The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.

对于多数病毒而言，仅需1至2种蛋白质即可介导细胞吸附与侵入：侵入过程可通过细胞质膜完成，或在低pH条件下经由内吞作用实现。与之相反，牛痘病毒（vaccinia virus, VACV）可通过中性及低pH两种途径侵入细胞：4种蛋白质介导细胞吸附，另有12种嵌入膜复合物且在所有痘病毒中保守的蛋白质，专门介导侵入过程。本研究旨在明确细胞与病毒蛋白质在病毒侵入初始阶段——即成熟病毒体与宿主细胞膜的膜融合过程——中的具体作用。为分析细胞组分的功能，我们采用了经过充分表征的抑制剂，并通过三类实验开展检测：一是检测融合有核心蛋白的重组牛痘病毒体的细胞结合情况，该重组病毒体的核心蛋白携带有高斯荧光素酶（Gaussia luciferase）标签；二是利用病毒体膜中的自猝灭荧光探针，检测病毒膜与细胞膜的脂质混合过程；三是借助表达萤火虫荧光素酶（firefly luciferase）的重组牛痘病毒，结合电子显微镜技术观测病毒核心的侵入过程。实验结果显示，酪氨酸蛋白激酶、发动蛋白GTP酶以及肌动蛋白动力学的抑制剂，对病毒体与细胞的吸附过程几乎无影响，但会损害膜融合过程；而部分胆固醇耗竭处理，以及内体酸化、膜起泡抑制剂，则会在病毒核心侵入的后期阶段产生显著抑制效果。为明确病毒蛋白质的功能，我们从10株条件致死诱导突变体感染的细胞中，纯化得到了缺失单个膜组分的牛痘病毒体。所有缺失侵入相关蛋白质的病毒体，其感染性均大幅下降；除A28、L1及L5外，其余突变体的膜融合过程均受到严重损害。此外，一种强效中和性L1单克隆抗体，可在膜脂质混合完成后的步骤阻断病毒侵入过程。综上，本研究结果支持痘病毒双阶段侵入模型，并揭示了痘病毒侵入过程中，参与病毒-宿主细胞膜融合启动的信号转导与肌动蛋白重排相关的大量病毒蛋白质与细胞组分，这一发现此前尚无先例。