Pal R, Barenholz Y, Wagner R R
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Biochemistry. 1988 Jan 12;27(1):30-6. doi: 10.1021/bi00401a006.
We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.
我们正在利用病毒膜中的荧光内源性磷脂来研究与受体膜相互作用时促进融合的因素。为此，在幼仓鼠肾（BHK - 21）细胞中生长的水泡性口炎病毒（VSV）用荧光脂质进行生物标记，这些荧光脂质主要是源自芘脂肪酸的磷脂酰胆碱和磷脂酰乙醇胺。病毒粒子中存在的芘脂质呈现出典型的芘荧光光谱，有强烈的单体峰和宽的激基缔合物峰。芘脂质标记的VSV与血清脂蛋白相互作用导致包膜中少量芘脂肪酸的自发快速转移（半衰期小于或等于7分钟），随后芘磷脂从病毒粒子膜的转移则要慢得多（半衰期大于或等于12小时）。用磷脂酰丝氨酸小单层囊泡孵育芘磷脂标记的VSV，在低pH（pH 5.0）下会导致融合，这可通过激基缔合物/单体荧光强度比的变化来测量。融合动力学很快，在pH 5.0和37℃下4分钟后达到平台期。在中性pH或4℃时仅观察到可忽略不计的融合。用荧光脂质进行生物标记得到的完全有感染性的病毒粒子为研究细胞 - 病毒相互作用机制以及与病毒膜糖蛋白反应的特异性单克隆抗体对病毒感染性的中和作用提供了一个有用的工具。
