The Abundant Tegument Protein pUL25 of Human Cytomegalovirus Prevents Proteasomal Degradation of pUL26 and Supports Its Suppression of ISGylation
The Abundant Tegument Protein pUL25 of Human Cytomegalovirus Prevents Proteasomal Degradation of pUL26 and Supports Its Suppression of ISGylation
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DOI：
10.1128/jvi.01180-18
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发表时间：
2018-10
影响因子：
5.4
通讯作者：
C. Zimmermann;Nicole Büscher;Steffi Krauter;N. Krämer;U. Wolfrum;E. Sehn;S. Tenzer;B. Plachter
中科院分区：
文献类型：
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作者：
C. Zimmermann;Nicole Büscher;Steffi Krauter;N. Krämer;U. Wolfrum;E. Sehn;S. Tenzer;B. Plachter
人巨细胞病毒 (HCMV) 表达许多干扰细胞内在和先天防御机制的外皮蛋白。 HCMV 感染对干扰素刺激的基因 15 蛋白 (ISG15) 的初始诱导以及蛋白与 ISG15 的结合 (ISGylation) 随后会因病毒 IE1、pUL50 和 pUL26 蛋白的表达而减弱。这项研究添加了 pUL25 作为另一个有助于抑制 ISGylation 的因素。外皮蛋白与 pUL26 相互作用并防止其被蛋白酶体降解。通过这样做，它支持其对 ISGylation 的限制性影响。此外，pUL25 的缺乏会提高游离 ISG15 的水平，表明皮膜蛋白可能会在与 pUL26 相互作用以外的水平上干扰干扰素反应。这项研究中获得的知识拓宽了我们对 HCMV 免疫逃避的理解，也可能为使用 pUL25 阴性菌株生产疫苗提供新途径。摘要 人巨细胞病毒 (HCMV) 病毒粒子的外皮含有干扰内在免疫和先天免疫的蛋白质。 pUL25 是一种迄今为止功能未知的蛋白质。病毒突变体（Towne-ΔUL25）中 pUL25 的缺失对病毒体和亚病毒致密体的释放或病毒体形态发生没有影响。蛋白质组学分析显示细胞外颗粒的整体蛋白质组成几乎没有变化。然而，令人惊讶的结果是，病毒粒子和 Towne-ΔUL25 致密体中几乎完全不存在 pUL26，并且突变病毒感染细胞中大亚型 pUL26-p27 减少。 pUL26 已被证明可以抑制与干扰素刺激的基因 15 蛋白 (ISG15) 的蛋白缀合，从而支持 HCMV 复制。为了测试 pUL25 和 pUL26 之间的功能关系，我们研究了 pUL26 的稳态水平，发现它们在 Towne-ΔUL25 感染的细胞中降低。免疫共沉淀实验证明了 pUL25 和 pUL26 之间存在相互作用。令人惊讶的是，Towne-ΔUL25 感染的细胞中整体蛋白质 ISG 化增强，从而模仿了 pUL26 缺失的 HCMV 突变体的表型。 Towne-ΔUL25 的复制对 β 干扰素更敏感，证实了这一点的功能相关性。在感染缺乏皮膜蛋白 pp65 的突变体的细胞中也观察到蛋白质 ISGylation 的增加。重新测试后，我们发现当 pp65 不可用时，pUL26 的降解也会增加。我们的实验表明，pUL25 和 pp65 均调节 pUL26 降解和 pUL26 依赖性 ISGylation 减少，并添加 pUL25 作为另一种干扰宿主细胞内在免疫的 HCMV 外被蛋白。重要性 人巨细胞病毒 (HCMV) 表达许多干扰细胞内在和先天防御机制的外皮蛋白。 HCMV 感染对干扰素刺激的基因 15 蛋白 (ISG15) 的初始诱导以及蛋白与 ISG15 的结合 (ISGylation) 随后会因病毒 IE1、pUL50 和 pUL26 蛋白的表达而减弱。这项研究添加了 pUL25 作为另一个有助于抑制 ISGylation 的因素。外皮蛋白与 pUL26 相互作用并防止其被蛋白酶体降解。通过这样做，它支持其对 ISGylation 的限制性影响。此外，pUL25 的缺乏会提高游离 ISG15 的水平，表明皮膜蛋白可能会在与 pUL26 相互作用以外的水平上干扰干扰素反应。这项研究中获得的知识拓宽了我们对 HCMV 免疫逃避的理解，也可能为使用 pUL25 阴性菌株生产疫苗提供新途径。
Human cytomegalovirus (HCMV) expresses a number of tegument proteins that interfere with the intrinsic and the innate defense mechanisms of the cell. Initial induction of the interferon-stimulated gene 15 protein (ISG15) and conjugation of proteins with ISG15 (ISGylation) by HCMV infection are subsequently attenuated by the expression of the viral IE1, pUL50, and pUL26 proteins. This study adds pUL25 as another factor that contributes to suppression of ISGylation. The tegument protein interacts with pUL26 and prevents its degradation by the proteasome. By doing this, it supports its restrictive influence on ISGylation. In addition, a lack of pUL25 enhances the levels of free ISG15, indicating that the tegument protein may interfere with the interferon response on levels other than interacting with pUL26. Knowledge obtained in this study widens our understanding of HCMV immune evasion and may also provide a new avenue for the use of pUL25-negative strains for vaccine production. ABSTRACT The tegument of human cytomegalovirus (HCMV) virions contains proteins that interfere with both the intrinsic and the innate immunity. One protein with a thus far unknown function is pUL25. The deletion of pUL25 in a viral mutant (Towne-ΔUL25) had no impact on the release of virions and subviral dense bodies or on virion morphogenesis. Proteomic analyses showed few alterations in the overall protein composition of extracellular particles. A surprising result, however, was the almost complete absence of pUL26 in virions and dense bodies of Towne-ΔUL25 and a reduction of the large isoform pUL26-p27 in mutant virus-infected cells. pUL26 had been shown to inhibit protein conjugation with the interferon-stimulated gene 15 protein (ISG15), thereby supporting HCMV replication. To test for a functional relationship between pUL25 and pUL26, we addressed the steady-state levels of pUL26 and found them to be reduced in Towne-ΔUL25-infected cells. Coimmunoprecipitation experiments proved an interaction between pUL25 and pUL26. Surprisingly, the overall protein ISGylation was enhanced in Towne-ΔUL25-infected cells, thus mimicking the phenotype of a pUL26-deleted HCMV mutant. The functional relevance of this was confirmed by showing that the replication of Towne-ΔUL25 was more sensitive to beta interferon. The increase of protein ISGylation was also seen in cells infected with a mutant lacking the tegument protein pp65. Upon retesting, we found that pUL26 degradation was also increased when pp65 was unavailable. Our experiments show that both pUL25 and pp65 regulate pUL26 degradation and the pUL26-dependent reduction of ISGylation and add pUL25 as another HCMV tegument protein that interferes with the intrinsic immunity of the host cell. IMPORTANCE Human cytomegalovirus (HCMV) expresses a number of tegument proteins that interfere with the intrinsic and the innate defense mechanisms of the cell. Initial induction of the interferon-stimulated gene 15 protein (ISG15) and conjugation of proteins with ISG15 (ISGylation) by HCMV infection are subsequently attenuated by the expression of the viral IE1, pUL50, and pUL26 proteins. This study adds pUL25 as another factor that contributes to suppression of ISGylation. The tegument protein interacts with pUL26 and prevents its degradation by the proteasome. By doing this, it supports its restrictive influence on ISGylation. In addition, a lack of pUL25 enhances the levels of free ISG15, indicating that the tegument protein may interfere with the interferon response on levels other than interacting with pUL26. Knowledge obtained in this study widens our understanding of HCMV immune evasion and may also provide a new avenue for the use of pUL25-negative strains for vaccine production.