Phosphorylation of Vif and its role in HIV-1 replication.
Phosphorylation of Vif and its role in HIV-1 replication.
复制标题
Vif 的磷酸化及其在 HIV-1 复制中的作用。
DOI：
10.1074/jbc.271.17.10121
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Gabuzda,D
中科院分区：
文献类型：
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作者：
Yang,X;Goncalves,J;Gabuzda,D
VIF是由人类免疫缺陷病毒1型(HIV-1)编码的一种23 kDa的蛋白质，对病毒粒子的感染性具有重要作用。在这里，我们描述了HIV-1 Vif的磷酸化及其在HIV-1复制中的作用。在活体研究中，Vif在丝氨酸和苏氨酸残基上高度磷酸化。为了鉴定Vif蛋白的磷酸化位点并鉴定Vif蛋白(S)的特性，我们在大肠杆菌中表达了Vif蛋白，并将其纯化后作为底物用于玻璃体蛋白激酶的检测。纯化的Vif蛋白被胞浆和膜部分中存在的一种激酶(S)在胞外丝氨酸和苏氨酸残基中磷酸化。佛波酯可刺激Vif的磷酸化，而星形孢子素和金丝桃素则可抑制Vif的磷酸化。Vif激酶(S)对蛋白激酶C、cAMP依赖的激酶和cGMP依赖的激酶的抑制剂具有抵抗力，这表明它不同于这些酶。用V8酶消化2P标记的Vif，用反相高效液相色谱分离多肽，以确定其磷酸化位点。放射性多肽测序确定了C末端的三个磷酸化位点，Ser144、Thr155和Thr188。二维胰酶磷酸肽图显示，这些位点在体内也被磷酸化。Ser144和Thr188都包含在丝氨酸/苏氨酸蛋白激酶所使用的识别基序(R/KXXS*/T*和R/KXXS*/T*)中，如cGMP依赖的激酶和PKC。Ser144存在于SLQXLA基序中，SLQXLA是慢病毒Vif蛋白中最保守的序列。Ser144突变为丙氨酸导致Vif活性丧失，HIV-1复制抑制90%。这些研究表明，丝氨酸/苏氨酸蛋白激酶(S)对Vif的磷酸化在调节HIV-1的复制和感染性方面发挥着重要作用。
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 vivostudies demonstrated that Vif is highly phosphorylated on serine and threonine residues. To identify phosphorylation sites and characterize the Vif kinase(s), Vif was expressed inEscherichia coliand purified for use as a substrate inin vitrokinase assays. The purified Vif protein was phosphorylatedin vitroon 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,2P-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 phosphorylatedin vivo. Both Ser144and Thr188are 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. Ser144is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins. Mutation of Ser144to 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.