Phosphorylation of Vif and Its Role in HIV-1 Replication

Xiaoyu Yang,Joao Goncalves,Dana Gabuzda

doi:10.1074/jbc.271.17.10121

Abstract

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.

Highlights

Vif, one of the human immunodeficiency virus type I (HIV1)1 accessory genes, encodes a 23-kDa protein which is impor
Several HIV-1 proteins, including p24gag [15,16,17], p17gag [18], Vpu [19, 20], Rev [21,22,23], and Nef (24 – 26), have been shown to be phosphorylated. p17gag, 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]
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

Summary

EXPERIMENTAL PROCEDURES

Materials—[␥-32P]ATP (3000 Ci/mmol) and [32P]orthophosphate (8500 Ci/mmol) were purchased from DuPont NEN. The vif gene fused to a 6-His tag was amplified by polymerase chain reaction from pD10Vif using the 5Ј and 3Ј primers (5ЈGGGGCCATGGGAGGATCGCATCACC-3Ј and 5Ј-GGGGGATCCTAGTGTCCATTCATT-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 transfected with 7.5 ␮g of pTM1/hVif using 45 ␮g of liposomes (DOTAP) (Boehringer Mannheim) according to the manufacturer’s instructions. Transfection of HeLa cells with pcDNAVif and pcDNAVifSer144 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. 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

Predicted Determined

DISCUSSION

TABLE II Vif phosphorylation sites identified by radioactive peptide sequencing