Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Virol. 2011 Sep;85(17):8725-37. doi: 10.1128/JVI.00497-11. Epub 2011 Jul 6.
TRIM5α(rh) is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5α(rh) is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5α exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5α(rh) restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5α(rh) RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5α and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5α RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5α(rh), such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5α molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5α self-ubiquitylation in the ability of TRIM5α to restrict HIV-1.
TRIM5α(rh) 是一种细胞质蛋白，在逆转录前强烈限制 HIV-1。TRIM5α(rh) 由四个不同的结构域组成：RING、B-box 2、卷曲螺旋和 B30.2(SPRY)。这些结构域中的每一个对限制作用的贡献都进行了广泛的研究，除了 RING 结构域。TRIM5α 的 RING 结构域具有 E3-泛素连接酶活性，但该活性对 HIV-1 限制的贡献尚不清楚。为了检验 RING 结构域的 E3-泛素连接酶活性调节 TRIM5α(rh) 限制 HIV-1 的假设，我们将一组 TRIM5α(rh) RING 结构域变体的 E3-泛素连接酶活性与这些突变蛋白限制 HIV-1 的能力相关联。为此，我们首先解决了 TRIM5α 的 RING 结构域的核磁共振结构，并通过与其他 RING 结构域的同源性定义了 RING 结构域的潜在功能区域。有了这些结构信息，我们对 RING 结构域区域进行了系统的诱变，并测试了 TRIM5α RING 结构域变体进行自我泛素化的能力。几个残基，特别是 RING 结构域的 E2 结合区域的残基，在自我泛素化能力上存在缺陷。为了将 HIV-1 限制与自我泛素化相关联，我们使用了在自我泛素化方面存在缺陷但保留了 TRIM5α(rh) 有效限制所需的重要性质的 RING 结构域突变蛋白，例如衣壳结合和高级自身缔合。从这些研究中，我们发现了一组残基，当它们发生突变时，TRIM5α 分子会失去有效限制 HIV-1 的能力及其自我泛素化活性。值得注意的是，所有这些变化都发生在 RING 结构域的 E2 结合区域的残基上。总的来说，这些结果表明 TRIM5α 自我泛素化在 TRIM5α 限制 HIV-1 的能力中起作用。
