Phosphorylation of Vif and its role in HIV-1 replication.

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, 32P-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, Ser144, Thr155, and Thr188. Two-dimensional tryptic phosphopeptide mapping indicated that these sites are also phosphorylated in vivo. Both Ser144 and Thr188 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. Ser144 is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins. Mutation of Ser144 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.

tant 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). 2 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 membraneassociated 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.
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 144 , is contained within the most highly conserved motif in Vif proteins from all lentiviruses. Mutation * This work was supported in part by National Institutes of Health Grants AI33837 and AI36186, a Claudia A. Barr Investigator Award, and the G. Harold and Leila Y. Mathers Charitable Foundation. We also acknowledge the Center for AIDS Research (Grant AI28691) and Center for Cancer Research (Grant AO6514) for supporting necessary core facilities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  of Ser 144 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.
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 5Ј-and 3Ј-primers: 5Ј-GGGGGGATCCGAAAACAGATGG-3Ј and 5Ј-GGGG-AAGCTTCTAGTGTCCATTCAT-3Ј 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 M isopropyl-1-␤-Dgalactopyranoside to log phase bacterial cultures (OD 600 ϭ 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 2ϩ -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 g/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 ϫ g for 30 min at 4°C. The soluble fraction was adjusted to 10% glycerol, and stored in aliquots at Ϫ70°C.
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 g/ml leupeptin, 10 g/ml aprotinin, 50 g/ml antipain, and 5 g/ml pepstatin on ice for 30 min. Lysates were cleared by centrifugation at 14,000 ϫ 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 l of kinase buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 5 mM MnCl 2 , 1 M ATP, 1.4 g of purified Vif, 2 g of cell lysate, and 5 Ci of [␥-32 P]ATP) for 30 min at room temperature. The reaction was stopped by addition of 1 volume of 2 ϫ 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 32 P-labeled proteins were detected by autoradiography. Phosphoamino Acid Analysis-Phosphoamino acid content was determined as described (34,35). Briefly, the 32 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 l of H 2 O, 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%). 32 P-Labeled phosphoamino acids were identified by autoradiography.
Proteolytic Digestion of 32 P-Labeled Vif and Radioactive Peptide Sequencing-Vif protein (90 g) was phosphorylated in vitro using [␥-32 P]ATP and resolved by SDS-PAGE. The Vif band was excised, washed extensively with H 2 O, 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 l/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 32 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 5Ј and 3Ј primers (5Ј-GGGGCCATGGGAGGATCGCATCACC-3Ј and 5Ј-GGGGGATCCTAG-TGTCCATTCATT-3Ј) 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 g of pTM1/hVif using 45 g of liposomes (DOTAP) (Boehringer Mannheim) according to the manufacturer's instructions. Cells were labeled for 12 h with [ 35 S]methionine at 100 Ci/ml at 24 h after transfection, or labeled for 4 h with [ 32 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 M). 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 ϫ g for 40 min and the supernatants were mixed with 30 l of Ni 2ϩ -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 144 was performed by incubating overnight with 2.5 g 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 H 2 O, 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 H 2 O and digested with 10 g of trypsin in 50 mM NH 4 HCO 3 , pH 8.0, for 3 h at 37°C, followed by addition of another 10 g of trypsin and incubation for 3 h. The supernatant was removed, clarified by centrifugation at 13,000 ϫ g, dried under a vacuum, and resuspended in 10 l of thin layer electrophoresis buffer (2.2% formic acid, 7.8% acetic acid in H 2 O, 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 H 2 O). The plate was dried and exposed to x-ray film at Ϫ70°C.

HIV-1 Replication Complementation Assay-
The activity of the Ser 144 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 144 plasmid which contains a serine to alanine substitution at position 144. Expression of the Ser 144 Vif mutant protein was confirmed by transfection of HeLa cells with pcDNAVifSer 144 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 pHXB-⌬vif⌬envCAT contains a deletion in vif in pHXB⌬envCAT (2). Plasmid pSVIIIenv expresses HIV-1 env and rev. Briefly, CEM cells were cotransfected by the DEAE-dextran method with 15 g of wild-type or mutant pcDNAVif, 1 g of either pHXB⌬envCAT or pHXB⌬vif⌬-envCAT, and 1 g 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.

RESULTS
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 [ 35 S]methionine (12 h) or [ 32 P]orthophosphate (4 h), and the histidine-tagged Vif protein was isolated by binding to Ni 2ϩ -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 [ 35 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 [ 32 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.
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 2ϩ -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 Ͼ95% 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 initia-tion 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 (Ͻ5%) as a 46-kDa dimer by overexposure of the autoradiograms (Fig. 2B, and not shown).
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 [␥-32 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 r 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.
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).

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 Ͼ90% (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 M 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 Ͼ95% (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 2ϩ , 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).
Identification of Vif Phosphorylation Sites-To identify the Vif phosphorylation sites, the purified protein was phosphorylated in vitro with [␥-32 P]ATP in the presence of CEM cell lysate and subjected to protease digestion followed by sequencing of the radioactive peptides. In initial experiments, 32 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, 32 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 I). 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 I). The identities of the two peptides were confirmed by limited N-terminal amino acid sequencing (Table I). Peptide I corresponds to the amino acid sequence at positions 172-192, and peptide II corresponds to the sequence at position 135-171.
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 I) 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 32 P was released predominantly at cycle 17, corresponding to Thr 188 (Fig. 6A). Radioactive sequencing of peptide II identified Ser 144 at cycle 10 and the Thr 155 at cycle 21 as the phosphorylated residues (Fig. 6B). 32 P release was not significant at cycles corresponding to other serine and threonine residues.
The preceding experiments identify three phosphorylation sites in Vif, all within the C terminus, Ser 144 , Thr 155 , and Thr 188 . Ser 144 is contained in the motif R/KXXS/T, a consensus phosphorylation site for PKC and PKG (Table II). Notably, Ser 144 is contained in the highly conserved motif SLQXLA, which is conserved among Vif sequences from all lentiviruses (10,47). Thr 188 , which is contained in the motif R/KXXXS/T recognized by PKG (Table II), is highly conserved among HIV-1 Vif sequences, but is not conserved in HIV-2 or SIV (47). Thr 155 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 32 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.
Ser 144 Is Required for Vif Activity and HIV-1 Replication-The data show that Ser 144 is a major phosphorylation site in Vif. This serine residue is the most highly conserved amino acid among the three phosphorylation sites identified. Therefore,

FIG. 5. Reverse-phase HPLC and phosphoamino acid analysis of 32 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.
Ser 144 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 144 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 144 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 144 showed that expression of the Ser 144 Vif mutant protein was similar to that of the wild-type protein (Fig. 8B). These results suggest that phosphorylation at Ser 144 is important for Vif function and HIV-1 replication.

DISCUSSION
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 phosphoryl-ation sites (Ser 144 , Thr 155 , and Thr 188 ) were identified, all within the C-terminal region of Vif. Importantly, Ser 144 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 144 with alanine results in loss of Vif activity and inhibits HIV-1 replication. Thr 188 is highly conserved among HIV-1 isolates, but not among other lentiviruses, while Thr 155 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 144 , Thr 155 , and Thr 188 were identified as phosphorylation sites. These sites correspond to consensus FIG. 6. Identification of phosphorylation sites in Vif. Radioactive sequencing of the 32 P-labeled peptides I (A) and II (B) generated by V8 protease digestion. The 32 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 32 P released at each cycle was determined by liquid scintillation counting.   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 2ϩ , 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. 2 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 proteinlipid 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 csk (50) and the membrane association of HIV-1 p17 gag (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 ϭ 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. 2 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 2ϩ -NTA-agarose, Vif was selectively isolated from a relatively small amount of sample under denaturing conditions. In contrast, only a small amount of Vif (Ͻ10% of the total) was immunoprecipitated from the same samples. 2 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.