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.

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.

PI(4,5)P2 binding sites in the Ebola virus matrix protein VP40 modulate assembly and budding.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P2. The PI(4,5)P2-induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P2. CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs.

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.

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness.

The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the ß-barrel. In particular, mCherryXL showed reduced nanosecond time scale breathing of the gap between the ß7 and ß10-strands, which was previously shown to be the most flexible region of mCherry. Together with experimental results, the simulations also reveal steric interactions of residue 161 and a network of hydrogen-bonding interactions of the chromophore with residues at positions 59, 143, and 163 that are critical in perturbing the chromophore electronic structure. Finally, we shed light on the conformational dynamics of the conserved residues R95 and S146, which are hydrogen-bonded to the chromophore, and provide physical insights into the observed photophysics. To the best of our knowledge, this is the first study that evaluates the conformational space for a set of closely related FPs generated by directed evolution.

A 5'-Flanking C/G Pair at the Core Region Enhances the Recognition and Binding of Kaiso to Methylated DNA.

Methyl CpG binding proteins (MBPs) are transcription factors that recognize the methylated CpG sites in DNA and mediate the DNA methylation signal into various downstream cellular processes. The C2H2 zinc finger (ZF) protein, Kaiso, also an MBP, preferentially binds to two symmetrically methylated CpG sites in DNA sequences via C-terminal C2H2 ZF domains and mediates the transcription regulation process. Investigation of the molecular mechanism of the recognition of methylated DNA (meDNA) by Kaiso is important to understand how this protein reads and translates this methylation signal into downstream transcription outcomes. Despite previous studies in Kaiso-meDNA interactions, detailed structural investigations on the sequence-specific interaction of Kaiso with the meDNA sequence are still lacking. In this work, we used molecular modeling and molecular dynamics (MD) simulation-based computational approaches to investigate the recognition of various methylated DNA sequences by Kaiso. Our MD simulation results show that the Kaiso-meDNA interaction is sequence specific. The recognition of meDNA by Kaiso is enhanced in the MeECad sequence compared to the MeCG2 sequence. Compared to the 5'-flanking T/A pair in MeCG2, both MeCG2_mutCG and MeECad sequences show that a C/G base pair allows GLU535 of Kaiso to preferably recognize and bind the core mCpG site. The core mCGmCG site is crucial for the recognition process and formation of a stable complex. Our results reveal that the 5'-flanking nucleotides are also important for the enhanced binding and recognition of methylated sites.

Immunoinformatic screening of Marburgvirus epitopes and computational investigations of epitope-allele complexes.

Marburgvirus (MARV), a member of the Filovirus family, causes severe hemorrhagic fever in humans. Currently, there are no approved vaccines or post exposure treatment methods available against MARV. With the aim of identifying vaccine candidates against MARV, we employ different sequence-based computational methods to predict the MHC-I and MHC-II T-cell epitopes as well as B-cell epitopes for the complete MARV genome. We analyzed the variations in the predicted epitopes among four MARV variants, the Lake Victoria, Angola, Musoke, and Ravn. We used a consensus approach to identify several epitopes, including novel epitopes, and narrowed down the selection based on different parameters such as antigenicity and IC50 values. The selected epitopes can be used in various vaccine constructs that give effective antibody responses. The MHC-I epitope-allele complexes for GP and NP with favorably low IC50 values were investigated using molecular dynamics computations to determine the molecular details of the epitope-allele complexes. This study provides information for further experimental validation of the potential epitopes and the design and development of MARV vaccines.

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.

Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

AT-hook peptides bind the major and minor groove of AT-rich DNA duplexes.

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.

Detecting Individual Proteins and Their Surface Charge Variations in Solution by the Potentiometric Nanoimpact Method.

Label-free detection and analysis of proteins in their natural form and their dynamic interactions with substrates at the single-molecule level are important for both fundamental studies and various applications. Herein, we demonstrate a simple potentiometric method to achieve this goal by detecting the native charge of protein in solution by utilizing the principle of single-entity electrochemistry techniques. When a charged protein moves near the vicinity of a floating carbon nanoelectrode connected to a high-impedance voltage meter, the distinct local electrostatic potential changes induced by the transient collision event of protein, also called the "nanoimpact" event, can be captured by the nanoelectrode as a potential probe. This potentiometric method is highly sensitive for charged proteins, and low-molecular-weight proteins less than 10 kDa can be detected in low-salt-concentration electrolytes. By analyzing the shape and magnitude of the recorded time-resolved potential change and its time derivative, we can reveal the charge and motion of the protein in the nonspecific protein-surface interaction event. The charge polarity variations of the proteins at different pH values were also successfully probed. Compared with synthetic spherical nanoparticles, the statistical analysis of many single-molecule nanoimpact events revealed a large variation in the recorded transient potential signals, which may be attributed to the intrinsic protein dynamics and surface charge heterogeneity, as suggested by the finite element method and molecular dynamic simulations.

Ebola virus protein VP40 binding to Sec24c for transport to the plasma membrane.

Outbreaks of the Ebola virus (EBOV) continue to occur and while a vaccine and treatment are now available, there remains a dearth of options for those who become sick with EBOV disease. An understanding at the atomic and molecular level of the various steps in the EBOV replication cycle can provide molecular targets for disrupting the virus. An important step in the EBOV replication cycle is the transport of EBOV structural matrix VP40 protein molecules to the plasma membrane inner leaflet, which involves VP40 binding to the host cell's Sec24c protein. Though some VP40 residues involved in the binding are known, the molecular details of VP40-Sec24c binding are not known. We use various molecular computational techniques to investigate the molecular details of how EBOV VP40 binds with the Sec24c complex of the ESCRT-I pathway. We employed different docking programs to identify the VP40-binding site on Sec24c and then performed molecular dynamics simulations to determine the atomic details and binding interactions of the complex. We also investigated how the inter-protein interactions of the complex are affected upon mutations of VP40 amino acids in the Sec24c-binding region. Our results provide a molecular basis for understanding previous coimmunoprecipitation experimental studies. In addition, we found that VP40 can bind to a site on Sec24c that can also bind Sec23 and suggests that VP40 may use the COPII transport mechanism in a manner that may not need the Sec23 protein in order for VP40 to be transported to the plasma membrane.

Lipid II Binding and Transmembrane Properties of Various Antimicrobial Lanthipeptides.

There has been an alarming rise in antibacterial resistant infections in recent years due to the widespread use of antibiotics, and there is a dire need for the development of new antibiotics utilizing novel modes of action. Lantibiotics are promising candidates to engage in the fight against resistant strains of bacteria due to their unique modes of action, including interference with cell wall synthesis by binding to lipid II and creating pores in bacterial membranes. In this study, we use atomic-scale molecular dynamics computational studies to compare both the lipid II binding ability and the membrane interactions of five lanthipeptides that are commonly used in antimicrobial research: nisin, Mutacin 1140 (MU1140), gallidermin, NVB302, and NAI107. Among the five peptides investigated, nisin is found to be the most efficient at forming water channels through a membrane, whereas gallidermin and MU1140 are found to be better at binding the lipid II molecules. Nisin's effectiveness in facilitating water transport across the membrane is due to the creation of several different water trajectories along with no significant water delay points along the paths. The shorter peptide deoxyactagardine B (NVB302) was found to not form a water channel. These detailed observations provide insights into the dual mechanisms of the action of lantibiotic peptides and can facilitate the design and development of novel lanthipeptides by strategic placement of different residues.

Mutation-induced changes in the receptor-binding interface of the SARS-CoV-2 Delta variant B.1.617.2 and implications for immune evasion.

Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant's receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding ß-loop-ß motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.

Cysteine Mutations in the Ebolavirus Matrix Protein VP40 Promote Phosphatidylserine Binding by Increasing the Flexibility of a Lipid-Binding Loop.

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.

Structural insights into the repair mechanism of AGT for methyl-induced DNA damage.

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- and post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex.

Structural and Dynamical Differences in the Spike Protein RBD in the SARS-CoV-2 Variants B.1.1.7 and B.1.351.

The novel coronavirus (SARS-CoV-2) pandemic that started in late 2019 is responsible for hundreds of millions of cases worldwide and millions of fatalities. Though vaccines are available, the virus is mutating to form new strains among which are the variants B.1.1.7 and B.1.351 that demonstrate increased transmissivity and infectivity. In this study, we performed molecular dynamics simulations to explore the role of the mutations in the interaction of the virus spike protein receptor binding domain (RBD) with the host receptor ACE2. We find that the hydrogen bond networks are rearranged in the variants and also that new hydrogen bonds are established between the RBD and ACE2 as a result of mutations. We investigated three variants: the wild-type (WT), B.1.1.7, and B.1.351. We find that the B.1.351 variant (also known as 501Y.V2) shows larger flexibility in the RBD loop segment involving residue K484, yet the RBD-ACE2 complex shows higher stability. Mutations that allow a more flexible interface that can result in a more stable complex may be a factor contributing to the increased infectivity of the mutated variants.

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.