Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein

Abstract

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.

The Dynamic Landscape of Capsid Proteins and Viral RNA Interactions in Flavivirus Genome Packaging and Virus Assembly

“…The ~30–100 aa 3D structure of the protein is composed of four α helices (α1–α4), which form three layers. The top layer contains α1 and an N-terminal unstructured domain, which is proposed to be involved in membrane association and RNA binding [ 60 , 61 ]. The middle layer is formed by α2, which is mainly hydrophobic and responsible for dimerization, and α3 and the connecting loop between α2 and α3 [ 56 ].…”

“…The middle layer is formed by α2, which is mainly hydrophobic and responsible for dimerization, and α3 and the connecting loop between α2 and α3 [ 56 ]. The bottom layer consists of the longest helix, α4, which is proposed to be involved in RNA binding and nuclear localization [ 46 , 61 ]. The length of the unstructured N-terminal region varies widely from TBEV (~18 aa) to ZIKV (~36 aa).…”

“…The fourth helix is on the opposite side of the protein relative to the proposed lipid binding region (α1–α2), and hence it has been suggested to face the interior of the NC, where genomic RNA is presumably located [ 52 , 54 , 72 ]. Recent studies from DENV and JEV have supported this model and further shown that C can interact simultaneously with membranes and nucleic acids, indicating that the interaction regions on C are structurally distinct [ 61 , 73 ]. While the surface-exposed, positively charged residues on α4 are implicated in RNA binding [ 46 , 74 ], interior-facing hydrophobic residues are important for dimerization [ 51 , 64 , 67 ].…”

“…Positively charged regions are likely involved in RNA binding in tandem with the α4, including two highly conserved residues (K31 and R32 DENV numbering), which, when mutated, were found to impact RNA binding and infectivity in both TBEV and DENV [63,64]. [46,61]. The length of the unstructured N-terminal region varies widely from TBEV (~18 aa) to ZIKV (~36 aa).…”

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation

“…The newly synthesized genomes bind to multiple copies of C to form nucleocapsids (NC), which seem to acquire an envelope by budding into the lumen of the endoplasmic reticulum (14)(15)(16)(17)(18). The envelope of immature TBEV particle contains 180 copies of each M protein precursor (prM) and E forming heterodimers.…”