Human cytomegalovirus UL97 kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis
Human cytomegalovirus UL97 kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis
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DOI：
10.1128/jvi.79.24.15494-15502.2005
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发表时间：
2005-12-01
影响因子：
5.4
通讯作者：
Kern, ER
中科院分区：
文献类型：
--
作者：
Prichard, MN;Britt, WJ;Kern, ER
不表达UL 97激酶活性的重组人巨细胞病毒表现出独特的斑块形态，其特征在于在感染后期形成高度可伸缩的体。在用UL 97激酶抑制剂Maribavir处理的感染细胞中也观察到了这些结构。核包涵体纯化至接近同质，并通过基质辅助激光解吸电离-飞行时间质谱鉴定组成蛋白。该分析表明，聚集体主要由被膜蛋白pp 65和ppUL 25形成，但也含有另外的病毒体结构蛋白，包括主要衣壳蛋白。免疫印迹实验证实了这些结果，并确定了一些额外的病毒蛋白存在于纯化的被膜聚集体。有趣的是，这些结构的形成似乎依赖于pp 65，因为在用该开放阅读框缺失的重组病毒感染的细胞中，pp 65不被诱导。形态学上相似的聚集体可以通过过度表达pp 65在未感染细胞的细胞核中复制，并且通过共表达UL 97激酶来防止它们的形成。用Maribavir抑制UL 97激酶活性或激酶中的一种必需氨基酸突变可消除其防止聚集体形成的能力。这些数据一起表明，UL 97激酶影响感染细胞核中pp 65的聚集。我们认为，激酶在细胞核中病毒体形态发生过程中的被膜获得中起着重要作用，这种活性代表了成熟病毒颗粒产生的重要步骤。
Recombinant human cytomegalovi ruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly retractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.