Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation
Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation
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DOI：
10.1128/jvi.02384-16
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发表时间：
2017-06-01
影响因子：
5.4
通讯作者：
Borst, Eva Maria
中科院分区：
文献类型：
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作者：
Neuber, Sebastian;Wagner, Karen;Borst, Eva Maria
人巨细胞病毒（HCMV）基因组的终止需要几种必需的病毒蛋白，其中包括构成病毒末端酶的pUL 56、pUL 89和最近描述的pUL 51。为了深入了解终止酶复合物的组装，我们研究了各个亚基之间的相互作用。对于病毒背景下的分析，使用在编码末端酶蛋白的开放阅读框中携带缺失的HCMV细菌人工染色体。这些实验通过瞬时转染测定与表达终止酶组分的质粒进行补充。我们发现，如果一个末端酶蛋白缺失，其他末端酶蛋白的水平显着减少，这可以克服蛋白酶体抑制或提供缺失的亚基在反式。这些数据意味着，隔离的单个亚基内的末端酶复合物保护他们从蛋白酶体营业额。只有当所有三个一起存在时，终止酶蛋白之间才发生有效的相互作用，这一发现让人想起了导致合作稳定性的结合后折叠原理。此外，尽管pUL 56自身易位到细胞核中，pUL 51和pUL 89的正确核定位同样需要所有三种末端酶成分。总而言之，这些特征表明HCMV末端酶是一种多蛋白复合物，其中三个参与者相互调节稳定性，亚细胞定位和组装成功能性三方全酶。重要性HCMV是免疫功能低下个体的主要危险因素，先天性CMV感染是导致长期后遗症的主要病毒原因，包括耳聋和智力低下。目前CMV疾病的治疗是基于具有相同机制的药物，即抑制病毒DNA复制，并且经常导致不良副作用和耐药病毒株的出现。最近，HCMV末端酶已经成为新型抗病毒药物的一个吉祥的靶点。一种抑制HCMV末端酶的新候选药物Letermovir在临床试验中表现出优异的效力;然而，其确切的作用方式尚未了解。在这里，我们描述了HCMV末端酶成分的相互依赖性，它们组装成一个功能性的末端酶复合物。除了提供新的基本见解终止酶的形成，这些结果将是有价值的，当研究作用机制的药物靶向HCMV终止酶和开发其他物质干扰病毒基因组的终止。
Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans. These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.