Mechanisms of poxvirus entry into cells

痘病毒进入细胞的分子机制仍然令人困惑。我们的目标是解剖 这些机制的重点是涉及三种正痘病毒进入的包膜蛋白：牛痘 (VACV)、天花(VARV)和猴痘(MPXV)。VARV的意外或故意释放，加上 鉴于MPXV有可能成为一种更有效的人类病原体，强调有必要进一步 调查这些病毒的生物防御重要性和作为新出现的感染源。 痘病毒的细胞受体(S)和病毒受体结合蛋白(S)尚不清楚。我们的预赛 数据表明，MV蛋白L1是一种受体结合蛋白，可能通过以下方式触发融合 病毒融合复合体。我们发现，L1的一种可溶形式独立地与细胞结合 糖胺多聚糖并阻止病毒进入，表明它与病毒粒子相关的L1竞争细胞 受体。在目标1中，我们将绘制L1上对其进入功能至关重要的区域，并识别细胞 它所结合的受体(S)。我们将确定L1的哪些残基与其阻止病毒的能力有关 使用基于结构的突变的条目。类似的研究将与以下网站的同事合作进行 用于MPXV和VARV的CDC。我们将使用两种方法来确定L1的细胞受体(S)。首先，我们将 利用L1本身试图从敏感细胞中“捞出”受体。第二，我们将采取更加公正的 使用RNAi筛选在VACV进入过程中重要的蛋白质，尤其是那些蛋白质 可能以L1为目标。在目标2中，我们将研究MV包膜蛋白如何在痘病毒进入过程中发挥作用。我们会 使用一种新的共焦-FACS技术，使用分析来跟踪受体结合后发生的事件。我们 将使用双分子荧光互补来研究VACV包膜之间的相互作用 完整细胞中的蛋白质，目的是将它们与VACV进入/融合联系起来。我们的研究应该提供 与痘病毒进入有关的病毒和细胞因子的分子细节。此外，我们可能会 确定开发痘病毒进入的小分子抑制剂的新靶点。

The molecular mechanisms involved in poxvirus entry into cells remain perplexing. Our goal is to dissect these mechanisms by focusing on the envelope proteins involved in entry of three orthopoxviruses: vaccinia (VACV), variola (VARV) and monkeypox (MPXV). The accidental or intentional release of VARV, coupled with the potential of MPXV to become a more efficient human pathogen, underscore the need for further investigation of these viruses in terms of their biodefense importance and as emerging infectious agents. The cell receptor(s) and the viral receptor-binding protein(s) for poxviruses are unknown. Our preliminary data suggest that the MV protein L1 is a receptor binding protein that may trigger fusion that is carried out by the virus fusion complex. We found that a soluble form of L1 binds to cells independently of glycosaminoglycans and blocks virus entry, suggesting it competes with virion associated L1 for a cell receptor. In Aim 1, we will map regions on L1 critical for its function in entry and identify the cellular receptor(s) to which it binds. We will determine which residues of L1 are involved in its ability to block virus entry using structure based mutagenesis. Similar studies will be done in collaboration with colleagues at CDC for MPXV and VARV. We will use two approaches to identify the cell receptor(s) for L1. First, we will use L1 itself to try to "fish out" the receptor from susceptible cells. Second, we will take a more unbiased approach using RNAi to screen for proteins that are important in VACV entry, focusing particularly on ones that may target L1. In Aim 2 we will study how the MV envelope proteins function in poxvirus entry. We will use assays to follow events that occur after receptor binding using a novel confocal-FACS technology. We will employ bimolecular fluorescence complementation to study interactions between VACV envelope proteins in intact cells with the goal of relating them to VACV entry/fusion. Our studies should provide the molecular details of both the viral and cellular factors involved in poxvirus entry. Additionally, we may identify novel targets for developing small molecule inhibitors of poxvirus entry.