HMPV Fusion protein-host cell interactions

描述（由申请人提供）：人偏肺病毒（HMPV）是最近发现的一种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。我们建立了研究HMPV融合(F)糖蛋白促进膜融合的有效方法。有趣的是，HMPV F蛋白介导的细胞-细胞融合仅在转染细胞用胰蛋白酶处理（蛋白水解处理HMPV F蛋白）并暴露于低pH时发生。正如先前研究的副粘病毒F蛋白在中性pH下促进质膜融合，低pH触发HMPV F蛋白融合活性表明该蛋白在副粘病毒中以独特的方式起作用。我们的研究结果还表明，HMPV F在缺乏附着(G)蛋白的情况下能够促进融合，这与缺乏HMPV G蛋白的重组HMPV病毒在细胞培养、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，并且这种相互作用对于启动F蛋白以促进低ph诱导的膜融合是必要的。为了解决这一假设，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高效报告基因测定系统地确定HMPV F蛋白促进膜融合所需的靶细胞成分。其次，我们将分析对HMPV F蛋白的处理对促进膜融合的影响，包括先前的低pH处理，F蛋白的蛋白水解处理或添加可溶性糖胺聚糖的影响。最后，我们将利用HMPV F蛋白的一种可溶形式来分析HMPV F蛋白的结合并鉴定其特定的细胞受体。完成这些目标将提供与HMPV F蛋白相互作用的细胞成分的关键信息，并可能很好地鉴定副粘病毒融合蛋白的第一个受体。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，并为未来分析这一重要病毒蛋白的抑制剂奠定基础。人偏肺病毒（HMPV）是最近发现的一种人类病原体，可导致严重的呼吸道疾病，特别是在婴儿中。本研究将解决有关与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.