Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Asuka Nanbo,Ayato Takada,Yoichiro Fujioka,Shinya Nishide,Masaki Imai,Yusuke Ohba,Michiko Ujie,Junki Maruyama,Yoshihiro Kawaoka,Thomas Geisbert

doi:10.1371/journal.ppat.1006848

Abstract

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

Highlights

Ebola virus (EBOV), a member of the family Filoviridae, is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates [1]
Phosphatidylserine normally distributes in the inner layer of the cell surface membrane, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the membrane in infected cells for Ebola virus to acquire it
Because the mechanism by which phosphatidylserine changes its orientation in Ebola virus-infected cells is poorly understood, we studied and identified a cellular enzyme, XK-related protein 8 (Xkr8), as a responsible factor involved in this process

Summary

Introduction

Ebola virus (EBOV), a member of the family Filoviridae, is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates [1]. The viral entry process is one of the targets for antiviral development. Several early studies investigated the mechanism by which EBOV enters host cells, focusing on the interaction of GP with cell surface attachment factors. EBOV entry is initiated by the binding of GP to attachment factors (e.g., C-type lectins), which induces macropinocytosis-mediated uptake of the virions [3,4,5]. The internalized virions are trafficked in the endosomal pathway, during which GP is processed by cathepsins in low pH-dependent manner. Proteolysis of GP allows the putative receptor-binding region to be exposed and subsequently interact with a cholesterol transporter, Niemann-Pick C1 (NPC1), which initiates fusion of the viral envelope with the endosomal membrane [6,7,8]

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