Justman J, Klimjack M R, Kielian M
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
J Virol. 1993 Dec;67(12):7597-607. doi: 10.1128/JVI.67.12.7597-7607.1993.
The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.
甲病毒塞米基森林病毒（SFV）以及许多其他包膜动物病毒通过内吞小泡内低pH引发的膜融合反应感染细胞。除了有低pH要求外，SFV融合和感染还严格依赖于宿主细胞膜中胆固醇的存在。低pH处理后，SFV刺突蛋白会发生一些构象变化，包括E1-E2二聚体的解离、E1和E2亚基的构象变化以及E1寡聚形成同源三聚体。为了确定这些事件的顺序，我们利用接近融合阈值的pH处理和低温孵育来减缓融合反应，比较了这些构象变化与融合的动力学。二聚体解离、E1构象变化和E1三聚化均发生在病毒与细胞膜混合之前。对在20摄氏度孵育的细胞的研究表明，与病毒融合一样，E1三聚化发生在内体中，然后才转运至溶酶体。然而，与严格依赖胆固醇的膜融合反应不同，E1同源三聚体在病毒摄取过程中，可由胆固醇缺失的细胞在体内产生，或在体外通过在有或无胆固醇的人工脂质体存在下对病毒进行低pH处理产生。对纯化的、不含脂质的刺突蛋白玫瑰花结进行分析，以确定E1同源三聚体形成过程中对病毒膜胆固醇的需求。发现刺突蛋白玫瑰花结在暴露于低pH和靶脂质体时会发生E1寡聚化，并且含胆固醇的膜会增强寡聚化。E1同源三聚体可能代表一种灌注复合体，需要胆固醇来完成病毒膜与靶膜的最终融合。
