Markosyan Ruben M, Kielian Margaret, Cohen Fredric S
Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1653 W. Congress Pkwy., Chicago, IL 60612, USA.
J Virol. 2007 Oct;81(20):11218-25. doi: 10.1128/JVI.01256-07. Epub 2007 Aug 8.
Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.
表达塞姆利基森林病毒(SFV)低pH触发的II类病毒融合蛋白E1的细胞与靶细胞融合。通过电容和水溶性染料测量来监测融合情况。电压钳测量表明,在酸化后,SFV E1诱导的细胞-细胞融合在靶膜上出现跨膜负电位(即靶细胞内部为负电位)时迅速发生,但跨膜正电位会消除所有融合。使用离子载体来控制大量细胞的电位,证实了融合对电压极性的依赖性。相比之下,人类免疫缺陷病毒、禽肉瘤白血病病毒和流感病毒的I类融合蛋白诱导的融合与靶细胞上的电压极性无关。对于I类蛋白,初始孔径和孔生长也与电压极性无关。通过在低温下短暂酸化产生了SFV E1诱导融合的一个中间体。在此中间体状态下膜处于半融合状态,在中性pH下升高温度可使完全融合发生。电容测量表明,维持跨膜正电位肯定会在半融合中间体形成后的步骤中阻止融合,并且可能在先前步骤中就抑制了融合。有人提出,在内体膜上的跨膜负电压在SFV E1发生低pH诱导的构象变化后促进融合。