Structural Heterogeneity of the Rabies Virus Virion.

Rabies virus (RABV) is among the first recognized viruses of public health concern and has historically contributed to the development of viral vaccines. Despite these significances, the three-dimensional structure of the RABV virion remains unknown due to the challenges in isolating structurally homogenous virion samples in sufficient quantities needed for structural investigation. Here, by combining the capabilities of cryogenic electron tomography (cryoET) and microscopy (cryoEM), we determined the three-dimensional structure of the wild-type RABV virion. Tomograms of RABV virions reveal a high level of structural heterogeneity among the bullet-shaped virion particles encompassing the glycoprotein (G) trimer-decorated envelope and the nucleocapsid composed of RNA, nucleoprotein (N), and matrix protein (M). The structure of the trunk region of the virion was determined by cryoEM helical reconstruction, revealing a one-start N-RNA helix bound by a single layer of M proteins at an N:M ratio of 1. The N-M interaction differs from that in fellow rhabdovirus vesicular stomatitis virus (VSV), which features two layers of M stabilizing the N-RNA helix at an M:N ratio of 2. These differences in both M-N stoichiometry and binding allow RABV to flex its N-RNA helix more freely and point to different mechanisms of viral assembly between these two bullet-shaped rhabdoviruses.

Insights into the specific feature of the electrostatic recognition binding mechanism between BM2 and BM1: a molecular dynamics simulation study.

Matrix protein 2 (M2) and matrix protein 1 (M1) of the influenza B virus are two important proteins, and the interactions between BM2 and BM1 play an important role in the process of virus assembly and replication. However, the interaction details between BM2 and BM1 are still unclear at the atomic level. Here, we constructed the BM2-BM1 complex system using homology modelling and molecular docking methods. Molecular dynamics (MD) simulations were used to illustrate the binding mechanism between BM2 and BM1. The results identify that the eight polar residues (E88B, E89B, H119BM1, E94B, R101BM1, K102BM1, R105BM1, and E104B) play an important role in stabilizing the binding through the formation of hydrogen bond networks and salt-bridge interactions at the binding interface. Furthermore, based on the simulation results and the experimental facts, the mutation experiments were designed to verify the influence of the mutation of residues both within and outside the effector domain. The mutations directly or indirectly disrupt interactions between polar residues, thus affecting viral assembly and replication. The results could help us understand the details of the interactions between BM2 and BM1 and provide useful information for the anti-influenza drug design.

Membrane curvature sensing and symmetry breaking of the M2 proton channel from Influenza A.

The M2 proton channel aids in the exit of mature influenza viral particles from the host plasma membrane through its ability to stabilize regions of high negative Gaussian curvature (NGC) that occur at the neck of budding virions. The channels are homo-tetramers that contain a cytoplasm-facing amphipathic helix (AH) that is necessary and sufficient for NGC generation; however, constructs containing the transmembrane spanning helix, which facilitates tetramerization, exhibit enhanced curvature generation. Here, we used all-atom molecular dynamics (MD) simulations to explore the conformational dynamics of M2 channels in lipid bilayers revealing that the AH is dynamic, quickly breaking the fourfold symmetry observed in most structures. Next, we carried out MD simulations with the protein restrained in four- and twofold symmetric conformations to determine the impact on the membrane shape. While each pattern was distinct, all configurations induced pronounced curvature in the outer leaflet, while conversely, the inner leaflets showed minimal curvature and significant lipid tilt around the AHs. The MD-generated profiles at the protein-membrane interface were then extracted and used as boundary conditions in a continuum elastic membrane model to calculate the membrane-bending energy of each conformation embedded in different membrane surfaces characteristic of a budding virus. The calculations show that all three M2 conformations are stabilized in inward-budding, concave spherical caps and destabilized in outward-budding, convex spherical caps, the latter reminiscent of a budding virus. One of the C2-broken symmetry conformations is stabilized by 4 kT in NGC surfaces with the minimum energy conformation occurring at a curvature corresponding to 33 nm radii. In total, our work provides atomistic insight into the curvature sensing capabilities of M2 channels and how enrichment in the nascent viral particle depends on protein shape and membrane geometry.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane.

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

In silico exploration of deep-sea fungal metabolites as inhibitor of Ebola and Marburg VP35 and VP40.

VP30 and VP40 proteins of Ebola and Marburg viruses have been recognized as potential targets for antiviral drug development due to their essential roles in the viral lifecycle. Targeting these proteins could disrupt key stages of the viral replication process, inhibiting the viruses' ability to propagate and cause disease. The current study aims to perform molecular docking and virtual screening on deep-sea fungal metabolites targeting Marburg virus VP40 Dimer, matrix protein VP40 from Ebola virus Sudan, Ebola VP35 Interferon Inhibitory Domain, and VP35 from Marburg virus. The top ten compounds for each protein target were chosen using the glide score. All the compounds obtained indicate a positive binding interaction. Furthermore, AdmetSAR was utilized to investigate the pharmacokinetics of the inhibitors chosen. Gliotoxin was used as a ligand with Marburg virus VP40 Dimer, Austinol with matrix protein VP40 from Ebola virus Sudan, Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) with Ebola VP35 Interferon Inhibitory Domain, and Dehydroaustinol with VP35 from Marburg virus. MD modeling and MMPBSA studies were used to provide a better understanding of binding behaviors. Pre-clinical experiments can assist validate our in-silico studies and assess whether the molecule can be employed as an anti-viral drug.

The impact of transmembrane peptides on lipid bilayer structure and mechanics: A study of the transmembrane domain of the influenza A virus M2 protein.

Transmembrane peptides play important roles in many biological processes by interacting with lipid membranes. This study investigates how the transmembrane domain of the influenza A virus M2 protein, M2TM, affects the structure and mechanics of model lipid bilayers. Atomic force microscopy (AFM) imaging revealed small decreases in bilayer thickness with increasing peptide concentrations. AFM-based force spectroscopy experiments complemented by theoretical model analysis demonstrated significant decreases in bilayer's Young's modulus (E) and lateral area compressibility modulus (KA). This suggests that M2TM disrupts the cohesive interactions between neighboring lipid molecules, leading to a decrease in both the bilayer's resistance to indentation (E) and its ability to resist lateral compression/expansion (KA). The large decreases in bilayer elastic parameters (i.e., E and KA) contrast with small changes in bilayer thickness, implying that bilayer mechanics are not solely dictated by bilayer thickness in the presence of transmembrane peptides. The observed significant reduction in bilayer mechanical properties suggests a softening effect on the bilayer, potentially facilitating membrane curvature generation, a crucial step for M2-mediated viral budding. In parallel, our Raman spectroscopy revealed small but statistically significant changes in hydrocarbon chain vibrational dynamics, indicative of minor disordering in lipid chain conformation. Our findings provide useful insights into the complex interplay between transmembrane peptides and lipid bilayers, highlighting the significance of peptide-lipid interactions in modulating membrane structure, mechanics, and molecular dynamics.

Structure and dynamics of the proton-selective histidine and the gating tryptophan in an inward rectifying hybrid influenza B and A virus M2 proton channel.

The M2 proteins of influenza A and B viruses form acid-activated proton channels that are essential for the virus lifecycle. Proton selectivity is achieved by a transmembrane (TM) histidine whereas gating is achieved by a tryptophan residue. Although this functional apparatus is conserved between AM2 and BM2 channels, AM2 conducts protons exclusively inward whereas BM2 conducts protons in either direction depending on the pH gradient. Previous studies showed that in AM2, mutations of D44 abolished inward rectification of AM2, suggesting that the tryptophan gate is destabilized. To elucidate how charged residues C-terminal to the tryptophan regulates channel gating, here we investigate the structure and dynamics of H19 and W23 in a BM2 mutant, GDR-BM2, in which three BM2 residues are mutated to the corresponding AM2 residues, S16G, G26D and H27R. Whole-cell electrophysiological data show that GDR-BM2 conducts protons with inward rectification, identical to wild-type (WT) AM2 but different from WT-BM2. Solid-state NMR 15N and 13C spectra of H19 indicate that the mutant BM2 channel contains higher populations of cationic histidine and neutral τ tautomers compared to WT-BM2 at acidic pH. Moreover, 19F NMR spectra of 5-19F-labeled W23 resolve three peaks at acidic pH, suggesting three tryptophan sidechain conformations. Comparison of these spectra with the tryptophan spectra of other M2 peptides suggests that these indole sidechain conformations arise from interactions with the C-terminal charged residues and with the N-terminal cationic histidine. Taken together, these solid-state NMR data show that inward rectification in M2 proton channels is accomplished by tryptophan interactions with charged residues on both its C-terminal and N-terminal sides. Gating of these M2 proton channels is thus accomplished by a multi-residue complex with finely tuned electrostatic and aromatic interactions.

Assembly and activation of EBV latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

High-throughput virtual screening of Streptomyces spp. metabolites as antiviral inhibitors against the Nipah virus matrix protein.

Nipah virus (NiV) remains a significant global concern due to its impact on both the agricultural industry and human health, resulting in substantial economic and health consequences. Currently, there is no cure or commercially available vaccine for the virus. Therefore, it is crucial to prioritize the discovery of new and effective treatment options to prevent its continued spread. Streptomyces spp. are rich sources of metabolites known for their bioactivity against certain diseases; however, their potential as antiviral drugs against the Nipah virus remain unexplored. In this study, 6524 Streptomyces spp. metabolites were screened through in silico methods for their inhibitory effects against the Nipah virus matrix (NiV-M) protein, which assists in virion assembly of Nipah virus. Different computer-aided tools were utilized to carry out the virtual screening process: ADMET profiling revealed 913 compounds with excellent safety and efficacy profiles, molecular docking predicted the binding poses and associated docking scores of the ligands in their respective targets, MD simulations confirmed the binding stability of the top ten highest-scoring ligands in a 100 ns all-atom simulation, PCA elucidated simulation convergence, and MMPB(GB)SA calculations estimated the binding energies of the final candidate compounds and determined the key residues crucial for complex formation. Using in silico methods, we identified six metabolites targeting the main substrate-binding site and five targeting the dimerization site that exhibited excellent stability and strong binding affinity. We recommend testing these compounds in the next stages of drug development to confirm their effectiveness as therapeutic agents against Nipah virus.

Identification of Prospective Ebola Virus VP35 and VP40 Protein Inhibitors from Myxobacterial Natural Products.

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.

Conformations of influenza A M2 protein in DOPC/DOPS and E. coli native lipids and proteins.

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IM2) protein reconstituted in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPS) bilayers to those in isolated Escherichia coli (E. coli) membranes, having preserved its native proteins and lipids. IM2 is a single-pass transmembrane protein known to assemble into a homo-tetrameric proton channel. To represent this channel, we made a construct containing the IM2's TMD region flanked by the juxtamembrane residues. The single cysteine substitution, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the electron spin resonance (ESR) study. For this particular residue, we probed the conformations of the spin-labeled IM2 reconstituted in DOPC/DOPS and isolated E. coli membranes using continuous-wave ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The continuous-wave ESR spectra corresponded to very slow spin-label motion in both environments. In all cases, the DEER data were reconstructed into distance distributions with well-resolved peaks at 1.68 and 2.37 nm in distance and amplitude ratios of 1.41 ± 0.2 and 2:1, respectively. This suggests four nitroxide spin labels located at the corners of a square, indicative of an axially symmetric tetramer. The distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IM2 TMD tetramer in agreement with the model. Thus, we can conclude that, under conditions of pH 7.4 used in this study, IM2 TMD has similar conformations in model lipid bilayers and membranes made of native E. coli lipids and proteins of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

A top-down approach for studying the in-silico effect of the novel phytocompound tribulusamide B on the inhibition of Nipah virus transmission through targeting fusion glycoprotein and matrix protein.

The proteins of Nipah virus ascribe to its lifecycle and are crucial to infections caused by the virus. In the absence of approved therapeutics, these proteins can be considered as drug targets. This study examined the potential of fifty-three (53) natural compounds to inhibit Nipah virus fusion glycoprotein (NiV F) and matrix protein (NiV M) in silico. The molecular docking experiment, supported by the principal component analysis (PCA), showed that out of all the phytochemicals considered, Tribulusamide B had the highest inhibitory potential against the target proteins NiV F and NiV M (-9.21 and -8.66 kcal mol-1, respectively), when compared to the control drug, Ribavirin (-7.01 and -6.52 kcal mol-1, respectively). Furthermore, it was found that Tribulusamide B pharmacophores, namely, hydrogen donors, acceptors, aromatic and hydrophobic groups, contributed towards the effective residual interactions with the target proteins. The molecular dynamic simulation further validated the results of the docking studies and concluded that Tribulusamide B formed a stable complex with the target proteins. The data obtained from MM-PBSA study further explained that the phytochemical could strongly bind with NiV F (-31.26 kJ mol-1) and NiV M (-40.26 kJ mol-1) proteins in comparison with the control drug Ribavirin (-13.12 and -13.94 kJ mol-1, respectively). Finally, the results indicated that Tribulusamide B, a common inhibitor effective against multiple proteins, can be considered a potential therapeutic entity in treating the Nipah virus infection.

Comparing Multifunctional Viral and Eukaryotic Proteins for Generating Scission Necks in Membranes.

Deterministic formation of membrane scission necks by protein machinery with multiplexed functions is critical in biology. A microbial example is M2 viroporin, a proton pump from the influenza A virus that is multiplexed with membrane remodeling activity to induce budding and scission in the host membrane during viral maturation. In comparison, the dynamin family constitutes a class of eukaryotic proteins implicated in mitochondrial fission, as well as various budding and endocytosis pathways. In the case of Dnm1, the mitochondrial fission protein in yeast, the membrane remodeling activity is multiplexed with mechanoenzyme activity to create fission necks. It is not clear why these functions are combined in these scission processes, which occur in drastically different compositions and solution conditions. In general, direct experimental access to changing neck sizes induced by individual proteins or peptide fragments is challenging due to the nanoscale dimensions and influence of thermal fluctuations. Here, we use a mechanical model to estimate the size of scission necks by leveraging small-angle X-ray scattering structural data of protein-lipid systems under different conditions. The influence of interfacial tension, lipid composition, and membrane budding morphology on the size of the induced scission necks is systematically investigated using our data and molecular dynamic simulations. We find that the M2 budding protein from the influenza A virus has robust pH-dependent membrane activity that induces nanoscopic necks within the range of spontaneous hemifission for a broad range of lipid compositions. In contrast, the sizes of scission necks generated by mitochondrial fission proteins strongly depend on lipid composition, which suggests a role for mechanical constriction.

Structure-guided mutagenesis targeting interactions between pp150 tegument protein and small capsid protein identify five lethal and two live-attenuated HCMV mutants.

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.

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.

Cooperative Membrane Binding of HIV-1 Matrix Proteins.

The HIV-1 assembly process begins with a newly synthesized Gag polyprotein being targeted to the inner leaflet of the plasma membrane of the infected cells to form immature viral particles. Gag-membrane interactions are mediated through the myristoylated (Myr) N-terminal matrix (MA) domain of Gag, which eventually multimerize on the membrane to form trimers and higher order oligomers. The study of the structure and dynamics of peripheral membrane proteins like MA has been challenging for both experimental and computational studies due to the complex transient dynamics of protein-membrane interactions. Although the roles of anionic phospholipids (PIP2, PS) and the Myr group in the membrane targeting and stable membrane binding of MA are now well-established, the cooperative interactions between the MA monomers and MA-membrane remain elusive in the context of viral assembly and release. Our present study focuses on the membrane binding dynamics of a higher order oligomeric structure of MA protein (a dimer of trimers), which has not been explored before. Employing time-lagged independent component analysis (tICA) to our microsecond-long trajectories, we investigate conformational changes of the matrix protein induced by membrane binding. Interestingly, the Myr switch of an MA monomer correlates with the conformational switch of adjacent monomers in the same trimer. Together, our findings suggest complex protein dynamics during the formation of the immature HIV-1 lattice; while MA trimerization facilitates Myr insertion, MA trimer-trimer interactions in the immature lattice can hinder the same.

The Potential of Cyclodextrins as Inhibitors for the BM2 Protein: An In Silico Investigation.

The influenza BM2 transmembrane domain (BM2TM), an acid-activated proton channel, is an attractive antiviral target due to its essential roles during influenza virus replication, whereas no effective inhibitors have been reported for BM2. In this study, we draw inspiration from the properties of cyclodextrins (CDs) and hypothesize that CDs of appropriate sizes may possess the potential to act as inhibitors of the BM2TM proton channel. To explore this possibility, molecular dynamics simulations were employed to assess their inhibitory capabilities. Our findings reveal that CD4, CD5, and CD6 are capable of binding to the BM2TM proton channel, resulting in disrupted water networks and reduced hydrogen bond occupancy between H19 and the solvent within the BM2TM channel necessary for proton conduction. Notably, CD4 completely obstructs the BM2TM water channel. Based on these observations, we propose that CD4, CD5, and CD6 individually contribute to diminishing the proton transfer efficiency of the BM2 protein, and CD4 demonstrates promising potential as an inhibitor for the BM2 proton channel.

In Situ Spectroscopic Detection of Large-Scale Reorientations of Transmembrane Helices During Influenza A M2 Channel Opening.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2's transmembrane α-helices in situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state nuclear magnetic resonance-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.