Abstract
VP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly at the plasma membrane and subsequent virus like particle formation. Additionally, PI(4,5)P2 was required for formation of extensive oligomers of VP40, suggesting PS and PI(4,5)P2 have different roles in VP40 assembly where PS regulates formation of hexamers from VP40 dimers and PI(4,5)P2 stabilizes and/or induces extensive VP40 oligomerization at the plasma membrane.
Highlights
VP40 is a peripheral protein containing 326 amino acids with a N-terminal domain (NTD) that mediates dimerization[12] and oligomerization[12,15,16] and C-terminal domain (CTD) that mediates membrane binding[12,17,18]
An EGFP fusion tagged version of VP40 localized to the inner leaflet of the plasma membrane of COS-7 cells where virus like particles (VLPs) are formed
As previously reported[24], plasma membrane localization of KRΦ was significantly reduced upon sphingosine treatment, compared to vehicle (Fig. 1B,C) but no significant decrease in VP40 plasma membrane localization was observed with either sphingosine treatment or vehicle control (Fig. 1B,C)
Summary
VP40 is a peripheral protein containing 326 amino acids with a N-terminal domain (NTD) that mediates dimerization[12] and oligomerization[12,15,16] and C-terminal domain (CTD) that mediates membrane binding[12,17,18]. The PS exposure is most abundant on VLPs; this observation was expected as PS exposure expedites entry into host immune cells through interaction with the PS binding TIM-1 receptor[25,26,27] This finding, raised some questions regarding how VP40 maintains the structural integrity of the viral particle since the VP40 interaction partner PS becomes exposed on the exoplasmic leaflet of VLPs. This finding, raised some questions regarding how VP40 maintains the structural integrity of the viral particle since the VP40 interaction partner PS becomes exposed on the exoplasmic leaflet of VLPs This lead us to hypothesize that other lipids at the plasma membrane may be involved in coordinating and maintaining the VP40-plasma membrane interactions. Results demonstrate that VP40 associates with PI(4,5)P2 in the plasma membrane and in vitro vesicles, and this interaction is a prerequisite for extensive VP40 oligomerization at the plasma membrane and VLP formation
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