A cyclophilin homology domain-independent role for Nup358 in HIV-1 infection.
A cyclophilin homology domain-independent role for Nup358 in HIV-1 infection.
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DOI：
10.1371/journal.ppat.1003969
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发表时间：
2014-02
期刊：
影响因子：
6.7
通讯作者：
Poeschla EM
中科院分区：
文献类型：
--
作者：
Meehan AM;Saenz DT;Guevera R;Morrison JH;Peretz M;Fadel HJ;Hamada M;van Deursen J;Poeschla EM
大的核孔蛋白Nup358/RanBP2形成从核孔突出到细胞质中的八个细丝，在那里它们作为核质转运受体的对接平台。RNAi筛选表明Nup358参与了HIV-1的生命周期。该3，224个氨基酸的蛋白质的164个C-末端氨基酸是亲环蛋白同源结构域（Nup358Cyp），其具有结合HIV-1衣壳并调节病毒整合进程的潜力。在这里，我们研究了Nup358在条件性敲除小鼠细胞和RNAi耗尽的人CD4 + T细胞中的病毒学作用。Cre介导的基因敲除具有毒性，并降低了HIV-1的感染性。然而，如果在基因失活之前使用转座子表达所有Nup 358或仅表达含有三个FG重复序列和一个Ran结合结构域的N末端1340个氨基酸，则细胞健康和HIV-1易感性将得到协调保留。HIV-1，而不是N74D衣壳蛋白突变体HIV-1，对TNPO 3消耗显著敏感，但它们感染1 - 1340片段互补的Nup358敲除细胞的效果相当。人类和小鼠CypA都在CypA基因−/− Jurkat细胞中拯救了HIV-1，并且来自每个物种的TRIM-Nup358Cyp融合物具有同样的抗病毒性;每个都抑制WT和N74D病毒。在人CD4 + T细胞系SupT1中，突然的Nup358耗竭降低了病毒复制，但稳定的Nup358耗竭细胞正常复制HIV-1。因此，人CD4 + T细胞可以适应Nup358的丢失并保持HIV-1易感性。用环孢菌素、具有不结合亲环蛋白的衣壳的病毒和生长停滞的实验没有揭示病毒对Nup358的C-末端结构域的依赖性。我们的数据加强了TNPO 3的病毒学重要性，并表明Nup358支持细胞内稳态和HIV-1核输入重要的核转运功能。然而，这些结果并没有表明Nup358亲环蛋白或SUMO E3连接酶结构域在核转位前与HIV-1衣壳接合中的直接作用。Nup358/RanBP 2的亲环蛋白同源结构域（CHD）在体外可以与组装的HIV-1衣壳相互作用，这表明在细胞中，进入的病毒核心可以在穿过核孔之前被CHD功能性地接合。由于细胞核质转运的全面改变以及缺乏重新表达控制（由于蛋白质的大小而难以实现），Nup 358敲除的解释变得复杂。我们提出了Nup358敲除细胞和Nup358耗尽的人CD4 + T细胞中HIV-1生命周期的第一次分析，以及结构域要求和再表达控制的第一次详细研究。我们发现Nup358的N-末端部分含有三个FG重复而不是C-末端部分足以在分裂和非分裂基因敲除细胞中保持HIV-1感染易感性。CypA和Nup358Cyp的小鼠和人类版本在功能上是等同的，并且TNPO 3在两个物种的细胞中显示出显著的依赖性因子活性。人CD4 + T细胞系可以稳定地缺乏Nup358而不丧失HIV-1过氧化物酶。这些数据支持Nup358在HIV-1生命周期中的重要作用，尽管亲环蛋白同源结构域独立，并证明了人类和小鼠细胞之间HIV-1早期事件途径的保守性。
The large nucleoporin Nup358/RanBP2 forms eight filaments that project from the nuclear pore into the cytoplasm where they function as docking platforms for nucleocytoplasmic transport receptors. RNAi screens have implicated Nup358 in the HIV-1 life cycle. The 164 C-terminal amino acids of this 3,224 amino acid protein are a cyclophilin homology domain (Nup358Cyp), which has potential to bind the HIV-1 capsid and regulate viral progress to integration. Here we examined the virological role of Nup358 in conditional knockout mouse cells and in RNAi-depleted human CD4+ T cells. Cre-mediated gene knockout was toxic and diminished HIV-1 infectivity. However, cellular health and HIV-1 susceptibility were coordinately preserved if, prior to gene inactivation, a transposon was used to express all of Nup358 or only the N-terminal 1340 amino acids that contain three FG repeats and a Ran-binding domain. HIV-1, but not N74D capsid-mutant HIV-1, was markedly sensitive to TNPO3 depletion, but they infected 1–1340 segment-complemented Nup358 knockout cells equivalently. Human and mouse CypA both rescued HIV-1 in CypA gene −/− Jurkat cells and TRIM-Nup358Cyp fusions derived from each species were equally antiviral; each also inhibited both WT and N74D virus. In the human CD4+ T cell line SupT1, abrupt Nup358 depletion reduced viral replication but stable Nup358-depleted cells replicated HIV-1 normally. Thus, human CD4+ T cells can accommodate to loss of Nup358 and preserve HIV-1 susceptibility. Experiments with cylosporine, viruses with capsids that do not bind cyclophilins, and growth arrest did not uncover viral dependency on the C-terminal domains of Nup358. Our data reinforce the virological importance of TNPO3 and show that Nup358 supports nuclear transport functions important for cellular homeostasis and for HIV-1 nuclear import. However, the results do not suggest direct roles for the Nup358 cyclophilin or SUMO E3 ligase domains in engaging the HIV-1 capsid prior to nuclear translocation. The purified cyclophilin homology domain (CHD) of Nup358/RanBP2 can interact with assembled HIV-1 capsids in vitro, which suggests that, in cells, the incoming virus core could be engaged functionally by the CHD prior to nucleopore traverse. Interpretations of Nup358 knockdowns have been complicated by toxicity due to globally altered cellular nucleocytoplasmic transport and by a lack of re-expression controls, which are difficult because of the protein's size. We present the first analyses of the HIV-1 life cycle in Nup358 knockout cells and in Nup358-depleted human CD4+ T cells, and the first detailed studies of domain requirements and re-expression controls. We find that the N-terminal portion containing three FG repeats rather than the C-terminal portion of Nup358 is sufficient to preserve HIV-1 infection susceptibility in dividing and non-dividing gene knockout cells. Mouse and human versions of CypA and Nup358Cyp are functionally equivalent and TNPO3 displays marked dependency factor activity in cells of both species. A human CD4+ T cell line can be stably deficient in Nup358 without loss of HIV-1 permissivity. The data support an important, though cyclophilin homology domain-independent, role for Nup358 in the HIV-1 life cycle and demonstrate conservation of HIV-1 early event pathways between human and mouse cells.
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DOI：
10.1073/pnas.0505659102
发表时间：
2005-10-11
影响因子：
11.1
作者：
Berthoux, L;Sebastian, S;Luban, J
通讯作者：
Luban, J
影响因子：
11.4
作者：
Braaten, D;Luban, J
通讯作者：
Luban, J
影响因子：
3.3
作者：
De Iaco A;Luban J
通讯作者：
Luban J
影响因子：
3.3
作者：
Fricke T;Valle-Casuso JC;White TE;Brandariz-Nuñez A;Bosche WJ;Reszka N;Gorelick R;Diaz-Griffero F
通讯作者：
Diaz-Griffero F
影响因子：
3.5
作者：
Joseph, Jomon;Dasso, Mary
通讯作者：
Dasso, Mary