Paramyxovirus F protein mediated membrane fusion

描述(由申请人提供)：膜融合是病毒进入的关键过程，由副粘病毒融合(F)蛋白促进。所有的F蛋白都包含一些在融合中起基础作用的共同特征。然而，与蛋白激活和促进膜融合相关的F蛋白之间存在着显著的差异，这些重要的病毒蛋白促进融合的机制仍然存在许多关键问题。我们研究的长期目标是了解副粘病毒F蛋白促进膜融合的确切机制(S)。我们的总体假设是，对融合促进至关重要的结构域的功能将在不同的F蛋白中保守，但这些结构域中的相互作用将调节触发机制。为了解决这一重要假设，我们将追求以下具体目标：1)我们将在我们令人兴奋的初步数据的基础上，证明分离的Hendra F跨膜(TM)结构域形成三聚体，以确定TM-TM相互作用在糖蛋白折叠和融合中的作用。因此，我们将描述TM-TM相互作用的关键残基，并评估这种相互作用在Hendra F折叠、运输和融合中的作用；分析HRB或细胞质尾部添加对TM-TM相互作用稳定性的影响；比较PIV5和HMPV F蛋白与Hendra F TM的TM-TM相互作用；并确定副粘病毒附着蛋白的TM结构域是与自身相互作用，还是与F蛋白TM结构域相互作用；2)我们将明确低pH和内吞作用在HMPV病毒进入中的作用，并描述HMPV F在低pH诱导的构象变化和重组病毒进入中HRB连接区的静电排斥作用。我们将分析稳定HRA和F2保守区域之间相互作用的作用，我们对PIV5 F的研究表明，通过定义PIV5 F中该区域的关键相互作用，评估受影响的融合阶段，分析该区域突变对病毒进入的影响，以及分析突变对其他F蛋白中该区域的影响，可以在触发该区域的过程中发挥作用。实现这些目标将提供有关F蛋白促进的膜融合过程中涉及的分子事件控制区域的关键信息，并可能确定抗病毒治疗的新靶点。副粘病毒家族既包括已有的人类病原体，如麻疹病毒和呼吸道合胞病毒，也包括新出现的人类病原体，包括高致病性Hendra和Nipah病毒，以及最近发现的人类偏肺病毒(HMPV)。对融合机制的详细了解可能会导致新的抗病毒试剂的开发。

DESCRIPTION (provided by applicant): Membrane fusion, a process critical for viral entry, is promoted by the paramyxovirus fusion (F) proteins. All F proteins contain a number of common features that play fundamental roles in fusion. However, significant variations exist between F proteins related to protein activation and promotion of membrane fusion, and many critical questions remain concerning the mechanism by which fusion is promoted by these important viral proteins. The long-term objective of our research is to understand the precise mechanism(s) of paramyxovirus F protein-promoted membrane fusion. Our overall hypothesis is that the function of domains critical for fusion promotion will be conserved in diverse F proteins, but that interactions in these domains will modulate triggering mechanisms. To address this important hypothesis we will pursue the following specific aims: 1.) We will build on our exciting preliminary data which demonstrates trimer formation of isolated Hendra F transmembrane (TM) domains to define the role of TM-TM interactions in glycoprotein folding and fusion. We will therefore delineate the residues critical for TM-TM interactions, and evaluate the role of this interaction in Hendra F folding, trafficking and fusion; analyze the effect of HRB or cytoplasmic-tail additions on stability of TM-TM interactions; compare TM-TM interactions for the PIV5 and HMPV F proteins to those observed for the Hendra F TM; and determine if TM domains from paramyxovirus attachment proteins interact with themselves or with the F protein TM domains; 2.) We will define the role of low pH and endocytosis in HMPV viral entry and delineate the role of electrostatic repulsion in the HRB linker region in HMPV F low pH-induced conformational changes and entry of recombinant viruses and; 3.) We will analyze the role of stabilizing interactions between HRA and a conserved region of F2, which our studies of PIV5 F indicate can play a role in triggering, by defining key interactions in this domain in PIV5 F, assessing the stage of fusion which is affected, analyzing the effect of mutations in this region on viral entry, and analyzing the effect of mutations to this region in other F proteins. Accomplishing these goals will provide crucial information on the regions that control the molecular events involved in the F protein-promoted membrane fusion process, and potentially identify new targets for antiviral therapy. PUBLIC HEALTH RELEVANCE The paramyxovirus family contains both established human pathogens, such as measles virus and respiratory syncytial virus, and newly emerged human pathogens, including the highly pathogenic Hendra and Nipah viruses and the recently identified human metapneumovirus (HMPV). A detailed understanding of the fusion mechanism could lead to development of new antiviral reagents.