The late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell type-dependent manner
The late domain of human immunodeficiency virus type 1 p6 promotes virus release in a cell type-dependent manner
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DOI：
10.1128/jvi.76.1.105-117.2002
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发表时间：
2002-01-01
影响因子：
5.4
通讯作者：
Freed, EO
中科院分区：
文献类型：
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作者：
Demirov, DG;Orenstein, JM;Freed, EO
人类免疫缺陷病毒I型（HIV-1）的p6结构域位于Gag前体蛋白Pr 55（Gag）的C末端。先前的研究表明，p6在HIV-1颗粒从表达病毒的HeLa细胞出芽中起关键作用。在这项研究中，我们进行了详细的突变分析的N末端的p6映射所需的序列有效的病毒释放。我们观察到，位于p6的N末端附近的高度保守的P-T/S-A-P基序是非常敏感的变化，甚至在这个序列中的保守突变造成深刻的病毒释放缺陷的HeLa细胞。相比之下，P-T/S-A-P基序外的单氨基酸和双氨基酸取代对颗粒释放没有显著影响。在HeLa细胞中，在P-T/S-A-P基序之外的一个或两个残基引入终止密码子显著损害病毒粒子释放，而在P-T/S-A-P之外截短四个残基对颗粒产生没有影响。通过研究p6突变的影响，在生物和生化分析，并通过电子显微镜，我们定义了p6在颗粒释放和病毒复制在一个面板的T细胞和贴壁细胞系，并在初级淋巴细胞和单核细胞衍生的巨噬细胞的作用。我们证明，p6突变对病毒复制的影响是显着的细胞类型依赖性。有趣的是，即使在p6突变阻断病毒复制的T细胞系和原代淋巴细胞中，这些变化对颗粒释放的影响很小或没有影响。然而，在T细胞系和原代淋巴细胞中产生的p6突变体颗粒在病毒体-病毒体脱离中表现出缺陷，导致病毒体的拴系链的产生。单核细胞源性巨噬细胞中的病毒释放被p6突变显著抑制。为了进一步检查HeLa细胞与T细胞中的细胞类型特异性病毒释放缺陷，在HeLa细胞和Jurkat T细胞系之间产生瞬时异核体。这些异核体显示出T细胞样表型，相对于颗粒释放中对p6的要求。这里描述的结果定义了p6在广泛的细胞类型中病毒复制中的作用，并揭示了在病毒颗粒出芽中对p6的强烈的细胞类型依赖性要求。
The p6 domain of human immunodeficiency virus type I (HIV-1) is located at the C terminus of the Gag precursor protein Pr55(Gag). Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.