(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, 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.
Vif, one of the human immunodeficiency virus type I
(HIV-1) ()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) . (
)
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(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.
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, 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.
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 [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.
Figure 2:
Purification of Vif from E. coli.
Vif was expressed in E. coli using the pD10Vif bacterial
expression plasmid. A, denaturing Ni-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.
Figure 3:
Phosphorylation of Vif in vitro. In vitro kinase assays were performed using purified Vif
protein and [-
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.
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
[-
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.
Figure 5:
Reverse-phase HPLC and phosphoamino acid
analysis of 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.
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 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.
Figure 6:
Identification of phosphorylation sites in
Vif. Radioactive sequencing of the 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.
The preceding experiments
identify three phosphorylation sites in Vif, all within the C terminus,
Ser, 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.
Figure 7:
Tryptic phosphopeptide mapping of Vif
phosphorylated in vitro and in vivo.A, Vif
phosphorylated in vitro using
[-
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.
Figure 8:
Activity of Vif Ser 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 pHXB
envCAT (open bars) or
pHXB
vif
envCAT (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.
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, 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.
Vif contains several potential phosphorylation sites,
including consensus sites for PKC, PKA, PKG, and casein kinase II.
Among these sites, Ser, 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.
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. 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.
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. 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.
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.