Development of Cellular Assays of Dengue Virus Infection

登革病毒（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传播风险的地区。尽管如此，目前还没有针对登革热病毒有效的抗病毒药物，也没有保护性疫苗。病毒感染可以看作是与宿主细胞生化平衡相协调的一系列外源生化反应。大多数情况下，支持登革热病毒感染的特定细胞因素尚未确定。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。与特定细胞靶点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热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。该提案旨在开发识别小分子的方法，这些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。我们设计了一个系统，荧光共振能量转移（FRET）用于监测登革热病毒酶的活性，作为病毒感染、基因表达、复制和宿主细胞传播的标记。本提案的目标是将该系统发展成为一种高通量细胞测定方法，使我们能够识别干扰登革热病毒-宿主细胞感染周期的化合物。优化后的实验将用于筛选已知的生物活性文库。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的化合物集合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分析来验证屏幕上的“命中”。这将包括(1)在存在和不存在登革热病毒感染的情况下测量复合细胞毒性；(2)测定对感染性登革热病毒滴度的影响；(3)测定对病毒蛋白翻译和加工的影响；(4)测定对病毒基因组复制的影响；(5)电镜观察复合效应对登革热感染细胞和模拟感染细胞的超微结构影响；(6)通过测量抗登革热化合物对一组病毒（包括代表登革热病毒所有四种血清型的分离株，以及其他黄病毒和黄病毒科以外的病毒）的作用，表征抗登革热化合物的病毒特异性。总的来说，这些实验将提供能够可靠地检测小分子的工具，这些小分子可用于在分子水平上探测登革热病毒与其宿主细胞之间的相互作用。一般策略也可以扩展到检查其他病毒与其宿主之间的相互作用。登革病毒每年感染1亿人，造成多达50万例可能致命的登革出血热病例；然而，目前还没有针对登革热病毒有效的抗病毒药物，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求生存，但我们对登革热如何与宿主相互作用知之甚少——它需要什么或它是如何引起疾病的。我们正在开发工具，使我们能够识别小的、类似药物的分子，这些分子可以通过与宿主细胞中的目标相互作用来抑制病毒。这些抑制剂可用于研究登革热病毒如何与其宿主相互作用，它们也可能有助于开发治疗登革热病毒感染的疗法。

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.