Evidence for an Activation Domain at the Amino Terminus of Simian Immunodeficiency Virus Vpx
Evidence for an Activation Domain at the Amino Terminus of Simian Immunodeficiency Virus Vpx
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DOI：
10.1128/jvi.01437-09
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发表时间：
2010-02-01
影响因子：
5.4
通讯作者：
Landau, Nathaniel R.
中科院分区：
文献类型：
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作者：
Gramberg, Thomas;Sunseri, Nicole;Landau, Nathaniel R.
Vpx和Vpr是相关的慢病毒辅助蛋白，可增强巨噬细胞和树突状细胞中的病毒复制。这两种蛋白都被包装成病毒粒子，并通过与含有DCAF1和DDB1的E3泛素连接酶的相互作用来介导它们在靶细胞中的作用。当以病毒样颗粒的形式引入原代巨噬细胞和树突状细胞时，Vpx可以提高后续感染的效率。本研究通过使用单周期报告病毒和用含有Vpx的病毒样颗粒预处理细胞，证实了Vpx能够使巨噬细胞感染猿猴免疫缺陷病毒（SIV）和人类免疫缺陷病毒1型（HIV-1）的能力提高100倍。Vpx在分化的THP-1细胞中也有活性，但在其他细胞系中无活性。I型干扰素在巨噬细胞中诱导抗病毒状态显著放大了Vpx对HIV-1感染的作用，表明Vpx有助于病毒克服可诱导的细胞内限制。新感染巨噬细胞中SIV和HIV-1逆转录物的定量PCR定量分析表明，该阻断处于逆转录的早期阶段。尽管结构相似，Vpr却无活性。这种差异使我们能够用Vpr/Vpx嵌合体面板绘制Vpx的功能域。嵌合体分析表明Vpx的氨基末端结构域对增强感染具有重要作用。该区域的精细定位表明，在位置9、12、15至17需要氨基酸。尽管突变体不能增强感染，但它们保留了与DCAF1相互作用的能力。这些发现表明Vpx氨基端包含一个激活域，作为细胞限制因子的结合位点。
Vpx and Vpr are related lentiviral accessory proteins that enhance virus replication in macrophages and dendritic cells. Both proteins are packaged into virions and mediate their effects in the target cell through an interaction with an E3 ubiquitin ligase that contains DCAF1 and DDB1. When introduced into primary macrophages and dendritic cells in viruslike particles, Vpx can enhance the efficiency of a subsequent infection. Here, we confirm the ability of Vpx to enhance simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) infection of macrophages up to 100-fold by using single-cycle reporter viruses and by pretreatment of the cells with Vpx-containing viruslike particles. Vpx was also active in differentiated THP-1 cells but not in other cell lines. Induction of an antiviral state in macrophages with type I interferon significantly magnified the effect of Vpx on HIV-1 infection, suggesting that Vpx helps the virus to overcome an inducible intracellular restriction. Quantitative PCR quantitation of SIV and HIV-1 reverse transcripts in newly infected macrophages showed that the block was at an early step in reverse transcription. In spite of its structural similarity, Vpr was inactive. This difference allowed us to map the functional domains of Vpx with a panel of Vpr/Vpx chimeras. Analysis of the chimeras demonstrated that the amino-terminal domain of Vpx is important for the enhancement of infection. Fine mapping of the region indicated that amino acids at positions 9, 12, and 15 to 17 were required. Although the mutants failed to enhance infection, they retained their ability to interact with DCAF1. These findings suggest that the Vpx amino terminus contains an activation domain that serves as the binding site for a cellular restriction factor.