A/B型禽偏肺病毒吸附蛋白的抗融合机制研究

Avian metapneumovirus(aMPV), which is designated subtypes A, B, C and D, is an important member in family Paramyxoviridae. Paramyxoviruses entry host cells by direct fusion between the viral envelope and the cell membrane, or via endocytosis then followed by fusion between the membrane of the virion and the endosome to release the nucleocapsid into the cytoplasm. This fusion requires both attachment protein and fusion protein. However, the fusion triggered by aMPV(types A and B) in Paramyxoviruses displayed different mechanism in that the attachment protein is not helpful, but has inhibitory activity for fusion. This is new biological phenomenon that is not able to be explained by the traditional fusion models for Paramyxovirus. Here, we will localize the region that is responsible for the inhibitory activity by construction and evaluation of a series of chimeric proteins composed of domains from the attachment proteins with or without inhibitory activity. Meanwhile, we will identify the receptor(s) of the attachment proteins of aMPV, and evaluate the potential roles of the receptor(s) in fusion. We will elaborate the fusion inhibitory mechanism of the attachment proteins of avian metapneumovirus(types A / B ) from two aspects of internal factors (the functional domain in the attachment protein) and external factors (receptor). This study can not only enrich the knowledge of fusion mechanisms of paramyxovirus, but also will broaden the traditional scope of membrane fusion mediated by enveloped virus.

禽偏肺病毒是副粘病毒科的重要成员，分为A、B、C和D四型。副粘病毒侵染是通过病毒囊膜与靶细胞膜或与内体膜相互融合，进而将遗传物质释放到靶细胞中，这一融合过程通常由吸附蛋白和融合蛋白共同完成。我们最新研究发现A/B型禽偏肺病毒吸附蛋白不仅对融合没有促进作用，反而显著抑制膜融合，这一现象打破了人们对副粘病毒介导膜融合的固有理解。本研究通过构建一系列具有抑制活性和不具备抑制活性吸附蛋白的嵌合体来定位发挥融合抑制作用的功能域，在分子水平上确立吸附蛋白融合抑制的具体途径；同时，鉴定禽偏肺病毒吸附蛋白受体，并研究其在融合抑制中的作用，最终从吸附蛋白内部因素（自身功能域）和外部因素(蛋白受体)两个方面阐述其融合抑制的微观分子基础。本研究不仅可以丰富副粘病毒膜融合机制的知识体系，也将拓宽人们对囊膜病毒介导膜融合的认知边界。

本研究通过构建禽偏肺病毒吸附蛋白基因的一系列缺失体，并将各缺失体与融合蛋白基因真核表达载体共转染细胞，分别用定性和定量两种方法检测细胞融合抑制情况，定位吸附蛋白中发挥融合抑制作用的区域。将A/B型aMPV 吸附蛋白基因真核表达载体与融合蛋白基因共转染细胞，检测细胞表面蛋白表达情况。同时对禽偏肺病毒蛋白类受体和非蛋白类受体在细胞融合中的作用进行研究。研究结果表明抑制细胞融合的区域位于吸附蛋白的第29-271位氨基酸，A/B型禽偏肺病毒吸附蛋白通过抑制其融合蛋白的细胞表面表达实现对细胞融合的抑制。进一步通过对融合蛋白潜在受体结合域和糖基化位点进行突变研究发现，在这些位点的突变可显著抑制细胞融合，说明受体在细胞融合中起着重要作用。本研究确立了A/B型禽偏肺病毒细胞融合抑制的途径，初步阐明了A/B型禽偏肺病毒中抑制细胞融合的分子机制。该研究结果为开发新型抗病毒药物或对病毒进行突变至弱开发新型弱毒疫苗相提供了理论基础。