Role of Calcium Signaling in HSV-2 Invasion

描述（由申请人提供）：目前迫切需要阻断单纯疱疹病毒（HSV）和其他性传播病原体传播的局部杀微生物剂。硫酸肝素蛋白聚糖作为附着受体的发现推动了磺化聚合物的发展，这些聚合物目前正在临床试验中作为候选杀微生物剂。鉴定靶向后结合步骤的新制剂需要在分子水平上阐明病毒包膜与细胞膜融合所需的信号通路以及病毒衣壳的核转运。初步研究表明，病毒入侵需要激活钙（Ca2+）信号和酪氨酸磷酸化局灶黏附激酶（FAK）和其他细胞蛋白。Ca2+和磷酸化信号通路的激活是与其他微生物（最明显的是HIV）入侵相关的常见机制。本申请中提出的研究建立在这些观察的基础上。这些研究的重点是HSV血清2型（HSV-2），这是生殖器疱疹的主要原因，这些研究将检查病毒入侵所需的细胞信号通路以及病毒包膜糖蛋白在这一过程中发挥的作用。大多数关于病毒进入的研究都是使用永久性非极化上皮细胞系进行的。然而，上皮细胞的极性是其结构和功能的基础，并影响对感染的易感性。初步研究表明，极化上皮细胞感染HSV- 2在根尖暴露后比基底外侧暴露更有效。因此，病毒入侵极化细胞培养系统的机制也将被探讨。在极化细胞中观察将更准确地反映体内情况。这些研究结果将加强对HSV入侵分子生物学的理解，并有助于合理设计新的局部抗菌剂，以防止HSV的传播和获得。

DESCRIPTION (provided by applicant): Topical microbicides that block transmission of herpes simplex virus (HSV) and other sexually transmitted pathogens are urgently needed. The observation that heparan sulfate proteoglycans serve as attachment receptors propelled the development of sulfonated polymers, which are now in clinical trial, as candidate microbicides. Identification of new agents that target steps post-binding requires elucidation, at a molecular level, of the signaling pathways required for fusion of the viral envelope with the cell plasma membrane and nuclear transport of viral capsids. Preliminary studies indicate that viral invasion requires activation of calcium (Ca2+) signaling and tyrosine phosphorylation of focal adhesion kinase (FAK) and other cellular proteins. Activation of Ca2+ and phosphorylation signaling pathways are common mechanisms associated with invasion by other microbes, most notably HIV. The studies proposed in this application build on these observations. Focusing on HSV serotype 2 (HSV-2), the predominant cause of genital herpes, these studies will examine the cellular signaling pathways required for viral invasion and the role viral envelope glycoproteins play in this process. Most studies of viral entry have been conducted using permanent non-polarized epithelial cell lines. However, the polarity of epithelial cells is fundamental to their structure and function and influences susceptibility to infection. Preliminary work indicates that HSV- 2 infection of polarized epithelial cells is more efficient following apical compared to basolateral exposure. Therefore, the mechanism of viral invasion in polarized cell culture systems will also be explored. Observations in polarized cells will more accurately reflect the conditions in vivo. Results of these studies will enhance understanding of the molecular biology of HSV invasion and should facilitate the rational design of new topical antimicrobials to prevent HSV transmission and acquisition.