Smita P. Soni

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

Background: The Ebola virus matrix protein (VP40) regulates the plasma membrane assembly and egress of the Ebola virus. Results: The plasma membrane induces membrane penetration of the VP40 C-terminal domain. Conclusion: Membrane penetration by VP40 is important for VP40 cellular localization, oligomerization, and viral budding. Significance: A better understanding of VP40-membrane interactions will help us to understand Ebola virus assembly and budding. Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu213, Ile293, Leu295, and Val298 demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

ABSTRACT Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCE The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.

Background: Ebola virus VP40 matrix protein is able to produce virus-like particles from cells. Results: VP40 induces negative membrane curvature and scission from in vitro membranes. Conclusion: Phosphatidylserine plays an important role in VP40-induced vesiculation. Significance: Elucidation of VP40 interactions with lipids could lead to a better understanding of the Ebola virus replication cycle. Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission.