Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation.

The Ebola virus (EBOV) is a lipid-enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus-like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL-4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics-based generalized Born with Poisson-Boltzmann surface area (MM-GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha-helical interface (between residues 106-117) as well as in a loop region (between residues 52-61) below this alpha-helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Minor changes in electrostatics robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine (PS) and PI(4,5)P2 in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles (VLPs). In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Elucidating Residue-Level Determinants Affecting Dimerization of Ebola Virus Matrix Protein Using High-Throughput Site Saturation Mutagenesis and Biophysical Approaches.

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer-dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV 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.

Lipid-protein interactions in virus assembly and budding from the host cell plasma membrane.

Lipid enveloped viruses contain a lipid bilayer coat that protects their genome to help facilitate entry into the new host cell. This lipid bilayer comes from the host cell which they infect. After viral replication, the mature virion hijacks the host cell plasma membrane where it is then released to infect new cells. This process is facilitated by the interaction between phospholipids that make up the plasma membrane and specialized viral matrix proteins. This step in the viral lifecycle may represent a viable therapeutic strategy for small molecules that aim to block enveloped virus spread. In this review, we summarize the current knowledge on the role of plasma membrane lipid-protein interactions on viral assembly and budding.

Engineering modular diterpene biosynthetic pathways in Physcomitrella patens.

MAIN CONCLUSION: Modular assembly and heterologous expression in the moss Physcomitrella patens of pairs of diterpene synthases results in accumulation of modern land plant diterpenoids. Physcomitrella patens is a representative of the ancient bryophyte plant lineage with a genome size of 511 Mb, dominant haploid life cycle and limited chemical and metabolic complexity. For these plants, exceptional capacity for genome editing through homologous recombination is met with recently demonstrated in vivo assembly of multiple heterologous DNA fragments. These traits earlier made P. patens an attractive choice as a biotechnological chassis for photosynthesis-driven production of recombinant peptides. The lack of diterpene gibberellic acid phytohormones in P. patens combined with the recent targeted disruption of the single bifunctional diterpene synthase yielded lines devoid of endogenous diterpenoid metabolites and well-suited for engineering of terpenoid production. Here, we mimicked the modular nature of diterpene biosynthetic pathways found in modern land plants by developing a flexible pipeline to install three combinations of class II and class I diterpene synthases in P. patens to access industrially relevant diterpene biomaterials. In addition to a well-established neutral locus for targeted integration, we also explored loci created by a class of Long Terminal Repeat Retrotransposon present at moderate number in the genome of P. patens. Assembly of the pathways and production of the enzymes from the neutral locus led to accumulation of diterpenes matching the reported activities in the angiosperm sources. In contrast, insights gained with the retrotransposon loci indicate their suitability for targeting, but reveal potentially inherent complications which may require adaptation of the experimental design.