Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle.
Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle.
复制标题
DOI：
10.1016/j.antiviral.2014.10.011
复制
发表时间：
2015-01
影响因子：
7.6
通讯作者：
Tortorella, Domenico
中科院分区：
文献类型：
--
作者：
Gardner, Thomas J.;Cohen, Tobias;Redmann, Veronika;Lau, Zerlina;Felsenfeld, Dan;Tortorella, Domenico
关键词：
人类巨细胞病毒（CMV）是一种潜伏且持久的病毒，其增殖会增加免疫功能低下个体的发病率和死亡率。目前针对病毒 DNA 聚合酶或主要立即早期 (MIE) 基因位点的抗 CMV 疗法在限制 CMV 相关疾病方面有些有效。然而，由于生物利用度低、毒性严重以及长期治疗后出现耐药CMV菌株，目前的抗CMV疗法还不够。为了帮助解决这一不足，我们建立了一种高内涵测定法来识别针对 CMV 进入和感染早期步骤的抑制剂。用表达嵌合 IE2-黄色荧光蛋白 (YFP) (AD169IE2-YFP) 的 CMV 实验室毒株 AD169 变体感染原代人成纤维细胞，为高内涵测定提供了基础。 AD169IE2-YFP 感染后不久，IE2-YFP 定位到细胞核，诱导了强烈的荧光信号，并使用共聚焦显微镜对其进行了量化。该测定经过优化，可实现出色的测定适应性和高 Z' 分数。然后，我们筛选了由 2080 种化合物组成的生物活性化学库，并根据 IE2-YFP 核表达的荧光信号的减少鉴定了命中化合物。这些命中化合物可能针对感染早期步骤中涉及的各种细胞过程，包括衣壳运输、染色质重塑和病毒基因表达。广泛的二次分析证实了一种热门化合物——铃兰毒素——能够抑制实验室和临床 CMV 毒株的感染并限制病毒增殖。总的来说，数据表明我们已经建立了一个强大的高内涵筛选来识别限制 CMV 生命周期早期步骤的化合物，并且早期感染事件的新型抑制剂可以作为可行的 CMV 疗法。
Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169IE2-YFP) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z′ scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.
登录
查看更多内容
影响因子：
4.9
作者：
Kapoor, Arun;Cai, Hongyi;Arav-Boger, Ravit
通讯作者：
Arav-Boger, Ravit
影响因子：
4.8
作者：
Baker, Brooke M.;Tortorella, Domenico
通讯作者：
Tortorella, Domenico
影响因子：
--
作者：
Gardner, Thomas J.;Bolovan-Fritts, Cynthia;Tortorella, Domenico
通讯作者：
Tortorella, Domenico
影响因子：
2.7
作者：
Chen, SW;Xuan, T;Tu, YQ
通讯作者：
Tu, YQ
影响因子：
13.5
作者：
Mercorelli B;Lembo D;Palù G;Loregian A
通讯作者：
Loregian A