Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate nucleocapsid assembly by characterizing the interactions of the wild-type and truncated capsid proteins with membranes by using biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

蜱传脑炎病毒（Tick-borne encephalitis virus）是黄病毒科（Flaviviridae）黄病毒属（Flavivirus）内一种有包膜的致病性RNA病毒。当由RNA基因组与多拷贝衣壳蛋白组成的核衣壳（nucleocapsid）经内质网膜出芽并获得病毒包膜及相关蛋白后，即形成病毒粒子。核衣壳组分向组装与出芽位点的协调机制目前仍未明确。本研究借助生物物理方法与模型膜系统，表征野生型及截短型衣壳蛋白与膜的相互作用，以此探究核衣壳的组装过程。研究发现，衣壳蛋白最初通过与带负电荷脂质的静电相互作用结合膜结构，随后发生膜插入。此外，本研究证实膜结合状态的衣壳蛋白可募集病毒基因组RNA。本研究通过质谱（mass spectrometry）检测发现，纯化的病毒粒子中含有带负电荷脂质，从而验证了上述生物物理研究结果的生物学相关性。本研究结果表明，核衣壳的组装由内质网膜上的带负电荷膜斑所调控，且衣壳蛋白介导了核衣壳与膜之间的直接接触。