Contribution of Ebola Virus Glycoprotein, Nucleoprotein, and VP24 to Budding of VP40 Virus-Like Particles

Abstract

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i) VP40 L domains are not required for GP-mediated enhancement of budding; (ii) the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii) NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv) the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.Ebola virus is a member of the Filoviridae family and is associated with recurrent outbreaks of deadly hemorrhagic fevers (8). Currently, there are no approved vaccines, nor are there antiviral therapeutics to prevent or treat individuals infected with Ebola virus (16). A better understanding of the molecular aspects of Ebola virus replication will be necessary for successful development of specific treatments for Ebola virus infection.The VP40 matrix protein of Ebola virus is the most abundant virion protein and plays a key role in virus assembly and budding (12,14,28). For example, VP40 can bud as a filamentous virus-like particle (VLP) from mammalian cells in the absence of any other viral protein (12,20). The ability of VP40 to bud as a VLP is due, in part, to the presence of L domains present at the N terminus of the protein (12,14,17). Viral L domains are thought to serve as docking sites for interactions with specific cellular proteins, and the resultant virus-host interactions are believed to facilitate efficient virus budding (for a review, see reference 9). Although VP40 and other viral matrix proteins (e.g., Gag and M proteins of retroviruses and rhabdoviruses, respectively) can bud independently from cells, additional viral proteins are undoubtedly important for the budding process. For example, the G protein of vesicular stomatitis virus (VSV) has been shown to be important for efficient budding. More specifically, the membrane-proximal stem, transmembrane domain, and cytoplasmic tail of VSV G appear to confer this efficiency (21, 25). In addition, alterations to the cytoplasmic tails of glycoproteins of other RNA viruses, such as influenza A, simian virus 5, and rabies virus, appear to result in poor budding despite an intact matrix protein (15,19,23).In addition to VP40, the surface glycoprotein (GP), the nucleoprotein (NP), and the minor matrix protein (VP24) of Ebola virus have been implicated in virus assembly and budding (1,11,12)...

Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

“…Because tetherin-restricted budding of Ebola VLPs was efficiently rescued by HIV Vpu, we next addressed whether Ebola virus encodes a protein that possesses a Vpu-like function that could overcome tetherin activity. Ebola encodes 7 genes, 4 of which, GP, nucleoprotein (NP), VP24, and VP35, have been shown previously to influence Ebola virus budding (9,10). Plasmids encoding these 4 Ebola genes were transfected with VP40 into 293T cells in the presence or absence of tetherin to test their effects upon budding.…”

Induction of Humoral and CD8+ T Cell Responses Are Required for Protection against Lethal Ebola Virus Infection

“…Ebola VLP (eVLP) and Marburg VLP self-assemble and bud from cellular lipid rafts following expression of GP and VP40 in mammalian cells (27)(28)(29)(30)(31)(32). Mice and guinea pigs vaccinated with eVLPs are completely protected from lethal EBOV challenge (28,29,33); therefore, eVLPs represent a promising vaccine candidate for prevention of lethal EBOV infections.…”

Filovirus Replication and Transcription

“…It is a peripheral membrane protein located on the inner side of the membrane and plays a key role in the budding of viral particles (see below) [10,12]. The viral protein VP24 is presumed to be a minor matrix protein involved in nucleocapsid formation and assembly [13][14][15][16][17]. Since VP24 plays a crucial role in host tropism determination and is able to counteract the type I interferon response, it is considered to be an important virulence factor [18][19][20].…”