Viral Lipid Envelope Research Articles

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 used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.

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.

Low-Temperature Adaptive Single-Atom Iron Nanozymes against Viruses in the Cold Chain.

Outbreaks of viral infectious diseases, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV), pose a great threat to human health. Viral spread is accelerated worldwide by the development of cold chain logistics; Therefore, an effective antiviral approach is required. In this study, it is aimed to develop a distinct antiviral strategy using nanozymes with low-temperature adaptability, suitable for cold chain logistics. Phosphorus (P) atoms are added to the remote counter position of Fe-N-C center to prepare FeN4P2-single-atom nanozymes (SAzymes), exhibiting lipid oxidase (OXD)-like activity at cold chain temperatures (-20, and 4°C). This feature enables FeN4P2-SAzymes to disrupt multiple enveloped viruses (human, swine, and avian coronaviruses, and H1-H11 subtypes of IAV) by catalyzing lipid peroxidation of the viral lipid envelope. Under the simulated conditions of cold chain logistics, FeN4P2-SAzymes are successfully applied as antiviral coatings on outer packaging and personal protective equipment; Therefore, FeN4P2-SAzymes with low-temperature adaptability and broad-spectrum antiviral properties may serve as key materials for developing specific antiviral approaches to interrupt viral transmission through the cold chain.

Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans

Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

Inactivation of the enveloped virus phi6 with hydrodynamic cavitation

The COVID −19 pandemic reminded us that we need better contingency plans to prevent the spread of infectious agents and the occurrence of epidemics or pandemics. Although the transmissibility of SARS-CoV-2 in water has not been confirmed, there are studies that have reported on the presence of infectious coronaviruses in water and wastewater samples. Since standard water treatments are not designed to eliminate viruses, it is of utmost importance to explore advanced treatment processes that can improve water treatment and help inactivate viruses when needed. This is the first study to investigate the effects of hydrodynamic cavitation on the inactivation of bacteriophage phi6, an enveloped virus used as a SARS-CoV-2 surrogate in many studies. In two series of experiments with increasing and constant sample temperature, virus reduction of up to 6.3 logs was achieved. Inactivation of phi6 at temperatures of 10 and 20 °C occurs predominantly by the mechanical effect of cavitation and results in a reduction of up to 4.5 logs. At 30 °C, the reduction increases to up to 6 logs, where the temperature-induced increased susceptibility of the viral lipid envelope makes the virus more prone to inactivation. Furthermore, the control experiments without cavitation showed that the increased temperature alone is not sufficient to cause inactivation, but that additional mechanical stress is still required. The RNA degradation results confirmed that virus inactivation was due to the disrupted lipid bilayer and not to RNA damage. Hydrodynamic cavitation, therefore, has the potential to inactivate current and potentially emerging enveloped pathogenic viruses in water at lower, environmentally relevant temperatures.

Cyclic lipopeptides as membrane fusion inhibitors against SARS-CoV-2: New tricks for old dogs

With the resurgence of the coronavirus pandemic, the repositioning of FDA-approved drugs against coronovirus and finding alternative strategies for antiviral therapy are both important. We previously identified the viral lipid envelope as a potential target for the prevention and treatment of SARS-CoV-2 infection with plant alkaloids (Shekunov et al., 2021). Here, we investigated the effects of eleven cyclic lipopeptides (CLPs), including well-known antifungal and antibacterial compounds, on the liposome fusion triggered by calcium, polyethylene glycol 8000, and a fragment of SARS-CoV-2 fusion peptide (816–827) by calcein release assays. Differential scanning microcalorimetry of the gel-to-liquid-crystalline and lamellar-to-inverted hexagonal phase transitions and confocal fluorescence microscopy demonstrated the relation of the fusion inhibitory effects of CLPs to alterations in lipid packing, membrane curvature stress and domain organization. The antiviral effects of CLPs were evaluated in an in vitro Vero-based cell model, and aculeacin A, anidulafugin, iturin A, and mycosubtilin attenuated the cytopathogenicity of SARS-CoV-2 without specific toxicity.

Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin.

Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.

Antiviral Activity of Syzygium polyanthum Extract against Herpes Simplex Virus-Type 1 (HSV-1)

Herpes simplex virus type 1 (HSV-1) is part of the alpha subfamily of human herpesviruses. Several antiviral medications can be prescribed for treating HSV-1 which is usually first line antiviral agents such as acyclovir (ACV). But there are several cases that have been reported on ACV resistance to HSV, thus, researchers tend to develop the antiviral agent from plants extract. Syzygium polyanthum is one of the plants that has been traditionally used as a drug which is rich in bioactive compounds, including essential oils, terpenoids, tannins, and flavonoids [1]. These phytochemical compounds, which have strong antioxidant action, can aid in the inhibition of viral genome replication which disables the viral lipid envelope [2].
 
 The objective of this study is to determine the cytotoxic concentration (CC50) and antiviral activity of S. polyanthum methanolic extract. Cytotoxicity was performed using the method described by [3]. The absorbance readings were analysed using microplate reader (Infinite M200 Pro). The 50% cytotoxicity concentration (CC50) was defined as the sample concentration that able to reduce 50% of cell viability compared to the untreated cells. Antiviral activity was performed by using plaque reduction assay with three different treatments [3] which are post- treatment, pre-treatment and virucidal assay.
 
 Based on Figure 1, cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for extract was 0.135 mg/mL which considered not in the range of cytotoxic compounds. Any CC50 or IC50 of a substance less than 4 µg/mL was considered an active cytotoxic effect [4].
 Based on Figure 2, post treatment assay showed the most anti-HSV-1 activity of S. polyanthum extract which the EC50 and SI value were 0.015mg/mL and 9.03, respectively. The extract showed to be slightly effective in reducing HSV-1 replication after 2-hour incubation which might be due to the inhibition in the early phase of the attachment. For pre-treatment, EC50 and SI values were 0.019 mg/mL and 7.12, which the extract showed to be moderately effective in inhibiting the attachment of HSV-1 before the replication can occur. This might occur as Vero cells bind to the extract that could interfere with the cell membrane's glycoprotein receptors which prevented HSV-1 from attaching to the cell surface [5]. The EC50 and SI value for virucidal assay was 0.018 mg/mL and 7.28 as the result showed moderately effective inhibited the viral replication. S. polyanthum has been shown to directly inactivate HSV-1 virions within 30 minutes of exposure in this study. An SI value greater than 10 (SI>10) indicates that any antimicrobial compound has the potency to become an antiviral treatment agent [6].
 In conclusion, the findings of this study demonstrate that S. polyanthum extract seems to have moderate antiviral effects and non-toxic to Vero cells.

Human Claudin-Derived Peptides Block the Membrane Fusion Process of Zika Virus and Are Broad Flavivirus Inhibitors.

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses.IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.

Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1.

Borna disease virus 1 (BoDV-1) is a non-segmented, negative-strand RNA virus that is characterized by persistent infection in the nucleus and low production of progeny virions. This feature impedes not only the harvesting of infectious viral particles from infected cells but also the rescue of high titres of recombinant BoDV-1 (rBoDV-1) by reverse genetics. Here, we demonstrate that exogenous expression of both matrix protein (M) and glycoprotein (G), which are constituents of the viral lipid envelope, significantly facilitates the formation of infectious particles and propagation of BoDV-1 without affecting its viral RNA synthesis. Furthermore, simultaneous transfection of M and G expression plasmids with N, P and L helper plasmids by reverse genetics drastically enhances the rescue efficiency of rBoDV-1. On the other hand, we also show that overexpression of M induces obvious cytotoxicity similar to that of other Mononegaviruses. Together with our recent report showing that excess expression of G induces aberrant accumulation of immature G, a potential stimulator of the host innate immune response, it is conceivable that BoDV-1 may suppress excess expression of M and G to reduce the cytopathic effect, thereby leading to maintenance of persistent infection. Our results contribute not only to the establishment of an efficient method to recover high-titre BoDV-1 but also to understanding the unique mechanism of persistent BoDV-1 infection.

Is Lipid Specificity Key to the Potential Antiviral Activity of Mouthwash Reagent Chlorhexidine against SARS-CoV-2?

Chlorhexidine (CHX), a popular antibacterial drug, is widely used for oral health. Emerging pieces of evidence suggest that commercially available chlorhexidine mouthwash formulations are effective in suppressing the spread of SARS-CoV-2, possibly through destabilization of the viral lipid envelope. CHX is known for its membrane-active properties; however, the molecular mechanism revealing how it damages the viral lipid envelope is yet to be understood. Here we used extensive conventional and umbrella sampling simulations to quantify the effects of CHX on model membranes mimicking the composition of the SARS-CoV-2 outer lipid membrane as well as the host plasma membrane. Our results show that the lipid composition and physical properties of the membrane play an important role in binding and insertion, with CHX binding favorably to the viral membrane over the plasma membrane. Among the simulated lipids, CHX preferentially binds to anionic lipids, PS and PI, which are more concentrated in the viral membrane. The deeper and stable binding of CHX to the viral membrane results in more pronounced swelling of the membrane laterally with a thinning of the bilayer. The overall free energies of pore formation are strongly reduced for the viral membrane compared to the plasma membrane; however, CHX has a larger concentration-dependent effect on free energies of pore formation in the plasma membrane than the viral membrane. The results indicate that CHX is less toxic to the human plasma membrane at low concentrations. Our simulations reveal that CHX facilitates pore formation by the combination of thinning the membrane and accumulation at the water defect. This study provides insights into the mechanism underlying the anti-SARS-CoV-2 potency of CHX, supporting its potential for application as an effective and safe oral rinse agent for preventing viral transmission.

Understanding the Lipid‐dependent function of SARS‐CoV‐2 E Protein

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the pathogen responsible for COVID‐19, a disease which has resulted in the death of millions worldwide. COVID‐19 ranges in severity of symptoms which are related to the inflammatory response. Although the field has progressed significantly, there is still a critical gap in understanding virus‐host lipid interactions that contribute to viral assembly and budding of this lipid‐enveloped virus. There are four structural proteins encoded in the SARS‐CoV‐2 genome: membrane, envelope (E), nucleocapsid, and spike. These proteins give shape to the bilayer lipid coat that encapsulate the genomic core necessary for virus infection and replication. These proteins are essential for viral reproduction but how they interact with each other and how lipid species regulate their assembly is not well understood. It is established that inflammation is common during infection. One enzyme that participates in this process is ceramide kinase (CERK), which synthesizes the pro‐inflammatory lipid, ceramide‐1‐phosphate (C1P). CERK is in fact a therapeutic target for a variety of inflammatory disorders. My primary goal is to define the relationship between CERK activity and SARS‐CoV‐2 assembly. We aim to understand the impact that CERK activity has on the localization and assembly of SARS‐CoV‐2 structural proteins. I am interested in how CERK regulates E protein localization and formation of virus‐like particles and the process by which E‐lipid interactions contributes to membrane curvature changes necessary for formation of new viral particles. Overall, I anticipate that this study will help define the relationships between a host enzyme and host membranes and the assembly of SARS‐CoV‐2.

Oxysterols can act antiviral through modification of lipid membrane properties – The Langmuir monolayer study

In this paper we tested how oxysterols influence on fusion process between viral lipid envelope and host cells membranes. For this purpose, the Zika virus was selected, while dendritic cell (DC) and neural cell (NC) membranes were chosen as target membranes. The investigated systems were modeled as multicomponent Langmuir monolayers and characterized using surface manometry and imaging in micro- (Brewster angle microscopy, BAM) and nanoscale (Atomic Force Microscopy, AFM) to monitor local heterogeneity. The fusion process was conducted by mixing viral and host cell membranes devoid and in the presence of oxysterols: 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) as representatives of chain- and ring-oxidized oxysterols, respectively. Our results show that oxysterols hinder the fusion with host cell membranes by modifying their biophysical properties. Moreover, oxysterols applied to an already infected membrane reverse the changes caused by the infection. It could therefore be concluded that oxysterols may display antiviral activity in two ways: they prevent the healthy membrane from viral infection by blocking the fusion process; and protect already infected membrane from pathological changes induced by the virus.

Ratiometric imaging of viral lipid order utilizing a solvatochromic fluorescent probe

The complex interplay between lipid phase structure of the host cells and nanoscale dynamics at the viral lipid envelope is considered critical for the pathogen’s ability to interact and fuse with the host-cell membrane. Despite decades of research, details about the exact role played by viral lipids in regulating its’ infectivity remain unknown. In particular, a clear mechanistic picture of viral lipid phase structure, and its impact on viral fusion is still lacking. This is partially owing to a dearth of techniques that reveal viral lipid organization in a holistic fashion.

Influenza HA C-terminal tail promotes envelope phosphatidylinositol phosphates (PIPs) of influenza virus and VLP thereof

Influenza virus' spike protein is hemagglutinin (HA), a trans-membrane trimeric glycoprotein mediating receptor-binding and membrane fusion. HA features a highly conserved, 12 residue cytoplasmic tail (CT) inside the viral lipid envelope. HA clusters with restricted mobility at the plasma membrane (PM) when expressed in cultured cells, a cluster that co-localizes with PIP2 (2019 Biophysical J. 116:893). Here, we engineered viral-like particles (VLP) with an HA CT deletion mutant to test PIP2 trafficking to the VLP envelope.

Influenza A virus M1 protein induces membrane rearrangements during viral entry stage

Influenza A virus is an enveloped RNA virus, which could cause seasonal epidemics and global pandemics. It infects the cell through endocytosis. A major step in infection is the fusion of the viral and host cell membranes, catalyzed by hemagglutinin (HA). It is believed that acidification of the endosome results in activation of HA followed by fusion pore formation. However, release of the viral ribonucleoprotein (RNP) into the cytoplasm requires disintegration of the M1 matrix protein scaffold located beneath the viral lipid envelope.

Alcohol-Based Sanitizers: An Effective Means for Preventing the Spread of Contagious Viral Diseases Including COVID-19

Abstract The global community is struggling with the highly contagious COVID-19. Returning to \normal life" now poses risks, and the use of appropriate protective measures has become necessary to continue daily life and protect public health. The main protective measures to prevent transmission of COVID-19 are masks, soaps and disinfectants. Because coronavirus is a \lipid-enveloped virus", it is very sensitive to lipid-dissolving chemicals and can therefore be effectively removed by washing hands sufficiently with soap and water. However, using an alcohol-based disinfectant is a more viable option for outdoor use. Alcohol-based disinfectants are inexpensive, immediately effective, easy to use and better tolerated by the skin compared to other disinfectants. WHO recommends disinfectants containing 75% isopropanol or 80% ethanol as highly effective in inactivating the SARS-CoV-2-virus. The current review discusses the role of alcohol-based hand sanitizers (ABHS) in preventing the spread of viruses, their side effects on human health, and suggests the use of alcohol-based sanitizers as potentially effective in combating the current epidemic.

Ethanol Inactivation of Enveloped Viruses: Structural and Surface Chemistry Insights into Phi6.

Lipid-enveloped viruses, such as Ebola, influenza, or coronaviruses, are a major threat to human health. Ethanol is an efficient disinfectant that is widely used to inactivate these viruses and prevent their transmission. However, the interactions between ethanol and enveloped viruses leading to their inactivation are not yet fully understood. This study demonstrates the link between ethanol-induced viral inactivation and the nanostructural and chemical transformations of the model virus Phi6, an 85 nm diameter lipid-enveloped bacterial virus that is commonly used as surrogate for human pathogenic viruses. The virus morphology was investigated using small-angle X-ray scattering and dynamic light scattering and was related to its infectivity. The Phi6's surface chemistry was characterized by cryogenic X-ray photoelectron spectroscopy, and the modifications in protein structure were assessed by circular dichroism and fluorescence spectroscopy. Ethanol-triggered structural modifications were found in the lipid envelope, detaching from the protein capsid and forming coexisting nanostructures.

Rapid Detection of Viral Envelope Lipids Using Lithium Adducts and AP-MALDI High-Resolution Mass Spectrometry.

There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry. AP-MALDI combined with an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone spiked with lithium salt, resulted in a robust and high-throughput lipid detection platform, specifically geared to sphingolipid detection. Application of the developed workflow included the structural characterization of prominent sphingolipids and detection of over 130 lipid structures from Influenza A virions. Overall, we demonstrate a high-throughput workflow for the detection and structural characterization of total lipid extracts from enveloped viruses using AP-MALDI HRMS and lithium adduct consolidation.

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.