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 the interactions of the wild-type and truncated capsid proteins with membranes with 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.

Author summaryTick-borne encephalitis virus (TBEV) causes a life-threatening neurological disease. The incidence of the disease is increasing despite the availability of efficient vaccines. The virus particles contain a nucleocapsid consisting of the viral RNA genome and multiple copies of the capsid protein. The nucleocapsid is surrounded by an envelope derived from host membranes and two viral surface protein species are embedded into it. The assembly of TBEV virions is poorly understood despite its medical significance. Here, we have used a large combination of biophysical methods to study the interactions of the capsid protein with artificial lipid membranes. We show that the capsid protein requires negatively-charged lipids for membrane binding and that the capsid protein is capable of inserting into the membranes in the presence of such lipids. Once membrane-bound, the capsid protein is able to recruit TBEV genomic RNA onto the membrane. Our results suggest that the initial events of TBEV assembly are mediated by the specific binding of the capsid protein to negatively-charged lipids, allowing for the recruitment of the genome. This is further supported by our characterization of the lipid composition of purified TBEV particles, which shows that the TBEV envelope contains negatively-charged lipids.