HMPV Fusion protein-host cell interactions

描述（由申请方提供）：人偏肺病毒（HMPV）是一种最近发现的与严重呼吸道疾病相关的副粘病毒，特别是在婴儿中，其症状与呼吸道合胞病毒非常相似。我们已经建立了有效的检测方法，用于研究HMPV融合（F）糖蛋白促进的膜融合。有趣的是，HMPV F蛋白介导的细胞-细胞融合仅发生在转染细胞用胰蛋白酶处理（以蛋白水解处理HMPV F蛋白）并暴露于低pH时。如先前研究的副粘病毒F蛋白在质膜中性pH促进融合，低pH触发HMPV F蛋白融合活性表明，这种蛋白质以副粘病毒中独特的方式发挥作用。我们的结果还表明，HMPV F能够在不存在附着（G）蛋白的情况下促进融合，这与缺乏HMPV G蛋白的重组HMPV病毒在细胞培养物以及仓鼠和非人灵长类动物模型中有效复制的报道一致。我们的长期目标是了解副粘病毒的附着和进入。我们的具体假设是，HMPV F与一个或多个确定的细胞受体相互作用，这种相互作用是必要的总理F蛋白低pH诱导促进膜融合。为了解决这个问题，我们将追求三个研究目标。首先，我们将利用我们最近开发的，有效的报告基因检测系统地确定所需的HMPV F蛋白促进膜融合的靶细胞成分。其次，我们将分析HMPV F蛋白的处理对促进膜融合的影响，包括预先低pH处理、F蛋白的蛋白水解加工或添加可溶性糖胺聚糖的影响。最后，我们将利用HMPV F蛋白的可溶性形式来分析结合并鉴定HMPV F的特异性细胞受体。实现这些目标将提供与HMPV F蛋白相互作用的细胞组分的关键信息，并可能导致副粘病毒融合蛋白的第一个受体的鉴定。此外，这些研究将进一步阐明HMPV F蛋白促进感染关键早期步骤的机制，并为未来分析这种重要病毒蛋白的抑制剂奠定基础。人偏肺病毒（HMPV）是最近发现的一种导致严重呼吸道疾病（尤其是婴儿）的人类病原体。这项研究将解决有关与HMPV融合蛋白相互作用以促进病毒附着和进入细胞的细胞因子的重要问题。

DESCRIPTION (provided by applicant): Human metapneumovirus (HMPV) is a recently identified paramyxovirus associated with severe respiratory disease, particularly in infants, with symptoms that closely resemble those seen with respiratory syncytial virus. We have established efficient assays for the study of membrane fusion promoted by the HMPV fusion (F) glycoprotein. Interestingly, HMPV F protein-mediated cell-cell fusion only occurs when transfected cells are both treated with trypsin (to proteolytically process the HMPV F protein) and exposed to low pH. As previously studied paramyxovirus F proteins promote fusion at neutral pH at the plasma membrane, the triggering of HMPV F protein fusion activity by low pH suggests that this protein functions in a manner unique among the paramyxoviruses. Our results also demonstrate that HMPV F is competent to promote fusion in the absence of the attachment (G) protein, consistent with reports that recombinant HMPV viruses lacking the HMPV G protein replicate efficiently both in cell culture and in hamster and non-human primate animal model. Our long-term goal is to understand the attachment and entry of paramyxoviruses. Our specific hypothesis is that HMPV F interacts with one or more defined cellular receptors, and that this interaction is necessary to prime the F protein for low-pH induced promotion of membrane fusion. To address this hypothesis we will pursue three research aims. First, we will utilize our recently developed, efficient reporter gene assay to systematically define the target cell components required for HMPV F protein-promoted membrane fusion. Second, we will analyze the effect of treatments to the HMPV F protein on promotion of membrane fusion, including effects of prior low pH treatment, proteolytic processing of the F protein or addition of soluble glycosaminoglycans. Finally, we will utilize a soluble form of the HMPV F protein to analyze binding and identify the specific cellular receptor(s) for HMPV F. Accomplishing these goals will provide crucial information on the cellular components that interact with the HMPV F protein, and may well lead to identification of the first receptor for a paramyxovirus fusion protein. In addition, these studies will further clarify the mechanism by which the HMPV F protein promotes critical early steps in infection, and set the stage for future analysis of inhibitors of this important viral protein. Human metapneumovirus (HMPV) is a recently identified human pathogen responsible for significant levels of severe respiratory disease, particularly in infants. This research will address significant questions concerning the cellular factors which interact with the HMPV fusion protein to facilitate attachment of the virus and entry into cells.