Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture
Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture
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DOI：
10.1128/jvi.70.8.5572-5581.1996
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发表时间：
1996-08-01
影响因子：
5.4
通讯作者：
Jeang, KT
中科院分区：
文献类型：
--
作者：
Neuveut, C;Jeang, KT
人类免疫缺陷病毒1型（HIV-1）达特是病毒复制所必需的，是病毒基因表达的有效反式激活剂。有证据表明，达特也影响病毒的感染性和细胞病变。在亚基因组环境中，利用点突变和瞬时转染读出对达特进行了广泛的结构-功能研究。这些报告基因测定已经确定某些氨基酸残基对于报告基因质粒的反式激活是重要的。然而，它们没有直接涉及与病毒复制相关的功能。在这里，我们研究了达特结构功能的复制病毒的设置。我们的特点是突变出现在达特在HIV-1感染的T淋巴细胞。为了确保改变的选择压力是针对蛋白质功能的，我们构建了HIV-1，其中达特阅读框架不受与vpr，rev和env的阅读框架重叠的限制。当这些重组病毒在T细胞中传代时，从220个独立分离的克隆的测序中观察到26个新的tnt核苷酸变化。将这些变化重新克隆到pNL 4 -3分子背景中，允许表征达特中对病毒复制重要的残基。有趣的是，许多影响复制的变化，当他们在瞬时反式激活质粒报告被发现是相对中性的。我们的结论是，达特在病毒复制的结构功能是不完全反映的活性测量的基础上，仅在亚基因组瞬时转染。
Human immunodeficiency virus type 1 (HIV-1) Tat is essential for virus replication and is a potent trans activator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Extensive structure-function studies of Tat in subgenomic settings with point mutagenesis and transient transfection readouts have been performed. These reporter assays have defined certain amino acid residues as being important for trans activation of reporter plasmids. However, they have not directly addressed functions related to virus replication. Here, we have studied Tat structure-function in the setting of replicating viruses. We characterized mutations that emerged in Tat during HIV-1 infections of T lymphocytes. To ensure that the selection pressure for change was directed toward protein function, we constructed HIV-1s in which the Tat reading frame was freed from constraints exerted by overlapping with the reading frames of vpr, rev, and env. When these recombinant viruses were passaged in T cells, 26 novel nucleotide changes in tnt were observed from sequencing of 220 independently isolated clones. Recloning of these changes into a pNL4-3 molecular background allowed for the characterization of residues in Tat important for virus replication. Interestingly, many of the changes that affected replication when they were assayed in transient trans activation of plasmid reporters were found to be relatively neutral. We conclude that the structure-function of Tat in virus replication is incompletely reflected by activity measurements based only on subgenomic transient transfections.