized within the C-terminal region. This observation
suggests that the C-terminal region of Vif is likely to be exposed on
the surface of the molecule and thus be accessible to the Vif
kinase(s). Further support for this conclusion is provided by computer
analysis using hydrophilicity or surface probability plots, which show
that the Vif C terminus from positions 150 to 192 is likely to be an
exposed region of the molecule. Biochemical studies of Vif have been
hindered by its unusual biochemical properties. Vif is a very basic
protein (predicted pI = 10.7) (10) which is present in
HIV-1-infected cells at relatively low levels. We and others have found
that Vif is very inefficiently immunoprecipitated by Vif antiserum,
most likely due to its ability to form high molecular weight
complexes. Although Vif is required for HIV-1
replication in primary cells and certain T cell lines, its biochemical
mechanism of action remains unknown. Vif acts during the late stages of
the virus life cycle to permit correct assembly of virus
particles(6, 7) . A previous study suggested that Vif
may have cysteine protease activity which is involved in the processing
of the HIV-1 envelope glycoproteins(52) , but subsequent
studies have not confirmed this conclusion(2, 4) .
Recently, Vif was shown to affect the processing of the HIV-1 gag proteins in peripheral blood mononuclear cells(53) ,
possibly by affecting the gag or gagpol precursor
protein or the viral protease. One possibility is that phosphorylation
of Vif may serve to initiate or promote interactions with another
protein, such as one of the gag proteins, and thus allow Vif
to promote normal assembly of the virion core(7) . In this
regard, it is interesting to note that hypericin, a potent inhibitor of
Vif phosphorylation, has been shown to interfere with proper assembly
of the HIV-1 virion core(42) . Alternatively, phosphorylation
may induce a conformational change in the protein which is important
for modulation of its biological activity. Elucidating the mechanisms
by which Vif enhances HIV-1 infectivity continues to be a major
challenge. Further studies on Vif phosphorylation, particularly the
identification of the Vif kinase(s) and its specific biological role in
regulating Vif activity, may lead to a better understanding of the
complex regulation of HIV-1 replication and provide insights into new
therapeutic possibilities.
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10121-10129
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P-labeled Vif was digested by V8
protease and the peptides were resolved by reverse-phase high
performance liquid chromatography. Radioactive peptide sequencing
identified three phosphorylation sites within the C terminus,
Ser
, Thr
, and Thr
.
Two-dimensional tryptic phosphopeptide mapping indicated that these
sites are also phosphorylated in vivo. Both Ser and Thr are contained in the recognition motifs
(R/KXXS
/T and
R/KXXXS/T) used by serine/threonine
protein kinases such as cGMP-dependent kinase and PKC. Ser is present in the motif SLQXLA, which is the most highly
conserved sequence among all lentivirus Vif proteins. Mutation of
Ser to alanine resulted in loss of Vif activity and
>90% inhibition of HIV-1 replication. These studies suggest that
phosphorylation of Vif by a serine/threonine protein kinase(s) plays an
important role in regulating HIV-1 replication and infectivity.
)accessory genes, encodes a 23-kDa protein which is
important for virion infectivity. Vif is required for HIV-1 replication
in peripheral blood mononuclear cells, indicating that its function is
likely to be essential in
vivo(1, 2, 3, 4, 5) .
Previous studies have shown that Vif is required for correct assembly
of HIV-1 virus particles(6, 7) . In the absence of
Vif, HIV-1 virions are defective in their ability to synthesize
proviral DNA most likely due to its effect during virus
production(4, 6, 8) . The biochemical
mechanism and regulation of Vif function are unknown. Some immortalized
cell lines do not require Vif to produce fully infectious
virus(2, 4, 9) , suggesting that Vif may
compensate for a cell-specific factor or neutralize an inhibitory
factor which interferes with correct virus assembly. However, computer
data base searches have not revealed any significant homologies with
known cellular proteins (10) . (
)
(15, 16, 17) ,
p17(18) , Vpu(19, 20) ,
Rev(21, 22, 23) , and
Nef(24, 25, 26) , have been shown to be
phosphorylated. p17, Nef, and Rev are
phosphorylated on serine/threonine residues by protein kinase C (PKC) (18, 23, 24) , and Vpu is phosphorylated on
serine by casein kinase II (19) . Tyrosine phosphorylation of
p17 controls nuclear transport of the viral
preintegration complex in non-dividing cells (27) , while
serine phosphorylation regulates p17 membrane
targeting(28) . Phosphorylation of Vpu may alter HIV-1
cytopathicity by modulating the formation of syncytia(29) . Nef
phosphorylation on Thr
affects its ability to
down-regulate transcription factors(30) . In contrast, serine
phosphorylation of Rev appears to be dispensable for its
activity(21) . Thus far, phosphorylation of Vif has not been
examined., is contained within the most
highly conserved motif in Vif proteins from all lentiviruses. Mutation
of Ser to alanine results in loss of Vif activity,
suggesting that phosphorylation at this site plays an important role in
regulating HIV-1 replication and infectivity.
Materials
[
-P]ATP
(3000 Ci/mmol) and [P]orthophosphate (8500
Ci/mmol) were purchased from DuPont NEN. Endoprotease Glu-C (V8
protease, sequencing grade) and DOTAP transfection reagent were from
Boehringer Mannheim. Phosphoserine, phosphothreonine, phosphotyrosine,
phosphatidylserine, diolein, staurosporine, phorbol 12-myristate
13-acetate (PMA), TPCK-trypsin, and ATP were from Sigma. Immobilon-P
(PVDF) membranes and Sequelon AA membrane disks were from Millipore.
H7, H8, KT5823, A3, HA1004, K-252a, hypericin, and okadaic acid were
from Calbiochem. Cellulose thin layer plates were from Kodak.Cell Cultures
The T-cell line CEM was maintained
in RPMI medium containing 10% fetal calf serum. COS-1 and HeLa cells
were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum.Expression and Purification of Vif
The pD10Vif
bacterial expression plasmid was made by polymerase chain reaction
amplification of the vif gene from the HIV-1 proviral clone
pHXB2 (31) using the 5`- and 3`-primers:
5`-GGGGGGATCCGAAAACAGATGG-3` and 5`-GGGGAAGCTTCTAGTGTCCATTCAT-3`
containing BamHI and HindIII sites (underlined
sequences) and insertion of the amplified vif gene between the BamHI and HindIII sites in plasmid pD10
(pDS56/RSII-6HIS)(32) . In plasmid pD10Vif, a 6-His tag is
fused with the Vif N terminus lacking the initiation methionine codon
(MRGSHHHHHHGS-Vif). The plasmid was transformed into Escherichia
coli MC10611 and expression of Vif was induced by addition of 400
µM isopropyl-1-
-D-galactopyranoside to log
phase bacterial cultures (OD
= 0.6-0.8).
After induction for 4 h at 37 °C, the bacterial cells were lysed in
6 M guanidine HCl, 0.1 M sodium phosphate, pH 8.0, at
room temperature, and stirred overnight. Insoluble cell debris was
removed by centrifugation at 15,000 rpm in a SS-34 rotor for 30 min and
the supernatant was loaded onto a Ni
-NTA-agarose
column (Qiagen). The column was washed extensively with lysis buffer
and sequentially eluted with the same solution at decreasing pH values
(pH 6.5, pH 6.0, pH 5.8, pH 5.5, and pH 5.0). The fractions containing
Vif eluted at pH 5.5 were pooled, diluted to 200 µg/ml, and
successively dialyzed against 50 mM MOPS, 150 mM NaCl, pH 6.5, containing 3.0, 1.5, 0.75, 0.42, 0.21, and 0 M guanidine HCl. The protein was then concentrated with a
Centriprep-10 concentrator (Amicon) and insoluble aggregates were
removed by centrifugation at 100,000 
g for 30 min at 4
°C. The soluble fraction was adjusted to 10% glycerol, and stored
in aliquots at -70 °C.Immunoblotting
Proteins were resolved by SDS-PAGE
and transferred to PVDF membranes. After blocking with 5% nonfat milk
in Tris-buffered saline containing 0.05% Tween 20, membranes were
probed with rabbit anti-Vif serum (11) (1:2000 dilution) or Vif
monoclonal antibodies (AGMED) (1:2500 dilution) for 1 h at room
temperature. The bound immunocomplexes were detected with the ECL
detection system (Amersham) and autoradiography.Phosphorylation of Vif in Vitro
CEM and COS-1 cell
lysates for in vitro kinase assays were prepared by lysis in
buffer consisting of 10 mM Tris-Cl, pH 7.4, 1.0% Triton X-100,
0.5% Nonidet P-40, 150 mM NaCl, 50 mM sodium
fluoride, 50 mM potassium fluoride, 25 mM imidazole,
0.6 mM sodium orthovanadate, 25 mM -glycerophosphate, 1.0 mM EGTA, 1.0 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 50 µg/ml antipain, and 5
µg/ml pepstatin on ice for 30 min. Lysates were cleared by
centrifugation at 14,000 g for 15 min and the
supernatants were used for in vitro kinase assays. Purified
Vif protein was phosphorylated using the method of Hayashi et
al.(33) . The kinase reaction was performed in a total
volume of 25 µl of kinase buffer (50 mM Hepes, pH 7.5, 150
mM NaCl, 5 mM MgCl, 5 mM MnCl, 1 µM ATP, 1.4 µg of purified
Vif, 2 µg of cell lysate, and 5 µCi of
[-P]ATP) for 30 min at room temperature.
The reaction was stopped by addition of 1 volume of 2 SDS
Laemmli sample buffer followed by heating at 95 °C for 3 min. The
proteins were then separated on 15% SDS-polyacrylamide gels and
transferred to PVDF membrane in 25 mM Tris, pH 8.3, 192 mM glycine containing 20% methanol, 0.1% SDS. The P-labeled proteins were detected by autoradiography.Phosphoamino Acid Analysis
Phosphoamino acid
content was determined as described(34, 35) . Briefly,
the P-labeled bands were excised from PVDF membrane and
hydrolyzed directly in 6 N HCl for 2 h at 110
°C(36) . The samples were dried, resuspended in 50 µl
of HO, dried again, dissolved in phosphoamino acid standard
solution (p-Ser, p-Thr, p-Tyr, 1 mg/ml each), and then spotted onto a
cellulose thin layer plate. Phosphoamino acids were separated under
1000 volts using Pharmacia Metophor II with cooling in 5% acetic acid
and 0.5% pyridine, pH 3.5. The positions of unlabeled standards were
determined by staining with ninhydrin (2%). P-Labeled
phosphoamino acids were identified by autoradiography.Proteolytic Digestion of
Vif protein (90 µg)
was phosphorylated in vitro using
[P-Labeled Vif
and Radioactive Peptide Sequencing-P]ATP and resolved by SDS-PAGE. The Vif
band was excised, washed extensively with HO, and digested
with endoproteinase Glu-C (1:20) for 18 h at 37 °C in digestion
buffer (100 mM Tris-HCl, pH 8.0, 10% acetonitrile, and 1%
rehydrogenated Triton X-100)(37) . The recovery of
radioactivity from the membrane after digestion was 50-60%. The
peptides generated were separated by reverse phase HPLC on a C-18
column using a linear gradient from 0 to 100% acetonitrile in 0.1%
trifluoroacetic acid at a flow rate of 200 µl/min. The absorbance
was measured at 214 nm. Fractions (30 s) were collected and the
radioactivity of the fractions was determined by Cerenkov counting. The
molecular masses of the radioactive peptides were determined by laser
desorption mass spectrometry using a Lasermat mass spectrometer
(Finnigan Mat Ltd., Hemel Hempsted, United Kingdom). The sequences of
the radioactive peptides were confirmed by limited N-terminal
sequencing by the pulse-liquid phase method using an Applied Biosystems
model 477A sequenator. The radioactive peptides were covalently coupled
to Sequelon AA membrane according to the manufacturer's
instructions and sequenced on a solid phase sequenator by the method of
Wettenhall et al.(38) . The P released
from each Edman degradation cycle was determined by liquid
scintillation counting.In Vivo Expression and Phosphorylation of Vif in HeLa
Cells
Vif was expressed in HeLa cells using an efficient
vaccinia virus expression system(39) . The vif gene
fused to a 6-His tag was amplified by polymerase chain reaction from
pD10Vif using the 5` and 3` primers (5`-GGGGCCATGGGAGGATCGCATCACC-3`
and 5`-GGGGGATCCTAGTGTCCATTCATT-3`) containing NcoI and BamHI sites (underlined), and inserted between the NcoI and BamHI sites in pTM-1 under control of a T7
promoter (39) to make plasmid pTM1/hVif. HeLa cells at 80%
confluence in 100-mm plates were infected for 1 h with vaccinia virus
VV-T7 containing the T7 RNA polymerase gene at 2 plaque units/cell in
serum-free Dulbecco's modified Eagle's medium and then
transfected with 7.5 µg of pTM1/hVif using 45 µg of liposomes
(DOTAP) (Boehringer Mannheim) according to the manufacturer's
instructions. Cells were labeled for 12 h with
[
S]methionine at 100 µCi/ml at 24 h after
transfection, or labeled for 4 h with
[P]orthophosphate at 1 mCi/ml in phosphate-free
Dulbecco's modified Eagle's medium containing 1% dialyzed
fetal calf serum at 36 h after transfection, followed by stimulation
for 10 min with PMA (200 nM) and okadaic acid (0.5
µM). Cells were washed with ice-cold PBS and lysed with 6 M guanidine HCl, 0.1 M sodium phosphate, pH 8.0, for
5 min at room temperature. Lysates were centrifuged at 100,000 g for 40 min and the supernatants were mixed with 30 µl of
Ni-NTA-agarose in the presence of 20 mM imidazole for 2 h at room temperature. The beads were washed twice
with 6 M guanidine HCl, 0.1 M sodium phosphate, 20
mM imidazole, pH 7.5, twice with the same buffer at pH 7.0,
and once with the same buffer containing 30 mM imidazole. Vif
was eluted with 80 mM imidazole in the same buffer,
precipitated with trichloroacetic acid in the presence of bovine serum
albumin, resolved by SDS-PAGE, and transferred to PVDF membranes. The
radiolabeled Vif was visualized by autoradiography or immunoblotting.
Transfection of HeLa cells with pcDNAVif and pcDNAVifSer was performed by incubating overnight with 2.5 µg of plasmid
DNA using DOTAP (Boehringer Mannheim) in the presence of
Dulbecco's modified Eagle's medium containing 1% fetal calf
serum according to the manufacturer's instructions.Two-dimensional Tryptic Phosphopeptide
Mapping
Phosphopeptide mapping was performed as
described(34) . Briefly, Vif phosphorylated in vitro or in vivo was resolved by SDS-PAGE, and transferred to
nitrocellulose membrane. The Vif band was localized by autoradiography,
excised, washed with several changes of HO, and then
incubated with 0.5% polyvinylpyrrolidine 360 in 0.1 M acetic
acid for 30 min at 37 °C. The membrane slice was washed extensively
with HO and digested with 10 µg of trypsin in 50 mM NH
HCO
, pH 8.0, for 3 h at 37 °C,
followed by addition of another 10 µg of trypsin and incubation for
3 h. The supernatant was removed, clarified by centrifugation at 13,000
g, dried under a vacuum, and resuspended in 10 µl
of thin layer electrophoresis buffer (2.2% formic acid, 7.8% acetic
acid in HO, pH 1.9). The tryptic peptides were separated in
the first dimension on a nitrocellulose plate by electrophoresis at pH
1.9, and in the second dimension by thin layer chromatography in
phosphochromatography buffer (37.5% n-butanol, 25% pyridine,
7.5% acetic acid in HO). The plate was dried and exposed to
x-ray film at -70 °C.HIV-1 Replication Complementation Assay
The
activity of the Ser Vif mutant was determined by
measuring the ability to complement vif-defective HIV-1 in
trans during a single round replication as
described(11, 40) . Plasmid pcDNAVif (2) expresses the vif gene of the HXB2 HIV-1 proviral
clone under the control of the cytomegalovirus promoter (Invitrogen).
Site-directed mutagenesis (41) was performed to generate the
mutant pcDNAVifSer plasmid which contains a serine to
alanine substitution at position 144. Expression of the Ser Vif mutant protein was confirmed by transfection of HeLa cells
with pcDNAVifSer and immunoblotting. Plasmid
pHXB
envCAT contains an HIV-1 provirus with a deletion in env and a chloramphenicol acetyltransferase (CAT) gene in place of nef(40) . Plasmid pHXBvifenvCAT contains a
deletion in vif in pHXBenvCAT(2) . Plasmid
pSVIIIenv expresses HIV-1 env and rev. Briefly, CEM
cells were cotransfected by the DEAE-dextran method with 15 µg of
wild-type or mutant pcDNAVif, 1 µg of either pHXBenvCAT or
pHXBvifenvCAT, and 1 µg of pSVIIIenv. The ability of the
wild-type or mutant Vif expressor plasmid to complement a single round
of replication of the vif-negative virus in trans was
measured by assaying for CAT activity in the transfected culture at 9
or 10 days after transfection.
Expression and Phosphorylation of Vif in Vivo
To
determine whether Vif is phosphorylated in vivo, Vif was
expressed in HeLa cells using a recombinant vaccinia virus expression
system. HeLa cells were infected with recombinant vaccinia virus
harboring a T7 RNA polymerase gene, and then transfected with the Vif
expressor plasmid pTM-l/hVif which contains a T7 promoter. The
transfected cells were metabolically labeled with
[S]methionine (12 h) or
[P]orthophosphate (4 h), and the
histidine-tagged Vif protein was isolated by binding to
Ni-NTA-agarose. The radiolabeled proteins were
separated by SDS-PAGE, transferred to PVDF membrane, and detected by
autoradiography or immunoblotting. The data (Fig. 1A,
left) show that a 24-kDa protein was labeled with
[S] methionine in cells transfected with
pTM-1/hVif, but not in control cells transfected with the vector
plasmid pTM-1. The same 24-kDa protein was heavily labeled by
[P]orthophosphate (Fig. 1A,
left). This protein was specifically detected by immunoblotting
with rabbit anti-Vif serum (Fig. 1A, right), confirming
that the 24-kDa phosphorylated protein is the Vif protein. Phosphoamino
acid analysis showed that Vif is phosphorylated on serine and threonine
residues (Fig. 1B). These data indicate that Vif is
phosphorylated in vivo by a serine/threonine kinase.
S]methionine (12 h) (lanes 1 and 2) or [P]orthophosphate (4 h) (lanes 3 and 4) and treated with PMA (200
nM) and okadaic acid (0.5 µM) for 10 min. The
histidine-tagged Vif was isolated from the cell lysates under
denaturing conditions by binding to Ni-NTA-agarose.
The purified Vif was subjected to SDS-PAGE, transferred to PVDF
membrane, and detected by autoradiography (left panel) or
immunoblotting with rabbit anti-Vif polyclonal serum (right).
Shown on the left are molecular weights (kDa) of marker proteins. B, phosphoamino acid analysis of Vif phosphorylated in
vivo. The P-labeled Vif was hydrolyzed with 6 N HCl, separated by thin layer electrophoresis, and analyzed by
autoradiography. The positions of pSer, pThr, and pTyr are
indicated.
Expression and Purification of Vif from E. coli
To
obtain sufficient quantities of Vif for biochemical studies, the
protein was expressed in E. coli and purified for use as a
substrate for in vitro kinase assays. The HIV-1 vif gene was amplified by polymerase chain reaction and cloned into
the pD10 bacterial expression vector (32) which contains a
6-histidine coding sequence following the methionine initiation codon.
Following induction with
isopropyl-1-thio--D-galactopyranoside, Vif with an
apparent molecular mass of 24 kDa was expressed at high levels of up to
approximately 10% of the total protein (Fig. 2A). Vif
was not detected in the soluble fraction, but was found exclusively in
the insoluble inclusion body fraction. Initial experiments demonstrated
that Vif remained insoluble in high concentrations of Triton X-100 and
NaCl. Therefore, the bacterial cell lysates were denatured with 6 M guanidine HCl and Vif was bound to
Ni-NTA-agarose via binding of the histidine tag and
eluted with 6 M guanidine HCl at decreasing pH (Fig. 2A). Under these conditions, most Vif was eluted
at pH 5.5. The purified Vif protein was renatured by gradient dialysis
to obtain soluble protein (Fig. 2B). Analysis by
SDS-PAGE and Coomassie Blue staining demonstrated that Vif was purified
to >95% homogeneity. The N-terminal amino acid sequence was
determined by Edman degradation and the sequence obtained
(MRGSHHHHHHGSENRXWQVM) corresponded to the predicted 6-His tag and Vif
N terminus minus the normal initiation methionine. The purified Vif
protein was specifically recognized by rabbit anti-Vif serum and
anti-Vif monoclonal antibodies (Fig. 2B).
Immunoblotting detected a small fraction of Vif (<5%) as a 46-kDa
dimer by overexposure of the autoradiograms (Fig. 2B,
and not shown).
-NTA
chromatography. The insoluble inclusion body fraction containing Vif
was dissolved with 6 M guanidine HCl, pH 8.0, loaded onto a
Ni-NTA column, and eluted with 6 M guanidine
HCl, 0.1 sodium phosphate at decreasing pH. An aliquot of each fraction
was dialyzed, analyzed by SDS-PAGE, and stained with Coomassie Blue.
Molecular weights (kDa) of marker proteins are indicated. B,
purified Vif protein. Vif from fractions eluted at pH 5.5 was pooled
and refolded by gradient dialysis against 50 mM MOPS, 150
mM NaCl, pH 6.5. The soluble protein was concentrated,
insoluble aggregates were removed by centrifugation, and the protein
was analyzed by SDS-PAGE and Coomassie Blue staining (lane 1),
or by immunoblotting with rabbit anti-Vif serum (lane 2) or
three different Vif monoclonal antibodies (lanes 3, 4, and 5). Lanes 1, 2, and 3-5 are from
different gels. The minor bands near the top of the gel in lanes
3-5 correspond to a 46-kDa Vif
dimer.
Phosphorylation of Vif in Vitro
To examine the
phosphorylation of Vif in vitro, the purified protein was used
as a substrate for in vitro kinase assays. Vif was incubated
with [-P]ATP and CEM cell lysates. The
phosphorylated proteins were separated by SDS-PAGE, transferred to PVDF
membrane, and detected by autoradiography. In the presence of Vif, a
24-kDa phosphorylated protein corresponding to the apparent M
of Vif was observed (Fig. 3A).
This band was not observed in the absence of Vif or cell lysate,
suggesting that the phosphorylated protein was not derived from the
cell lysate and that the recombinant Vif preparation did not contain
autophosphorylation or contaminating Vif kinase activity. Under these
assay conditions, kinetic studies showed that phosphorylation of Vif
was linear within the first 30 min and then reached a maximum level at
45 min (data not shown). Thus, Vif is phosphorylated in vitro by kinase(s) in the cell lysate.
-P]ATP. A,
phosphorylation of Vif by lysate prepared from CEM cells. B,
Vif kinase activity in soluble cytosol (S200), cellular membrane (P50),
and microsomal membrane (P200) fractions from CEM cell lysates. C, phosphoamino acid analysis of Vif phosphorylated by CEM
total cell lysate (left) and subcellular fractions (right). The positions of pSer, pThr, and pTyr are
indicated.
Inhibition and Activation of Vif Kinase(s)
To
further characterize the Vif kinase(s), we examined the effects of
different kinase inhibitors and cofactors on Vif phosphorylation in
vitro. Staurosporine and the staurosporine analog K-252a, which
are potent inhibitors of many different kinases, inhibited Vif
phosphorylation by >90% (Fig. 4A). However, Vif
phosphorylation was insensitive to many other kinase inhibitors,
including inhibitors of protein kinase C, cAMP-dependent protein kinase
(PKA), and cGMP-dependent protein kinase (PKG) (H7, H8, A3, KT5823, and
HA1004) (Fig. 4A). In the presence of 100 µM H7 or H8, only 30% inhibition was observed as determined by gel
densitometry. Interestingly, hypericin, an aromatic polycyclic dione
which potently inhibits HIV-1 replication (42, 43, 44) and PKC(45) , inhibited
the Vif kinase(s) activity by >95% (Fig. 4A).
Hypericin has been shown to abolish HIV-1 replication or inactivate
HIV-1 virions in a light-dependent
manner(42, 43, 44, 46) . Similarly,
inhibition of Vif kinase(s) by hypericin was also light-dependent. When
samples were incubated in the dark, phosphorylation of Vif was not
affected by hypericin (data not shown). The relative insensitivity of
Vif phosphorylation to PKC, PKA, and PKG inhibitors suggests that these
enzymes are unlikely to be the main Vif kinase(s). Consistent with this
conclusion, the addition of PKC, PKA, or PKG cofactors, such as
Ca, phospholipids, cAMP, or cGMP, did not
significantly affect the activity of the Vif kinase(s) (Fig. 4B). However, stimulation of intact cells with
PMA (200 nM) increased Vif kinase activity by approximately
2-fold (Fig. 4C). These results together with the
observation that H7 and H8 inhibit Vif phosphorylation by 30% raise the
possibility that PKC may phosphorylate Vif at only one site or minor
sites or may regulate the Vif kinase(s).
-P]ATP in the presence of whole cell
lysates. Inhibitors or cofactors were added to the standard reaction
mixtures. A, effect of the protein kinase inhibitors
staurosporine (1 µM), KT5823 (2 µM), A3 (40
µM), H7 (100 µM), H8 (100 µM),
hypericin (1 µg/ml), HA1004 (50 µM), and K252a (1
µM). B, effect of cofactors Ca (5 mM), cAMP (4 µM), cGMP (4
µM), or lipids (10 µg/ml phosphophatidylserine and 1
µg/ml diolein in the presence of 5 mM Ca). C, stimulation of Vif kinase(s) by
PMA. COS cells were stimulated with PMA (200 nM) for 30 min
and used to prepare lysates for in vitro kinase
assays.
Identification of Vif Phosphorylation Sites
To
identify the Vif phosphorylation sites, the purified protein was
phosphorylated in vitro with
[-P]ATP in the presence of CEM cell lysate
and subjected to protease digestion followed by sequencing of the
radioactive peptides. In initial experiments, P-labeled
Vif was digested with trypsin. However, the small peptides generated
due to the relatively high content of basic residues could not be
resolved by HPLC. Therefore, P-labeled Vif was isolated by
SDS-PAGE and digested with V8 protease (endoproteinase Glu-C), since
Vif contains relatively few glutamic acid residues. The peptides
generated were separated by reverse phase HPLC (Fig. 5A). Two radioactive peaks, corresponding to the
peptides eluted at 16.5 and 34.5 min, respectively, were detected (Fig. 5B). Fractions corresponding to these radioactive
peaks were collected and the molecular masses of these peptides were
determined by matrix-assisted laser desorption mass spectrometry. Mass
spectrometry revealed that the first peak (peptide I) consisted of a
peptide of molecular mass 2485, and the second peak (peptide II)
consisted of a peptide of molecular mass 3996 (Table 1). After
inspecting the Vif amino acid sequence, two peptides located within the
C terminus were identified as having similar predicted molecular
weights after V8 protease digestion (Table 1). The identities of
the two peptides were confirmed by limited N-terminal amino acid
sequencing (Table 1). Peptide I corresponds to the amino acid
sequence at positions 172-192, and peptide II corresponds to the
sequence at position 135-171.
P-labeled Vif peptides generated by V8
protease digestion. A, reverse-phase HPLC of Vif peptides
generated by V8 protease digestion. Peptides were separated on a C-18
column with a 0-100% acetonitrile gradient in 0.1%
trifluoroacetic acid. mAu, milliabsorbance units. B,
radioactivity of reverse-phase HPLC fractions from A. Two
radioactive peptides (I and II) eluted at 16.5 and 34.5 min,
respectively, were detected. C, phosphoamino acid analysis of
radioactive peptides I and II. The positions of pSer, pThr, and pTyr
are indicated.
P was
released predominantly at cycle 17, corresponding to Thr (Fig. 6A). Radioactive sequencing of peptide II
identified Ser at cycle 10 and the Thr at
cycle 21 as the phosphorylated residues (Fig. 6B). P release was not significant at cycles corresponding to
other serine and threonine residues.
P-labeled peptides I (A) and II (B) generated by V8 protease digestion.
The P-labeled peptides shown in Fig. 5B were coupled to Sequelon AA membrane disks, followed by Edman
degradation. The amino acids were collected at each Edman cycle and the P released at each cycle was determined by liquid
scintillation counting.
, Thr, and Thr.
Ser is contained in the motif R/KXXS/T, a
consensus phosphorylation site for PKC and PKG (Table 2).
Notably, Ser is contained in the highly conserved motif
SLQXLA, which is conserved among Vif sequences from all
lentiviruses(10, 47) . Thr, which is
contained in the motif R/KXXXS/T recognized by PKG (Table 2), is highly conserved among HIV-1 Vif sequences, but is
not conserved in HIV-2 or SIV(47) . Thr is
contained in the motif S/TXR/K, another PKC consensus phosphorylation
site. This threonine residue is not conserved among different HIV-1
sequences.
Two-dimensional Tryptic Phosphopeptide Mapping
To
determine whether Vif is phosphorylated on the same sites in vitro and in vivo, tryptic phosphopeptide mapping was
performed. Vif phosphorylated either in vitro or in vivo was resolved by SDS-PAGE, transferred to nitrocellulose membrane,
and subjected to proteolysis by TPCK-trypsin. The migration patterns of
the P-labeled phosphopeptides thus generated were compared
following two-dimensional thin layer electrophoresis and chromatography (Fig. 7). As expected from the predicted cleavage pattern,
tryptic digestion of Vif phosphorylated in vitro generated two
major phosphopeptides (Fig. 7A, spots 1 and 2)
in addition to several minor phosphopeptides. These phosphopeptides
comigrated with the tryptic phosphopeptides generated from Vif
phosphorylated in vivo (Fig. 7B). However, one
major phosphopeptide (spot 1) was more intensely labeled in vitro and three minor phosphopeptides (spots 5, 6, and 7) were more
intensely labeled in vivo. These results suggest that the
phosphorylation sites identified in vitro are also
phosphorylated by the Vif kinase(s) in intact cells.
-P]ATP and CEM cell lysate. B,
Vif phosphorylated in vivo by metabolic labeling of HeLa cells
with [P]orthophosphate after infection with
recombinant vaccinia virus VV-T7 and transfection with pTM-1/hVif. In A and B, P-labeled Vif was separated by
SDS-PAGE and transferred to nitrocellulose membrane. The P-Vif bands were excised and digested in situ with TPCK-trypsin. Peptides were separated on nitrocellulose
plastic plates by electrophoresis (from left to right) in the first
dimension followed by chromatography (from bottom to top) in the second
dimension.
Ser
The data show that SerIs Required for Vif Activity and
HIV-1 Replication is a
major phosphorylation site in Vif. This serine residue is the most
highly conserved amino acid among the three phosphorylation sites
identified. Therefore, Ser was replaced with alanine by
site-directed mutagenesis to determine whether it is important for Vif
activity and HIV-1 replication. The biological activity of the
Ser mutant Vif protein during a single round of HIV-1
replication was determined in a transient complementation
assay(2) . The assay was performed in CEM cells, since Vif is
required for HIV-1 replication in this cell line(2) . In this
assay, the expression of wild-type Vif restores the replication of a vif-defective virus to the wild-type level(2) . The
Ser mutation reduced HIV-1 replication complementation by
Vif to 10% of the wild-type level above background (Fig. 8A). Transfection of HeLa cells with
pcDNAVifSer showed that expression of the Ser Vif mutant protein was similar to that of the wild-type protein (Fig. 8B). These results suggest that phosphorylation
at Ser is important for Vif function and HIV-1
replication.
mutant
in an HIV-1 replication complementation assay. A, trans-complementation of HIV-1 replication by the Ser Vif mutant protein. CEM cells were cotransfected with the pcDNA
vector, pcDNAVif, or pcDNAVifSer Vif mutant expressor
plasmid, either pHXBenvCAT (open bars) or
pHXBvifenvCAT (solid bars), and an HIV-1 Env
expressor plasmid. Replication complementation was determined by
measuring CAT activity in CEM cell lysates 9 or 10 days after
transfection. Values shown represent the percentage of replication
complementation relative to the value obtained for the wild-type Vif
expressor plasmid. Results shown are the means ± S.E. from three
independent experiments. B, expression of wild-type and
Ser mutant Vif proteins in HeLa cells transfected with
the pcDNA vector, pcDNAVif, or pcDNAVifSer. Vif was
detected in cell lysates at 24 h after transfection by SDS-PAGE and
immunoblotting with rabbit anti-Vif serum. CEM-Vif, a CEM cell line
which stably expresses HIV-1 Vif(55) , was used as a positive
control.
, Thr, and
Thr) were identified, all within the C-terminal region of
Vif. Importantly, Ser is contained in the motif
SLQXLA at positions 144-149, the most highly conserved
Vif sequence from all lentiviruses, including HIV-1, HIV-2, SIV, and
non-primate lentiviruses(10, 47) . Phosphorylation at
this site is likely to be important for Vif activity, since replacement
of Ser with alanine results in loss of Vif activity and
inhibits HIV-1 replication. Thr is highly conserved among
HIV-1 isolates, but not among other lentiviruses, while Thr is not highly conserved. The biological importance of the
threonine phosphorylation sites and other phosphorylation sites which
may be utilized in vivo (Fig. 7) remains to be
established., Thr, and
Thr were identified as phosphorylation sites. These sites
correspond to consensus phosphorylation sites recognized by PKC or PKG.
However, the Vif kinase(s) is relatively insensitive to PKC, PKG, and
PKA inhibitors such as H7, H8, A3, and HA1004, suggesting that these
kinases are not the main enzymes which phosphorylate Vif. Consistent
with this conclusion, we found that PKC, PKA, and PKG cofactors such as
Ca, phosphatidylserine and diolein, cAMP, and cGMP
did not significantly increase Vif kinase activity. Our results,
however, do not exclude the possibility that PKC or
nucleotide-dependent protein kinases may phosphorylate Vif at
relatively low levels. It is possible that one of the identified
phosphorylation sites is phosphorylated by PKC, which would explain the
30% inhibition of Vif phosphorylation by H7 and H8. It is also possible
that PKC may regulate the Vif kinase(s), since the PKC activator PMA
stimulated Vif phosphorylation in intact cells. However, many other
kinases are activated in response to stimuli such as PMA. Thus, the
identity of the Vif kinase(s) remains to be determined. The Vif
kinase(s) was distributed in both soluble cytosolic and membrane
fractions, similar to the distribution of Vif. The Vif kinase(s) in
these subcellular fractions may be the same enzyme(s), since these
fractions phosphorylated Vif on serine and threonine at phosphoamino
acid ratios similar to that of the total cell lysate. In previous
studies, the HIV-1 Nef and Tat proteins have been found to be
specifically associated with cellular
kinases(48, 49) . The similar distribution of Vif and
the Vif kinase(s) raises the possibility that Vif may be associated
with its kinase(s). However, further experiments are needed to address
this possibility. Thus, the relative
accessibility of the Vif C terminus may permit interactions between
this domain and other proteins, including protein kinases. We
previously showed that clusters of basic residues in the Vif C terminus
may interact electrostatically with a membrane-associated protein(s) to
anchor Vif to the membrane surface(14) . This observation
raises the possibility that phosphorylation of Vif may play a role in
modulating its association with membrane-associated protein(s) or
lipids by introducing negative charges into the molecule.
Phosphorylation-dependent protein-protein or protein-lipid interactions
have been shown to mediate targeting of some proteins to the plasma
membrane or membrane-associated cytoskeletal elements, such as the
interaction between p36 and p50
(50) and the
membrane association of HIV-1 p17
(28) . In
contrast, other proteins, such as the myristoylated alanine-rich
protein kinase C substrate protein(51) , are released from the
membrane into the cytosol when they are phosphorylated. Studies are in
progress to determine the role of phosphorylation in regulating
membrane targeting of Vif. Our initial attempts to examine the
phosphorylation of Vif in vivo in HIV-1 infected cells were
unsuccessful, most likely due to both the relatively low level of Vif
expression and low efficiency of immunoprecipitation. In this study, we
expressed Vif as a histidine-tagged protein in HeLa cells using a
highly efficient vaccinia virus expression system(39) , an
approach which allowed us to clearly demonstrate its phosphorylation in vivo. By using Ni-NTA-agarose, Vif was
selectively isolated from a relatively small amount of sample under
denaturing conditions. In contrast, only a small amount of Vif (<10%
of the total) was immunoprecipitated from the same samples. To our knowledge, this is the first study to utilize purification
of a histidine-tagged protein from intact cells under denaturing
conditions to study protein phosphorylation in vivo. This
method may be generally applicable to the detection of low level
protein phosphorylation.
))
We thank J. Lee for providing assistance with
radioactive peptide sequencing, X. Wu and J. Kappes for providing
plasmid pTM-1 and vaccinia virus VV-T7, Jay Raina for providing Vif
monoclonal antibodies, Bruno Spire for providing the CEM-Vif cell line,
and J. Sodroski and A. Engelman for critical reading of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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