Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication
Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication
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DOI：
10.1128/jvi.78.23.12735-12746.2004
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发表时间：
2004-12-01
影响因子：
5.4
通讯作者：
Engelman, A
中科院分区：
文献类型：
--
作者：
Lu, R;Limón, A;Engelman, A
整合酶与人类免疫缺陷病毒1型（HIV-1）的核输入有关。整合酶分析，但是，可以复杂的突变的多效性：而I类突变体的整合缺陷，II类突变体显示额外的组装和/或逆转录缺陷。我们以前确定，HIV-1（V165 A），最初报告为核输入缺陷，是一个II类突变体。在这里，我们分析了包含其他假定的核定位信号变化的突变体，包括（186）KRK（188/211）KELQKQITK（219）和Cys-130。以前的工作确定HIV-1（K186 Q），HIV-1（Q214 L/Q216 L）和HIV-1（C130 G）为复制缺陷型，但表型分类不清楚，未解决非分裂细胞中的核输入。与以前的报道一致，这里研究的大多数二分突变体是复制缺陷型的。这些突变体以及HIV-1（V165 A）合成的cDNA水平降低，但正常部分的突变cDNA定位于分裂和非分裂细胞核。有些令人惊讶的是，重组的II类突变体蛋白具有催化活性，并且II类Vpr-整合酶融合蛋白有效地补充I类突变体病毒。由于I类Vpr整合酶突变体有效地补充H类突变病毒的条件下，II类Vpr整合酶未能发挥作用，我们得出结论，I类和H定义两个不同的互补组，并建议II类突变体主要是有缺陷的HIV-1复制的核后进入步骤。HIV-1（C130 G）也有逆转录缺陷，但Vpr整合酶（C130 G）不能有效地补充I类突变体HIV-1。由于HIV-1（C130 A）的生长像野生型，我们得出结论，Cys-130是不是复制所必需的，并推测整合酶结构的扰动有助于多效性HIV-1（C130 G）表型。
Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1(V165A),originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including (186)KRK(188/211)KELQKQITK(219)and Cys-130. Previous work established HIV-1(K186Q), HIV-1(Q214L/Q216L,) and HIV-1(C130G), as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1(V165A) synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class H mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and H define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1(C130G) was also defective for reverse transcription, but Vpr-integrase(c130G) did not efficiently complement class I mutant HIV-1. Since HIV-1(C130A) grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1(C130G) phenotype.