The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein.

IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

抑制剂卡苯甲氧基 (Z)-d - Phe- l -Phe-Gly（融合抑制剂肽 [FIP]）和 4-硝基-2-苯乙酰基氨基苯甲酰胺 (AS-48) 在阻断膜融合和合胞体形成方面具有相似的功效由麻疹病毒（MeV）介导。其他同系物，例如 Z- d -Phe，效果较差，但可能通过相同的机制发挥作用。为了绘制这些抑制剂的作用位点，我们产生了对 Z-d-Phe-l-Phe - Gly的抑制作用具有抗性的突变病毒。这 10 个突变定位于融合蛋白的七肽重复 B (HRB) 区域，在病毒血凝素（受体附着蛋白）中未观察到任何变化。使用转染的融合和血凝素表达质粒，在基于荧光素酶的膜融合测定中验证突变，或在 Vero、Vero-SLAM 和 Vero-Nectin 4 细胞系中使用基于合胞体的测定。I452T、D458N、D458G/V459A、N462K、N462H、G464E 和 I483R 的变化赋予了对 FIP 和 AS-48 的抗性，而不影响膜融合。抑制剂不会阻断血凝素蛋白介导的与靶细胞的结合。埃德蒙斯顿疫苗/实验室和 IC323 野生型菌株同样受到抑制剂的影响。逃逸突变被映射到根据已发表的副流感病毒 5 融合蛋白晶体结构建模的三维 (3D) 结构上。最有效的突变位于球状头底部附近的区域及其与预融合蛋白的α螺旋柄的连接处。我们假设融合抑制剂可以干扰融合蛋白的融合前和融合后构象之间发生的结构变化。

重要性由于世界范围内疫苗接种失败导致局部疫情爆发，麻疹病毒 (MeV) 作为一种病原体重新获得了重要性。针对麻疹病毒的抗病毒药物尚未上市销售，但可与麻疹病毒根除疫苗计划结合使用，并作为溶瘤病毒治疗的保障措施。三十年前，小疏水肽 Z- d -Phe- l -Phe-Gly (FIP) 被证明可以阻断猴肾细胞系中的 MeV 感染和合胞体形成。其确切的作用机制尚未确定，但它似乎具有与另一种化学抑制剂 AS-48 类似的特性，AS-48 似乎会干扰病毒 F 蛋白的构象变化，而这种变化是引发膜所需的。融合。使用逃逸突变来绘制 FIP 的作用位点。从这些研究中获得的知识可以帮助设计新的麻疹病毒抑制剂，并提供有关病毒介导的膜融合机制的更多知识。