Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage
Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage
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DOI：
10.1128/jvi.02152-16
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发表时间：
2017-02-01
影响因子：
5.4
通讯作者：
Geraghty, Robert J.
中科院分区：
文献类型：
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作者：
Wang, Yan;Mao, Lili;Geraghty, Robert J.
人巨细胞病毒末端酶复合物在基因组包装和颗粒组装期间将多联体基因组DNA切割成单位长度。这个过程是一个有吸引力的药物靶点，因为在哺乳动物细胞DNA复制中不需要切割多联体DNA，这表明靶向末端酶复合物的药物可能是安全和选择性的。人巨细胞病毒末端酶复合物的一种组分pUL89提供了基因组切割的核酸内切活性，据报道负责的结构域具有RNA酶H样折叠。我们假设pUL89核酸内切酶活性被已知的RNase H抑制剂抑制。使用一种新的酶联免疫吸附试验（ELISA）格式作为筛选试验，我们发现，羟基吡啶酮羧酸化合物，以前报道是一种人类免疫缺陷病毒RNase H的抑制剂，抑制pUL89核酸内切酶活性在低微摩尔浓度。进一步的表征显示，这种pUL89核酸内切酶抑制剂在相对较晚的时间点阻断人巨细胞病毒复制，类似于其他报道的末端酶复合物抑制剂。重要的是，这种抑制剂还阻止了感染细胞中病毒基因组DNA的切割。两者合计，这些结果证实了我们的药效团假说，并验证我们的配体为基础的方法对确定新的抑制剂pUL89 endonuclease.IMPORTANCE人巨细胞病毒感染的个人缺乏一个功能齐全的免疫系统，如新生儿和移植患者，可以有严重和衰弱的后果。美国食品和药物管理局批准的抗人巨细胞病毒药物主要针对病毒聚合酶，对这些药物的耐药性已经出现。因此，需要使用来自新靶点的抗人巨细胞病毒药物来代替当前的聚合酶抑制剂或与当前的聚合酶抑制剂组合使用。pUL89是一种病毒ATP酶和核酸内切酶，是抗人巨细胞病毒药物开发的有吸引力的靶标。我们鉴定并表征了pUL89内切核酸酶活性的抑制剂，其也抑制细胞培养物中的人巨细胞病毒复制。因此，pUL89核酸内切酶应被探索作为抗人巨细胞病毒的抗病毒开发的潜在靶点。
The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease.IMPORTANCE Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus drug development. We identified and characterized an inhibitor of pUL89 endonuclease activity that also inhibits human cytomegalovirus replication in cell culture. pUL89 endonuclease, therefore, should be explored as a potential target for antiviral development against human cytomegalovirus.