Doms R W, Blumenthal R, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1990 Oct;64(10):4884-92. doi: 10.1128/JVI.64.10.4884-4892.1990.
The membrane fusion activities of the isolated single-envelope intracellular form of vaccinia virus (INV) and the double-envelope extracellular (EEV) form were studied by using a lipid-mixing assay based on the dilution of a fluorescent probe. Fluorescently labeled INV and EEV from both the IHD-J and WR strains of vaccinia virus fused with HeLa cells at neutral pH, suggesting that fusion occurs with the plasma membrane during virus entry. EEV fused more efficiently and with faster kinetics than INV: approximately 50% of bound EEV particles fused over the course of 1 h, compared with only 25% of the INV particles. Fusion of INV and EEV was strongly temperature dependent, being decreased by 50% at 34 degrees C and by 90% at 28 degrees C. A monoclonal antibody to a 14-kilodalton envelope protein of INV that has been implicated in the fusion reaction (J. F. Rodriguez, E. Paez, and M. Esteban, J. Virol. 61:395-404, 1987) completely suppressed the initial rate of fusion of INV but had no effect on the fusion activity of EEV, suggesting that vaccinia virus encodes two or more membrane fusion proteins. Finally, cells infected with the WR strain of vaccinia virus formed syncytia when briefly incubated at pH 6.4 or below, indicating that an acid-activated viral fusion protein is expressed on the cell surface. However, WR INV and EEV did not display increased fusion activity at acid pH, suggesting that the acid-dependent fusion factor is not incorporated into virions or that its activity there is masked.
利用基于荧光探针稀释的脂质混合试验，研究了痘苗病毒分离出的单包膜细胞内形式（INV）和双包膜细胞外形式（EEV）的膜融合活性。来自痘苗病毒IHD-J株和WR株的荧光标记的INV和EEV在中性pH条件下与HeLa细胞融合，这表明在病毒进入过程中，融合发生在质膜上。EEV比INV融合更高效且动力学更快：在1小时的过程中，约50%结合的EEV颗粒发生融合，而INV颗粒只有25%。INV和EEV的融合强烈依赖温度，在34℃时降低50%，在28℃时降低90%。一种针对与融合反应有关的INV 14千道尔顿包膜蛋白的单克隆抗体（J.F. Rodriguez、E. Paez和M. Esteban，《病毒学杂志》61:395-404，1987）完全抑制了INV的初始融合速率，但对EEV的融合活性没有影响，这表明痘苗病毒编码两种或更多种膜融合蛋白。最后，感染痘苗病毒WR株的细胞在pH 6.4或更低条件下短暂孵育时会形成多核细胞，这表明一种酸激活的病毒融合蛋白在细胞表面表达。然而，WR INV和EEV在酸性pH条件下并未显示出增加的融合活性，这表明酸依赖性融合因子没有整合到病毒粒子中，或者其在那里的活性被掩盖了。
