Fates of retroviral core components during unrestricted and TRIM5-restricted infection.
Fates of retroviral core components during unrestricted and TRIM5-restricted infection.
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DOI：
10.1371/journal.ppat.1003214
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发表时间：
2013-03
期刊：
影响因子：
6.7
通讯作者：
Bieniasz PD
中科院分区：
文献类型：
--
作者：
Kutluay SB;Perez-Caballero D;Bieniasz PD
TRIM5蛋白可以在病毒核心进入细胞质后不久限制逆转录病毒感染。然而，trim5α抑制感染的分子机制一直难以捉摸，部分原因是开发和执行检测逆转录病毒生命周期这一阶段的生化分析的难度。流行的模型表明，TRIM5α导致逆转录病毒衣壳的过早分解和/或蛋白酶体对衣壳的降解，但这些事件中是否有一个导致另一个尚不清楚。此外，TRIM5α如何影响病毒核心的基本成分，而不是衣壳，尚不清楚。为了解决这些问题，我们设计了一种生化分析方法，可以确定逆转录病毒核心的多种成分在感染期间的命运。我们利用可以有效感染VSV-G伪型逆转录病毒的细胞，并在同步感染后以线性梯度分离胞浆蛋白。然后监测衣壳蛋白和整合酶蛋白的命运，以及病毒基因组RNA和逆转录产物。我们发现，在非限制性条件下，MLV和HIV-1核心的成分形成了一个很大的复合体。相反，当病毒感染被人TRIM5RNA限制时，感染细胞的整合酶蛋白和反转录产物丢失，而衣壳和病毒α都被溶解。同样，当HIV-1感染被恒河猴TrIM5α或猫头猴TrIMCyp限制时，整合酶蛋白和逆转录产物丢失。然而，病毒RNA也丢失了，预先存在的高水平的可溶性CA阻止了CA是否被溶解的确定。值得注意的是，蛋白酶体抑制阻止了TRIM5α介导的限制的所有上述生化后果，但对其抗病毒效力没有影响。总之，我们的结果显示了TRIM5α如何影响逆转录病毒的各种核心成分，并表明蛋白酶体是TRIM5α诱导的核心破坏所必需的，而不是TRIM5α诱导的限制所必需的。在灵长类动物中发现的TRIM5蛋白是逆转录病毒感染的抑制物，在病毒核心进入细胞质后不久起作用。目前还很难阐明TRIM5蛋白是如何工作的，因为可以应用于病毒生命周期这一步骤的技术很繁琐。我们开发了一种实验方法，在感染后的早期，当TRIM5发挥作用时，我们可以监测TRIM5诱导的病毒核心的变化。具体地说，我们监测了病毒衣壳蛋白、整合酶和病毒基因组的命运。我们表明，TRIM5诱导这些核心成分中的每一个分解，当一些核心成分简单地解离时，另一些被降解。这些解离和降解事件似乎都依赖于蛋白酶体的活性。然而，我们也发现，这些TRIM5诱导的效应事件中的每一个都不是抑制所必需的。本文开发的检测方法为了解TRIM5α限制的机制提供了重要的见解，原则上可以应用于逆转录病毒生命周期中这一点上发生的其他重要过程。
TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5α inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5α causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5α affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5α, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5α or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5α-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5α affects various retroviral core components and indicate that proteasomes are required for TRIM5α-induced core disruption but not for TRIM5α-induced restriction. The TRIM5 proteins found in primates are inhibitors of retroviral infection that act soon after delivery of the viral core into the cytoplasm. It has been difficult to elucidate how TRIM5 proteins work, because techniques that can be applied to this step of the viral life cycle are cumbersome. We developed an experimental approach in which we can monitor TRIM5-induced changes in the viral core at early times after infection, when TRIM5 exerts its effects. Specifically, we monitored the fate of the viral capsid protein, the integrase enzyme and the viral genome. We show that TRIM5 induces disassembly of each of these core components, and while some core components simply dissociate, others are degraded. These dissociation and degradation events all appear to be dependent on the activity of the proteasome. However, we also find that each of these TRIM5-induced effects events are not necessary for inhibition. The assay developed herein provides important insight into the mechanism of TRIM5α restriction and can, in principle, be applied to other important processes that occur at this point in the retrovirus life cycle.
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作者：
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通讯作者：
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影响因子：
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在恒河体限制HIV-1时，可视化蛋白酶体的中间体。
影响因子：
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作者：
Campbell, Edward M.;Perez, Omar;Anderson, Jenny L.;Hope, Thomas J.
通讯作者：
Hope, Thomas J.