Site-Directed Mutagenesis of HIV-1 vpu Gene Demonstrates Two Clusters of Replication-Defective Mutants with Distinct Ability to Down-Modulate Cell Surface CD4 and Tetherin.
Site-Directed Mutagenesis of HIV-1 vpu Gene Demonstrates Two Clusters of Replication-Defective Mutants with Distinct Ability to Down-Modulate Cell Surface CD4 and Tetherin.
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DOI：
10.3389/fmicb.2010.00116
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发表时间：
2010
影响因子：
5.2
通讯作者：
Adachi A
中科院分区：
文献类型：
--
作者：
Nomaguchi M;Doi N;Fujiwara S;Fujita M;Adachi A
HIV-1 Vpu通过介导内质网中的CD 4降解对病毒感染性起积极作用，并通过抵消病毒体释放限制因子Tetherin来增强病毒体释放。为了确定Vpu活性对HIV-1复制的影响，我们已经产生了一系列位点特异性前病毒Vpu突变体。15个突变体检查，7个表现出复制缺陷类似的vpu缺失突变体在淋巴细胞系H9。这些突变聚集在跨膜结构域（TMD）和胞质结构域（CTD）内的狭窄区域。复制缺陷型突变体无一例外地表现出降低的增强病毒体从单层细胞系HEp 2释放的能力。在用Vpu表达载体转染后，TMD突变体和CTD突变体都不阻断另一单层细胞系MAGI中细胞表面的CD 4表达。虽然TMD突变体不能下调HEp 2细胞中的细胞表面拴系蛋白，但CTD突变体却相当有效。共聚焦显微镜分析揭示了TMD和CTD突变体细胞内定位的差异。总的来说，携带Vpu突变的HIV-1的复制能力与Vpu增强病毒体释放和阻碍CD 4的细胞表面表达的能力密切相关，但与下调细胞表面拴系蛋白的能力无关。我们的研究结果表明，有效的病毒复制不仅需要下调细胞表面tetherin，但也降解。
HIV-1 Vpu acts positively on viral infectivity by mediating CD4 degradation in endoplasmic reticulum and enhances virion release by counteracting a virion release restriction factor, tetherin. In order to define the impact of Vpu activity on HIV-1 replication, we have generated a series of site-specific proviral vpu mutants. Of fifteen mutants examined, seven exhibited a replication-defect similar to that of a vpu-deletion mutant in a lymphocyte cell line H9. These mutations clustered in narrow regions within transmembrane domain (TMD) and cytoplasmic domain (CTD). Replication-defective mutants displayed the reduced ability to enhance virion release from a monolayer cell line HEp2 without exception. Upon transfection with Vpu expression vectors, neither TMD mutants nor CTD mutants blocked CD4 expression at the cell surface in another monolayer cell line MAGI. While TMD mutants were unable to down-modulate cell surface tetherin in HEp2 cells, CTD mutants did quite efficiently. Confocal microscopy analysis revealed the difference of intracellular localization between TMD and CTD mutants. In total, replication capability of HIV-1 carrying vpu mutations correlates well with the ability of Vpu to enhance virion release and to impede the cell surface expression of CD4 but not with the ability to down-modulate cell surface tetherin. Our results here suggest that efficient viral replication requires not only down-regulation of cell surface tetherin but also its degradation.
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