Biology subjects

Stahelin, R. V.

Publications and source records attributed to Stahelin, R. V..

17 recordsLinked to original sources

bioRxiv2025

SARS CoV-2 spike adopts distinct conformational ensembles in situ

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

biophysics↗

bioRxiv2025

Heat Shock-Induced PI(4)P Increase Drives HSPA1A Translocation to the Plasma Membrane in Cancer and Stressed Cells through PI4KIII Alpha Activation

HSPA1A, a major heat shock protein, is known to translocate to the plasma membrane (PM) in response to cellular stress and cancer, where it plays protective roles in membrane integrity and stress resistance. Although phosphatidylinositol 4-phosphate [PI(4)P] is essential in this translocation, the signals that trigger and facilitate HSPA1As movement remain undefined.Given that membrane lipid composition dynamically shifts during stress, we hypothesized that heat shock-induced PI(4)P changes are crucial for HSPA1As PM localization. To test this hypothesis, we investigated the mechanisms driving PI(4)P changes and HSPA1A PM localization under heat shock. Lipidomic analysis, enzyme-linked immunosorbent assay (ELISA), and confocal imaging revealed a rapid PI(4)P increase at the PM post-heat shock, with levels peaking at 0 hours and declining by 8 hours. RNA sequencing and protein quantification indicated no transcriptional increase in PI4KIII alpha, the kinase responsible for PI(4)P synthesis, suggesting an alternative regulatory mechanism. Hypothesizing that heat shock enhances PI4KIII alpha activity, we performed ELISA coupled with immunoprecipitation, confirming a significant rise in PI4KIII alpha activity following heat shock. Functional analyses further demonstrated that RNAi-mediated PI4KIII alpha depletion or pharmacological PI(4)P reduction, using GSK-A1, impairs HSPA1As localization to the PM, confirming that HSPA1A translocation is PI(4)P-dependent. Our findings identify PI4KIII alpha activity as a key regulator of PI(4)P accumulation and subsequent HSPA1A recruitment to the PM in stressed and cancer cells. This lipid-mediated response offers new insights into stress adaptation and potentially modifiable pathways for therapeutic interventions to control HSPA1A function in cancer.

biochemistry↗

bioRxiv2024

Heat-Induced Phosphatidylserine Changes Drive HSPA1A's Plasma Membrane Localization

Heat shock protein A1A (HSPA1A) is a molecular chaperone crucial in cell survival. In addition to its cytosolic functions, HSPA1A translocates to heat-shocked and cancer cells plasma membrane (PM). In cancer, PM-localized HSPA1A (mHSPA1A) is associated with increased tumor aggressiveness and therapeutic resistance, suggesting that preventing its membrane localization could have therapeutic value. This translocation depends on HSPA1As interaction with PM phospholipids, including phosphatidylserine (PS). Although PS binding regulates HSPA1As membrane localization, the exact trigger for this movement remains unclear. Given that lipid modifications are a cancer hallmark, we hypothesized that PS is a crucial lipid driving HSPA1A translocation and that heat-induced changes in PS levels trigger HSPA1As PM localization in response to heat stress. We tested this hypothesis using pharmacological inhibition and RNA interference (RNAi) targeting PS synthesis, combined with confocal microscopy, lipidomics, and western blotting. Lipidomic analysis and PS-specific biosensors confirmed a heat shock-induced PS increase, peaking immediately post-stress. Inhibition of PS synthesis with fendiline and RNAi significantly reduced HSPA1As PM localization, while depletion of cholesterol or fatty acids had minimal effects, confirming specificity for PS. Further experiments showed that PS saturation and elongation changes did not significantly impact HSPA1As PM localization, indicating that the total PS increase, rather than specific PS species, is the critical factor. These findings reshape current models of HSPA1A trafficking, demonstrating that PS is a crucial regulator of HSPA1As membrane translocation during the heat shock response. This work offers new insights into lipid-regulated protein trafficking and highlights the importance of PS in controlling cellular responses to stress.

cell biology↗

bioRxiv2024

Direct lipid interactions control SARS-CoV-2 M protein conformational dynamics and virus assembly

M is the most abundant structural membrane protein in coronaviruses and is essential for the formation of infectious virus particles. SARS-CoV-2 M adopts two conformations, Mshort and Mlong, and regulated transition between states is hypothesized to coordinate viral assembly and budding. However, the factors that regulate M conformation and roles for each state are unknown. Here, we discover a direct M-sphingolipid interaction that controls M conformational dynamics and virus assembly. We show M binds Golgi-enriched anionic lipids including ceramide-1-phosphate (C1P). Molecular dynamics simulations show C1P interaction promotes a long to short transition and energetically stabilizes Mshort. Cryo-EM structures show C1P specifically binds Mshort at a conserved site bridging transmembrane and cytoplasmic regions. Disrupting Mshort-C1P interaction alters M subcellular localization, reduces interaction with Spike and E, and impairs subsequent virus-like particle cell entry. Together, these results show endogenous signaling lipids regulate M structure and support a model in which Mshort is stabilized in the early endomembrane system to organize other structural proteins prior to viral budding.

microbiology↗

bioRxiv2024

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.

microbiology↗

bioRxiv2023

The SARS-CoV-2 nucleoprotein associates with anionic lipid membranes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N) and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we find the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, lipid binding is shown to occur in the N protein C-terminal domain, which is supported by extensive in silico analysis. Anionic lipid binding occurs for both the free and N oligomeric forms suggesting N can associate with membranes in the nucleocapsid form. Herein we present a lipid-dependent model based on in vitro, cellular and in silico data for the recruitment of N to M assembly sites in the lifecycle of SARS-CoV-2.

microbiology↗

bioRxiv2023

Impact of Ebola virus nucleoprotein on VP40 virus-like particle production: a computational approach

Ebola virus (EBOV) protein VP40 can assemble and bud in the form of virus-like particles (VLPs) when expressed in the absence of other EBOV proteins in mammalian cells. When nucleoprotein (NP) is co-expressed, VLPs will contain inclusion-body (IB) cores, and VLP production can be increased. However, the mechanism of how NP impacts VLP production remains unclear. Here, we use a computational approach to study NP-VP40 interactions. We find that NP may enhance VLP production through stabilizing VP40 filaments and accelerating the VLP budding step. Also, both the relative timing and amount of NP expression compared to VP40 are important for the effective production of IB-containing VLPs. We further find that there exists an optimum NP/VP40 expression ratio, and that earlier expression of NP compared to VP40 will produce IB-containing VLPs more efficiently. We conclude that disrupting the relative timing and amount of NP and VP40 expression could provide new avenues to treat EBOV infection. This work provides quantitative insights into how EBOV proteins interact and how those interactions could impact virion generation and drug efficacy.

systems biology↗

bioRxiv2023

Evaluation of Fendiline Treatment in VP40 System with Nucleation-Elongation Process: A Computational Model of Ebola Virus Matrix Protein Assembly

Ebola virus (EBOV) infection is threatening human health, especially in Central and West Africa. Limited clinical trials and the requirement of biosafety level-4 (BSL-4) laboratories hinders experimental work to advance our understanding of EBOV and evaluation of treatment. In this work, we use a computational model to study the assembly and budding process of EBOV and evaluate the effect of fendiline on these processes. Our results indicate that the assembly of VP40 filaments may follow the nucleation-elongation theory, as it is critical to maintain a pool of VP40 dimer for the maturation and production of virus-like particles (VLPs). We further find that the nucleation-elongation process can also be influenced by phosphatidylserine (PS), which can complicate the efficacy of fendiline, a drug that lowers cellular PS levels. We observe that fendiline may increase VLP production at earlier time points (24 h) and under low concentrations ([≤] 2 M). But this effect is transient and does not change the conclusion that fendiline generally decreases VLP production. We also conclude that fendiline can be more efficient at the stage of VLP budding relative to earlier phases. Combination therapy with a VLP budding step-targeted drug may further increase the treatment efficiency of fendiline. Finally, we also show that fendiline has higher efficacy when VP40 expression is high. While these are single-cell level results based on the VP40 system, it points out a potential way of fendiline application affecting EBOV assembly, which can be further tested in experimental studies with multiple EBOV proteins or live virus. ImportanceEBOV infection can cause deadly hemorrhagic fever, which has a mortality rate around 90% without treatment. The recent outbreaks in Uganda and Democratic Republic of the Congo illustrate its treat to human health. Though two antibody-based treatments are approved, mortality rates in the last outbreak is still higher than 30%. This can partly be due to the requirement of advanced medical facilities for current treatments. As a result, it is very important to develop and evaluate new therapies for EBOV infection, especially those can be easily applied in the developing world. The significance of our research is that we evaluate the potential treatment effect of fendiline on EBOV infection in the VP40 system with a computational approach, which both greatly saves time and lowers cost compared to traditional experimental studies, and provides innovative new tools to study viral protein dynamics.

systems biology↗

bioRxiv2022

Integrity of membrane fluidity and fatty acid environment are necessary for Ebola virus VP40 assembly and release of viral particles

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation and the order between the two leaflets are important factors for the stability of VP40 binding and oligomerization at the PM. Use of FDA-approved drugs (dibucaine, propranolol and trifluoperazine) to fluidize the plasma membrane, destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Lastly, we show that VP40 can tether and cluster lipid vesicles upon protein enrichment at the membrane. This is a new characteristic of the protein, and it opens the door to new avenues of exploration to deepen our understanding of VP40 host interactions and EBOV assembly. Indeed, our findings support a complex assembly mechanism of the EBOV viral matrix that reaches beyond lipid headgroup specificity using ordered PM lipid regions independent of cholesterol.

microbiology↗

bioRxiv2022

Contribution of the Golgi apparatus in morphogenesis of a virus induced cytopathic vacuolar system

The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of peroxidase tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here we propose a model for the conversion of GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our result has implications for alphavirus virus induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

microbiology↗

bioRxiv2021

Molecular assembly of measles and Nipah virus: specific lipid binding drives conformational change and 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 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.

microbiology↗

bioRxiv2021

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 used a computational model to evaluate EBOV matrix assembly. Our model focused on the assembly kinetics of VP40, the matrix protein in EBOV, and its interaction with phosphatidylserine (PS) in the host cell membrane. Human cells transfected with VP40-expressing plasmids are capable of producing virus-like particles (VLPs) that closely resemble EBOV virions. We used data from this in vitro VP40 system to calibrate our computational model. PS levels in the host cell membrane had been shown to affect VP40 dynamics as well as VLP production through recruiting VP40 dimers to plasma membrane inner leaflet. Our computational results indicated that PS may have direct influence on VP40 filament growth and affect multiple steps in the assembly and budding of VP40 VLPs. We also proposed that the assembly of VP40 filaments may follow the nucleation-elongation theory where initialization and oligomerization of VP40 are two separate and distinct steps in the assembly process. This work illustrated how computational and experimental approaches can be combined to allow for additional analysis and hypothesis generation. Our findings advanced understanding of the molecular process of EBOV assembly and budding processes and may help the development of new EBOV treatments targeting VP40 matrix assembly.

systems biology↗

bioRxiv2021

The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

Phosphatidylserine (PS) has been shown to be 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 subsequently reducing virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were employed including imaging, viral budding and viral entry assays. Fendiline reduced the PS content in mammalian cells and PS in the plasma membrane, reducing the ability of VP40 to form new virus particles. Further, particles that do form from fendiline treated cells have altered particle morphology and decreased infectivity capacity. These complementary studies reveal the mechanism by which filovirus matrix proteins cluster PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

microbiology↗

bioRxiv2020

Lipid-specific protein oligomerization is regulated by two interfaces in Marburg virus matrix protein VP40

Marburg virus major matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane and undergo a dynamic and extensive self-oligomerization into the structural matrix layer which confers the virion shape and stability. Using a myriad of in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrated the importance of each interface in the mVP40 matrix assembly through protein trafficking to the plasma membrane and homo-multimerization that induced protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. A novel APEX-TEM method was employed to closely assess the ultrastructural localization of and formation of viral particles for wild type and mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly.

microbiology↗

bioRxiv2020

SARS-CoV-2 viral budding and entry can be modeled using virus-like particles

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form viruslike particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.

microbiology↗

bioRxiv2018

PI(4,5)P2 Binding Sites in the Ebola Virus Matrix Protein Modulate Assembly and Budding

Ebola virus (EBOV) causes sever hemorrhagic fever in humans, can cause death in a large percentage of those infected, and still lacks FDA approved treatment options. In this study, we investigated how the essential EBOV protein, VP40, forms stable oligomers to mediate budding and assembly from the host cell plasma membrane. An array of in vitro and cellular assays identified and characterized two lysine rich regions that bind to PI(4,5)P2 and serve distinct functions through the lipid binding and assembly of the viral matrix layer. We found that when VP40 binds PI(4,5)P2, VP40 oligomers become extremely stable and long lived. Together, this work characterizes the molecular basis of PI(4,5)P2 binding by VP40, which stabilizes formation of VP40 oligomers necessary for viral assembly and budding. Quercetin, a natural product that lowers PI(4,5)P2 in the plasma membrane, inhibited budding of VP40 VLPs and may inform future treatment strategies against EBOV.

microbiology↗

bioRxiv2018

Mutation of Ebola Virus Matrix Protein Cysteine Residues Increases Binding to Phosphatidylserine through Increased Flexibility of a Lipid Binding Loop

The Ebola virus (EBOV) is a genetically simple negative sense RNA virus with only 7 genes yet it causes severe hemorrhagic fever in humans. The matrix protein VP40 of EBOV is the main driver of viral budding through binding to host plasma membrane lipids and formation of the filamentous, pleomorphic virus particles. To better understand this dynamic and complex process we have asked what the role of two highly conserved cysteine residues are in the C-terminal domain of VP40. Here we report that the mutation of Cys311 to alanine increases VP40 membrane binding affinity for phosphatidylserine containing membranes. C311A has a significant increase in binding to PS compared to WT, has longer virus like particles, and displays evidence of increased budding. C314A also has an increase in PS binding compared to WT, however to a lesser extent. The double Cys mutant shares the phenotypes of the single mutants with increased binding to PS. Computational studies demonstrate these Cys residues, Cys311 in particular, restrain a loop segment containing Lys residues that interact with the plasma membrane. Mutation of Cys311 promotes membrane binding loop flexibility, alters internal VP40 H-bonding, and increases PS binding. To the best of our knowledge, this is the first evidence of mutations that increase VP40 affinity for biological membranes and the length of EBOV virus like particles. Together, our findings indicate these residues are important for membrane dynamics at the plasma membrane via the interaction with phosphatidylserine.

microbiology↗