Development of Cellular Assays of Dengue Virus Infection

登革热病毒（DENV）是当今影响人类的最重要的蚊媒病毒性疾病，估计有25亿人生活在有流行病传播风险的地区。尽管如此，目前还没有针对登革热病毒的有效抗病毒药物，也没有保护性疫苗。病毒感染可以被看作是一系列与宿主细胞的生化平衡相结合的外源性生化反应。支持登革病毒感染的特定细胞因子在大多数情况下尚未确定。同样，影响登革病毒复制周期的近端和远端生化途径也是未知的。与特定细胞靶标相互作用的小分子已成为探测细胞途径的有力途径，并可能提供表征与登革病毒感染宿主细胞相关的生化途径和潜在药理学干预点的重要手段。该提案旨在开发用于鉴定小分子的方法，所述小分子可用于以动力学可逆的方式探测DENV与其宿主细胞之间的相互作用。我们已经设计了一个系统，其中荧光共振能量转移（FRET）是用来监测活动的登革病毒酶作为病毒感染，基因表达，复制和传播在宿主细胞中的标记。该提案的目标是将该系统开发成高通量细胞检测，使我们能够识别干扰登革病毒宿主细胞感染周期的化合物。优化的试验将用于筛选已知生物活性物质的文库。然后，我们将使用RNA干扰来确认筛选“命中”的抗登革热效果确实是由已知被化合物集合的成员靶向的细胞因子介导的。此外，我们将使用一组二级和三级检测来验证筛选的“命中”。这将包括（1）在存在和不存在登革病毒感染的情况下测量化合物的细胞毒性;（2）测量对感染性登革病毒滴度的影响;（3）测量对病毒蛋白翻译和加工的影响;（4）测量对病毒基因组复制的影响;（5）通过电子显微镜对化合物对登革感染和模拟感染细胞的影响的超微结构表征;和（6）通过测量抗登革化合物对一组病毒的作用来表征抗登革化合物的病毒特异性，所述病毒包括代表所有四种DENV血清型的分离物，以及其他黄病毒和黄病毒科以外的病毒。总的来说，这些实验将提供能够可靠检测小分子的工具，这些小分子可用于在分子水平上探测登革病毒与其宿主细胞之间的相互作用。一般的策略也可以扩展到检查其他病毒和它们的宿主之间的相互作用。登革热病毒每年感染1亿人，并导致多达50万例可能致命的登革出血热病例;然而，目前没有有效对抗登革热病毒的抗病毒药物，也没有保护性疫苗。所有病毒都在宿主细胞内复制，并窃取资源以生存，但我们对登革热如何与宿主相互作用知之甚少它需要什么或如何引起疾病。我们正在开发工具，使我们能够识别小的药物样分子，这些分子可以通过与宿主细胞中的靶点相互作用来抑制病毒。这些抑制剂可用于研究登革病毒如何与宿主相互作用，它们也可用于开发治疗登革病毒感染的疗法。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Dengue virus (DENV) is the most important mosquito-borne viral disease affecting humans today, with an estimated 2.5 billion living in areas at risk for epidemic transmission. Despite this, there are presently no antiviral drugs effective against dengue virus nor is there a protective vaccine. Viral infection can be regarded as a coordinated series of exogenous biochemical reactions that integrate with the biochemical equilibria of the host cell. The specific cellular factors that support dengue virus infection have, for the most part, not been identified. Likewise, the proximal and distal biochemical pathways that impinge on the dengue virus replication cycle are unknown. Small molecules that interact with specific cellular targets have become a powerful way to probe cellular pathways and may provide an important means by which to characterize the biochemical pathways and potential pharmacological intervention points associated with dengue virus infection of the host cell. This proposal aims to develop methods for identifying small molecules that can be used to probe interactions between DENV and its host cell in a kinetically reversible manner. We have designed a system in which fluorescence resonance energy transfer (FRET) is used to monitor activity of a dengue viral enzyme as a marker for viral infection, gene expression, replication, and spread in host cells. The goal of this proposal is to develop this system into a high-throughput cellular assay that enables us to identify compounds that interfere with the dengue virus-host cell infection cycle. The optimized assay will be used to screen a library of known bioactives. We will then use RNA interference to confirm that the anti-dengue effects of screening "hits" are indeed mediated by the cellular factors known to be targeted by members of the compound collection. In addition, we will use a panel of secondary and tertiary assays to validate "hits" from the screen. This will include (1) measurement of compound cytotoxicity in the presence and absence of dengue virus infection; (2) measurement of effects on infectious dengue viral titers; (3) measurement of effects on viral protein translation and processing; (4) measurement of effects on viral genome replication; (5) ultrastructural characterization of compound effects on dengue-infected and mock- infected cells by electron microscopy; and (6) characterization of the viral specificity of anti-dengue compounds by measuring their effects against a panel of viruses that include isolates representative of all four DENV serotypes, as well as additional flaviviruses and viruses beyond the Flaviviridae family. Collectively, these experiments will provide tools that enable the reliable detection of small molecules that can be used to probe the interactions between dengue virus and its host cell at the molecular level. The general strategy may also be extended to examine interactions between other viruses and their hosts. Project Narrative Dengue virus infects 100 million people and causes up to half a million cases of potentially fatal dengue hemorrhagic fever annually; yet there are currently no antiviral drugs effective against dengue virus, nor is there a protective vaccine. All viruses replicate inside a host cell and steal resources in order to survive, but we know relatively little about how dengue interacts with its host-what it needs or how it causes disease. We are developing tools that enable us to identify small, drug-like molecules that can inhibit the virus by interacting with targets in the host cell. These inhibitors can be used to study how dengue virus interacts with its host, and they may also be useful in developing therapies for the treatment of dengue virus infections.