Role of phosphatidic acid lipids on plasma membrane association of the Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 is responsible for the formation of the viral matrix by localizing at the inner leaflet of the human plasma membrane (PM). Various lipid types, including PI(4,5)P2 (i.e. PIP2) and phosphatidylserine (PS), play active roles in this process. Specifically, the negatively charged headgroups of both PIP2 and PS interact with the basic residues of VP40 and stabilize it at the membrane surface, allowing for eventual egress. Phosphatidic acid (PA), resulting from the enzyme phospholipase D (PLD), is also known to play an active role in viral development. In this work, we performed a biophysical and computational analysis to investigate the effects of the presence of PA on the membrane localization and association of VP40. We used coarse-grained molecular dynamics simulations to quantify VP40 hexamer interactions with the inner leaflet of the PM. Analysis of the local distribution of lipids shows enhanced lipid clustering when PA is abundant in the membrane. We observed that PA lipids have a similar role to that of PS lipids in VP40 association due to the geometry and charge. Complementary experiments performed in cell culture demonstrate competition between VP40 and a canonical PA-binding protein for the PM. Also, inhibition of PA synthesis reduced the detectable budding of virus-like particles. These computational and experimental results provide new insights into the early stages of Ebola virus budding and the role that PA lipids have on the VP40-PM association.

Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding.

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

Measles and Nipah virus assembly: Specific lipid binding drives matrix polymerization.

Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress that is needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane. Using x-ray crystallography, electron microscopy, and molecular dynamics, we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

Mechanisms of phosphatidylserine influence on viral production: A computational model of Ebola virus matrix protein assembly.

Ebola virus (EBOV) infections continue to pose a global public health threat, with high mortality rates and sporadic outbreaks in Central and Western Africa. A quantitative understanding of the key processes driving EBOV assembly and budding could provide valuable insights to inform drug development. Here, we use a computational model to evaluate EBOV matrix assembly. Our model focuses on the assembly kinetics of VP40, the matrix protein in EBOV, and its interaction with phosphatidylserine (PS) in the host cell membrane. It has been shown that mammalian cells transfected with VP40-expressing plasmids are capable of producing virus-like particles (VLPs) that closely resemble EBOV virions. Previous studies have also shown that PS levels in the host cell membrane affects VP40 association with the plasma membrane inner leaflet and that lower membrane PS levels result in lower VLP production. Our computational findings indicate that PS may also have a direct influence on VP40 VLP assembly and budding, where a higher PS level will result in a higher VLP budding rate and filament dissociation rate. Our results further suggest that the assembly of VP40 filaments follow the nucleation-elongation theory, where initialization and oligomerization of VP40 are two distinct steps in the assembly process. Our findings advance the current understanding of VP40 VLP formation by identifying new possible mechanisms of PS influence on VP40 assembly. We propose that these mechanisms could inform treatment strategies targeting PS alone or in combination with other VP40 assembly steps.

Negative-sense RNA viruses: An underexplored platform for examining virus-host lipid interactions.

Viruses are pathogenic agents that can infect all varieties of organisms, including plants, animals, and humans. These microscopic particles are genetically simple as they encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope that surrounds their protein and genetic contents. Notably, the lipid envelope is formed from the host cell the virus infects. Lipid-enveloped viruses comprise a diverse range of pathogenic viruses, which often lead to high fatality rates and many lack effective therapeutics and/or vaccines. This perspective primarily focuses on the negative-sense RNA viruses from the order Mononegavirales, which obtain their lipid envelope from the host plasma membrane. Specifically, the perspective highlights the common themes of host cell lipid and membrane biology necessary for virus replication, assembly, and budding.

Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response.

We lack a mechanistic understanding of aging-mediated changes in mitochondrial bioenergetics and lipid metabolism that affect T cell function. The bioactive sphingolipid ceramide, induced by aging stress, mediates mitophagy and cell death; however, the aging-related roles of ceramide metabolism in regulating T cell function remain unknown. Here, we show that activated T cells isolated from aging mice have elevated C14/C16 ceramide accumulation in mitochondria, generated by ceramide synthase 6, leading to mitophagy/mitochondrial dysfunction. Mechanistically, aging-dependent mitochondrial ceramide inhibits protein kinase A, leading to mitophagy in activated T cells. This aging/ceramide-dependent mitophagy attenuates the antitumor functions of T cells in vitro and in vivo. Also, inhibition of ceramide metabolism or PKA activation by genetic and pharmacologic means prevents mitophagy and restores the central memory phenotype in aging T cells. Thus, these studies help explain the mechanisms behind aging-related dysregulation of T cells' antitumor activity, which can be restored by inhibiting ceramide-dependent mitophagy.

Lipid-specific oligomerization of the Marburg virus matrix protein VP40 is regulated by two distinct interfaces for virion assembly.

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.

Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane.

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

The Plasmodium falciparum MESA erythrocyte cytoskeleton-binding (MEC) motif binds to erythrocyte ankyrin.

The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5C domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif's primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

Interdomain salt-bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization.

The Ebola virus protein VP40 is a transformer protein that possesses an extraordinary ability to accomplish multiple functions by transforming into various oligomeric conformations. The disengagement of the C-terminal domain (CTD) from the N-terminal domain (NTD) is a crucial step in the conformational transformations of VP40 from the dimeric form to the hexameric form or octameric ring structure. Here, we use various molecular dynamics (MD) simulations to investigate the dynamics of the VP40 protein and the roles of interdomain interactions that are important for the domain-domain association and dissociation, and report on experimental results of the behavior of mutant variants of VP40. The MD studies find that various salt-bridge interactions modulate the VP40 domain dynamics by providing conformational specificity through interdomain interactions. The MD simulations reveal a novel salt-bridge between D45-K326 when the CTD participates in a latch-like interaction with the NTD. The D45-K326 salt-bridge interaction is proposed to help domain-domain association, whereas the E76-K291 interaction is important for stabilizing the closed-form structure. The effects of the removal of important VP40 salt-bridges on plasma membrane (PM) localization, VP40 oligomerization, and virus like particle (VLP) budding assays were investigated experimentally by live cell imaging using an EGFP-tagged VP40 system. It is found that the mutations K291E and D45K show enhanced PM localization but D45K significantly reduced VLP formation.