Proteasome-dependent disruption of the E3 ubiquitin ligase anaphase-promoting complex by HCMV protein pUL21a.
Proteasome-dependent disruption of the E3 ubiquitin ligase anaphase-promoting complex by HCMV protein pUL21a.
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DOI：
10.1371/journal.ppat.1002789
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Yu D
中科院分区：
文献类型：
--
作者：
Fehr AR;Gualberto NC;Savaryn JP;Terhune SS;Yu D
后期促进复合体(APC)是一种E3泛素连接酶，控制多种细胞周期调节蛋白的泛素化和降解。在感染过程中，人类巨细胞病毒(HCMV)不仅通过多功能病毒激酶pUL97使APC辅活化子CDH1磷酸化，还通过一种未知的机制促进APC亚基的降解。利用蛋白质组学方法，我们发现最近发现的一种HCMV蛋白pUL21a与APC相互作用。重要的是，我们确定pUL21a的表达是APC亚基APC4和APC5依赖于蛋白酶体降解的必要条件和充分条件。这导致了APC中断，并需要pUL21a与APC结合。我们已经确定pUL21a第109-110位的Pro-Arg氨基酸对其结合和调节APC的能力是关键的。一种点突变病毒(PR-AA)在野生型水平上生长，其中的Pro-精氨酸突变为丙氨酸(PR-AA)。然而，与UL97缺失病毒相比，PR-AA点突变和UL97缺失同时取消了病毒调节APC的能力的双突变病毒的减弱程度明显更大。这表明这些突变是合成致命的，并且HCMV利用两个病毒因子来确保成功地破坏APC，以克服其对病毒感染的限制。这项研究揭示了HCMV蛋白pUL21a作为一种新的APC调节蛋白，并揭示了一种独特的病毒机制来破坏APC活性。在这项研究中，我们报告了一种有趣的机制，人类巨细胞病毒(HCMV)用来调节细胞内的E3泛素连接酶，后期促进复合体(APC)。在许多病毒感染中，劫持泛素-蛋白酶体系统以调节蛋白质降解和操纵细胞周期以合成病毒基因组的能力是至关重要的。APC是一种主要的细胞周期调节器，针对蛋白酶体降解的许多调节蛋白。它可以阻止细胞进入S期，从而阻碍病毒强迫细胞进入有利于病毒DNA合成的细胞环境。我们已经鉴定出一种HCMV蛋白pUL21a，它使用一种似乎违反直觉的机制来调节APC。它与APC相互作用，以此泛素连接酶亚单位为靶点进行蛋白酶体降解。这会导致复合体的破坏，并减少其活动。此外，缺少pUL21a和pUL97(另一种HCMV编码的APC调节子)的病毒与单独失去UL97相比高度减弱，这表明HCMV使用两种蛋白质来完全解除APC的武装。这项研究确定了一种疱疹病毒蛋白，它使用一种独特的、蛋白酶体依赖的机制来调节这种突出的细胞E3泛素连接酶的活性。
The anaphase-promoting complex (APC) is an E3 ubiquitin ligase which controls ubiquitination and degradation of multiple cell cycle regulatory proteins. During infection, human cytomegalovirus (HCMV), a widespread pathogen, not only phosphorylates the APC coactivator Cdh1 via the multifunctional viral kinase pUL97, it also promotes degradation of APC subunits via an unknown mechanism. Using a proteomics approach, we found that a recently identified HCMV protein, pUL21a, interacted with the APC. Importantly, we determined that expression of pUL21a was necessary and sufficient for proteasome-dependent degradation of APC subunits APC4 and APC5. This resulted in APC disruption and required pUL21a binding to the APC. We have identified the proline-arginine amino acid pair at residues 109–110 in pUL21a to be critical for its ability to bind and regulate the APC. A point mutant virus in which proline-arginine were mutated to alanines (PR-AA) grew at wild-type levels. However, a double mutant virus in which the viral ability to regulate the APC was abrogated by both PR-AA point mutation and UL97 deletion was markedly more attenuated compared to the UL97 deletion virus alone. This suggests that these mutations are synthetically lethal, and that HCMV exploits two viral factors to ensure successful disruption of the APC to overcome its restriction on virus infection. This study reveals the HCMV protein pUL21a as a novel APC regulator and uncovers a unique viral mechanism to subvert APC activity. In this study, we report an intriguing mechanism used by human cytomegalovirus (HCMV) to regulate a cellular E3 ubiquitin ligase, the anaphase promoting complex (APC). The ability to hijack the ubiquitin-proteasome system for regulating protein degradation and to manipulate the cell cycle for viral genome synthesis is critical in many viral infections. The APC is a master cell cycle modulator that targets a number of regulatory proteins for proteasomal degradation. It can prevent cells from entry into S-phase, thus creating a hindrance for viruses needing to coerce cells into a cellular environment favorable for viral DNA synthesis. We have identified an HCMV protein, pUL21a, which uses a seemingly counterintuitive mechanism to regulate the APC. It interacts with the APC to target the subunits of this ubiquitin ligase for proteasomal degradation. This causes disruption of the complex and reduces its activity. Furthermore, a virus lacking pUL21a and pUL97, which is another HCMV-encoded APC regulator, was highly attenuated when compared to loss of UL97 alone, suggesting that HCMV uses two proteins to fully disarm the APC. This study identifies a herpesviral protein that uses a unique, proteasome-dependent mechanism to regulate the activity of this prominent cellular E3 ubiquitin ligase.
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