Host Cell Membrane Research Articles

Otitiglycomycins A and B: Glycolipids from the Strain Nocardia otitidiscavarum 20S-13 with Antiviral Activity against Zika Virus.

The Zika virus (ZIKV), an emerging orthoflavivirus, presents a significant public health threat due to its rapid dissemination and association with severe neurological complications. The urgent need for effective antiviral agents has driven research into novel bioactive compounds derived from unique natural sources. Microorganisms inhabiting extreme environments are particularly promising for such discoveries due to their potential to produce unique metabolites. In this study, we explored microorganisms from the underexplored French Polynesian microbial mats known as "Kopara" to identify new bioactive natural products. Using a molecular networking-based dereplication strategy, we investigated various culture and extraction techniques of the strain Nocardia otitidiscaviarum 20-S13, leading to the discovery of two novel glycoglycerolipids, otitiglycomycins A and B (1 and 2). Structure elucidation of these compounds was achieved through NMR spectroscopy, X-ray crystallography, and TDDFT-specific rotation prediction. We found that otitiglycomycin A (1), but not otitiglycomycin B (2), suppresses ZIKV infection at non cytotoxic concentrations without effects on cell viability. Time-of-drug addition assays along with virus inactivation and binding assays demonstrated that 1 neutralizes ZIKV infectivity by preventing the virus from attaching to the host cell membrane.

HSV-1 virions and related particles: biogenesis and implications in the infection.

Virion formation and egress are sophisticated processes that rely on the spatial and temporal organization of host cell membranes and the manipulation of host machineries involved in protein sorting, membrane bending, fusion, and fission. These processes result in the formation of infectious virions, defective particles, and various vesicle-like structures. In herpes simplex virus 1 (HSV-1) infections, virions and capsid-less particles, known as light (L)-particles, are formed. HSV-1 infection also stimulates the release of particles that resemble extracellular vesicles (EVs). In productively infected cells, most EVs are generated through the CD63 tetraspanin biogenesis pathway and lack viral components. A smaller subset of EVs, generated through the endosomal sorting complexes required for transport (ESCRT) pathway, contains both viral and host factors. Viral mechanisms tightly regulate EV biogenesis, including the inhibition of autophagy-a process critical for increased production of CD63+ EVs during HSV-1 infection. Mutant viruses that fail to suppress autophagy instead promote microvesicle production from the plasma membrane. Additionally, the viral protein ICP0 (Infected Cell Protein 0) enhances EV biogenesis during HSV-1 infection. The different types of particles can be separated by density gradients due to their distinct biophysical properties. L-particles and ESCRT+ EVs display a pro-viral role, supporting viral replication, whereas CD63+ EVs exhibit antiviral effects. Overall, these studies highlight that HSV-1 infection yields numerous and diverse particles, with their type and composition shaped by the ability of the virus to evade host responses. These particles likely shape the infectious microenvironment and determine disease outcomes.

P-2023. A ‘Variant-Proof’ Cocktail Of Two Heavy Chain-Only Antibodies Targets 3 Epitopes In The Spike Protein S1 And S2 Subunits And Broadly Neutralizes SARS-Cov-2 With Exceptional Potency

Abstract Background More than 7 million people have died from COVID-19 to date and SARS-CoV-2 continues to cause substantial disease around the world. High-risk patients like immunocompromised and elderly are often not able to elicit adequate immune responses to vaccination and strongly benefit from an additional layer of protection in the form of complementary antibody therapy. Monoclonal antibodies act immediately and can provide immune support for months. However, all previously authorized antibodies lost their neutralization potency against the currently circulating and constantly mutating SARS-CoV-2 variants, leaving a vast unmet medical need. The combination XVR012 consists of the molecules XVR013m and XVR014. Methods The combination XVR012 consists of the molecules XVR013m and XVR014 (Figure 1). XVR013m is a mono-specific VHH-Fc antibody that targets a unique epitope in the S2 subunit, XVR014 is a bi-specific bivalent VHHx-Fc-VHHy antibody construct, targeting 2 non-competing epitopes in the Receptor-Binding Domain of the spike (Figure 2). Both antibodies contain a human IgG1 Fc with LS mutations for half-life extension. Structural analysis of the XVR013m epitope Left panel: Structural analysis of the VHH R3DC23 (green) bound to the HR2 domain of the S2 unit of the SARS-CoV-2 spike protein, between the terminal N1194 glycan (orange) and the viral membrane. Right panel: One R3DC23 (green) binds the interface of two HR2 coils and each HR2 coil is bound by two R3DC23 VHHs. Results XVR012 delivers a triple mode of action in preventing infection of host cells, i.e., (1) sterical hinderance of ACE2 receptor binding, (2) induction of S1 subunit shedding thereby preventing viral attachment and (3) inhibition of the fusion process between the viral and host cell membrane. XVR012 broadly neutralizes SARS-CoV-1 and SARS-CoV-2 viruses in vitro and demonstrates an IC50 ranging from 4.8 to 8,7 ng/mL against all SARS-CoV-2 variants tested so far in a pseudovirus neutralization assay, including the currently circulating BA.2.86.1, HK.3, EG.5.1 and HV.1 (Table 1). In vivo, efficacy was demonstrated in the Syrian Golden Hamster model in a therapeutic setting, showing complete reduction of lung viremia for all XVR012 dose levels tested, even with a very low dose of 0,5 mg/kg for the XVR013m component. (Figure 3). PK parameters have been determined in a Tg32 SCID mouse model and support an envisioned duration of protection up to 6 months. Neutralization potency of VSV pseudo-typed with spike proteins of recent SARS-CoV-2 variants. Data are presented as mean IC50 based on 3 independent experiments, except for (*) 1 independent experiment. Conclusion In summary, this second-generation cocktail targeting three unique and highly conserved epitopes in the S1 and S2 subunits of the spike is ready to proceed to clinical testing and may provide a long-term solution to the populations at highest risk. Lung infectious viral titers in Syrian golden hamster post-infection challenge model (Wuhan strain) on day 4 post infection. Mean values + standard error of the mean (SEM) are reported. * Two animals in 2 mg/kg group of XVR013m and one animal in the 20 mg/kg group of XVR014 were experimentally confirmed in PK assays to not have been exposed to the drug. Dotted lines represent the lower limit of detection (LLOD) range. Disclosures Florence M. Herschke, PhD, ExeVir Bio: Patent inventor|ExeVir Bio: Employee Bert Schepens, PhD, Exevir: Bert Schepens is Inventor on patents that describe the nanobodies that are mentioned in the abstract and presentation Loes van Schie, PhD, Exevir: Grant/Research Support|Exevir: I am a co-inventor on the patents protecting the biologicals described in the abstract and presentation Xavier Saelens, PhD, ExeVir Bio: I am an inventor on patent applications WO2022/167666 A1 and WO2023/222825 A1 which incorporate discoveries and inventions described here.|ExeVir Bio: I am a scientific co-founder of ExeVir Bio and in receipt of ExeVir Bio share options

Proteomics Reveals the Response Mechanism of Embryonic Bovine Lung Cells to Mycoplasma bovis Infection.

Mycoplasma bovis (M. bovis) has caused huge economic losses to the cattle industry. The interaction between M. bovis and host cells is elucidated by screening and identifying the target protein of M. bovis adhesin on the surface of the host cell membrane. However, the response mechanism of embryonic bovine lung (EBL) cells to M. bovis infection is not yet fully understood. Additionally, it is necessary to further explore whether infection with M. bovis induces oxidative stress and mitochondrial damage in EBL cells. In this study, oxidation reaction, mitochondrial membrane potential, mitochondrial structure, and apoptosis ability of EBL cells infected with M. bovis were assessed at different times (12, 24, 48 h post-infection; hpi). Then, the differential proteomic analysis of M. bovis-infected EBL cells at 12 h and 24 h was performed with uninfected cells as the control. The results showed that M. bovis infection reduced the antioxidant capacity of EBL cells, increased ROS levels, and decreased mitochondrial membrane potential. The mitochondrial membrane of EBL cells was damaged, and the ridge arrangement was disordered after infection by transmission electron microscopy. With the increase in infection time, the mitochondrial matrix partially dissolved and spilled. The apoptosis rate of EBL cells increased with the increase in infection time of M. bovis. Furthermore, proteomic analysis identified 268 and 2061 differentially expressed proteins (DEPs) at 12 hpi and 24 hpi, respectively, compared with the uninfected cells. According to GO analysis, these DEPs were involved in the mitosis and negative regulation of cell growth. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to mitochondrial damage or cell growth regulation, including glycolysis/gluconeogenesis, pentose phosphate pathway, oxidative phosphorylation, AMPK, cGMP-PKG, cAMP, calcium, Wnt, Phospholipase D, apoptosis, MAPK, cell cycle, Ras, PI3K-Akt, mTOR, HIF-1. PPI results indicated that YWHAZ, PIK3CA, HSP90AB1, RAP1A, TXN, RAF1, MAPK1, PKM, PGK1, and GAPDH might be involved in mitochondrial pathway apoptosis induced by M. bovis infection. This study offers helpful data toward understanding the response of mitochondria of EBL cells to M. bovis infection.

Structure of the Kaposi's sarcoma-associated herpesvirus gB in post-fusion conformation.

Discovered in 1994 in lesions of an AIDS patient, Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus subfamily of the Herpesviridae family, which contains a total of nine that infect humans. These viruses all contain a large envelope glycoprotein, glycoprotein B (gB), that is required for viral fusion with host cell membrane to initial infection. Although the atomic structures of five other human herpesviruses in their postfusion conformation and one in its prefusion conformation are known, the atomic structure of KSHV gB has not been reported. Here, we report the first structure of the KSHV gB ectodomain determined by single-particle cryogenic electron microscopy (cryoEM). Despite a similar global fold between herpesvirus gB, KSHV gB possesses local differences not shared by its relatives in other herpesviruses. The glycosylation sites of gB are arranged in belts down the symmetry axis with distinct localization compared to that of other herpesviruses, which occludes certain antibody binding sites. An extended glycan chain observed in domain I (DI), located proximal to the host membrane, may suggest its possible role in host cell attachment. Local flexibility of domain IV (DIV) governed by molecular hinges at its interdomain junctions identifies a means for enabling conformational change. A mutation in the domain III (DIII) central helix disrupts incorporation of gB into KSHV virions despite adoption of a canonical fold in vitro. Taken together, this study reveals mechanisms of structural variability of herpesvirus fusion protein gB and informs its folding and immunogenicity.IMPORTANCEIn 1994, a cancer-causing virus was discovered in lesions of AIDS patients, which was later named Kaposi's sarcoma-associated herpesvirus (KSHV). As the latest discovered human herpesvirus, KSHV has been classified into the gammaherpesvirus subfamily of the Herpesviridae. In this study, we have expressed KSHV gB and employed cryogenic electron microscopy (cryoEM) to determine its first structure. Importantly, our structure resolves some glycans beyond the first sugar moiety. These glycans are arranged in a pattern unique to KSHV, which impacts the antigenicity of KSHV gB. Our structure also reveals conformational flexibility caused by molecular hinges between domains that provide clues into the mechanism behind the drastic change between prefusion and postfusion states.

Kaposi's sarcoma-associated herpesvirus (KSHV) gB dictates a low-pH endocytotic entry pathway as revealed by a dual-fluorescent virus system and a rhesus monkey rhadinovirus expressing KSHV gB.

Interaction with host cell receptors initiates internalization of Kaposi's sarcoma-associated herpesvirus (KSHV) particles. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of glycoproteins H (gH), L (gL), and B (gB), that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed dual-fluorescent recombinant KSHV strains that incorporate fluorescent protein-tagged glycoproteins and capsid proteins. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. We demonstrated lytic reactivation and infectivity of dual-fluorescent KSHV. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. Using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established KSHV fusion kinetics upon infection of different target cells with marked differences in the "time-to-fusion" between cell types. Inhibition of vesicle acidification prevented KSHV particle-cell fusion, implicating low vesicle pH as a requirement. These findings were corroborated by comparison of RRV-YFP wildtype reporter virus and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB and classical fusion assays, these novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion.

Glycyrrhetinic acid prohibiting fusion peptides from fusing with cell membrane

The inhibition of the fusion peptide (FP) of the spike protein of coronaviruses, which mediates interactions with the host cell membrane, plays a critical role in the membrane fusion process. In this study, we investigated the interactions between the FP and cell membranes and evaluated the effect of three drug molecules — neferine (Nef), glycyrrhetinic acid (GA), quercetin (Qct) on the membrane fusion process by combining molecular dynamic (MD) simulations and experimental methods. Our findings revealed that glycyrrhetinic acid exhibited strong binding ability towards the FP (residues 815‐828), with particularly interactions observed with key residues L821, L822, F823, and L828. Furthermore, we performed FITC fluorescence staining experiments on cells and assessed the inhibitory effects of glycyrrhetinic acid on FP. The results showed a significant reduction in fluorescence intensity in the FP‐FITC experiment group treated with glycyrrhetinic acid, indicating that glycyrrhetinic acid interferes with the binding ability of FP and disrupts its localization on the cell membrane. Overall, this research will contribute to a deeper understanding of the inhibition mechanism of FP’s membrane fusion process, which widely exists in the intrusion process between viruses and cells.

The Bacterial Type III Secretion System as a Broadly Applied Protein Delivery Tool in Biological Sciences.

The type III secretion system (T3SS) is a nano-machine that allows Gram-negative bacteria to alter eukaryotic host biology by directly delivering effector proteins from the bacterial cytoplasm. Protein delivery based on the bacterial T3SS has been widely used in research in biology. This review explores recent advancements in the structure and function of the T3SS. We explore the molecular underpinnings of the T3SS apparatus, which spans bacterial and host cell membranes, and discuss the intricate transport mechanisms of effector proteins. Furthermore, this review emphasizes the innovative applications of the T3SS in crop biology, where it has been leveraged to study plant-pathogen interactions. By summarizing the current knowledge and recent progress, we underscore the potential of the T3SS as a powerful tool in biological sciences and their implications for future research in plant pathology and agricultural biotechnology.

Insights Into Phylogeny, Diversity and Functional Potential of Poseidoniales Viruses.

Viruses infecting archaea play significant ecological roles in marine ecosystems through host infection and lysis, yet they have remained an underexplored component of the virosphere. In this study, we recovered 451 archaeal viruses from a subtropical estuary, identifying 63 that are associated with the dominant marine order Poseidoniales (Marine Group II Archaea). Phylogenetic analyses of a subset of complete and nearly-complete viral genomes assigned these viruses to the order Magrovirales, a lineage of Poseidoniales viruses, and identified a novel group of viruses distinct from Magrovirales. Utilising demarcation criteria established for the classification of archaeal tailed viruses, we propose two families within the order Magrovirales: Apasviridae (magrovirus group A), comprising the genera Agnivirus and Savitrvirus, and Krittikaviridae (magrovirus group E) encompassing the genus Velanvirus. Additionally, we propose a new order, distinct from Magrovirales, named Adrikavirales, which includes the genus Vyasavirus. Our detailed genomic characterisation of the new viral lineages revealed genes involved in viral assembly and egress, such as those responsible for creating holin rafts to lyse host cell membranes, a feature predominantly known from bacteriophages. Furthermore, we identified a broad spectrum of auxiliary metabolic genes, suggesting that these viruses can modulate host metabolism. Collectively, our findings substantially enhance the current understanding of the diversity and functional potential of Poseidoniales viruses.

Structure, Attachment and Transmembrane Internalisation of Peste Des Petits Ruminants Virus

ABSTRACTPeste des petits ruminants virus (PPRV), a single‐stranded negative‐sense RNA virus with an envelope, belongs to the Morbillivirus in the Paramyxoviridae family and is prevalent worldwide. PPRV infection causes fever, stomatitis, diarrhoea, pneumonia, abortion and other symptoms in small ruminants, with a high mortality rate that poses a significant threat to the sustainability and productivity of the small ruminant livestock sector. The PPRV virus particles have a diameter of approximately 400–500 nm and are composed of six structural proteins: nucleocapsid protein (N), phosphoprotein (P), envelope matrix protein (M), fusion protein (F), haemagglutinin protein (H) and large protein (L). Each protein has a distinct role in the virus's life cycle. Although the life cycle activities of PPRV have been widely reported, they are still limited. Research has demonstrated that PPRV has distinct adhesion factors on various cell surfaces, such as the epithelial cell adhesion factor nectin‐4 or the lymphocyte adhesion factor SLAM. After attaching to the cell, the F and H proteins on the PPRV membrane interact with each other, resulting in a conformational change in the F protein. This change allows the F protein to enter the cell through direct fusion with the host cell membrane. The virus enters the host cell via the outer vesicle endocytosis strategy and replicates and proliferates through the role of caveolin, actin, dynein and cholesterol on the host cell membrane. This review summarises the viral structure, attachment mechanism and transmembrane internalisation mechanism of PPRV. The aim of this review is to provide theoretical support for the development of PPRV inhibitors and the prevention and control of PPR.

A multivalent binding model infers antibody Fc species from systems serology.

Systems serology aims to broadly profile the antigen binding, Fc biophysical features, immune receptor engagement, and effector functions of antibodies. This experimental approach excels at identifying antibody functional features that are relevant to a particular disease. However, a crucial limitation of this approach is its incomplete description of what structural features of the antibodies are responsible for the observed immune receptor engagement and effector functions. Knowing these antibody features is important for both understanding how effector responses are naturally controlled through antibody Fc structure and designing antibody therapies with specific effector profiles. Here, we address this limitation by modeling the molecular interactions occurring in these assays and using this model to infer quantities of specific antibody Fc species among the antibodies being profiled. We used several validation strategies to show that the model accurately infers antibody properties and then applied the model to infer previously unavailable antibody fucosylation information from existing systems serology data. Using this capability, we find that COVID-19 vaccine efficacy is associated with the induction of afucosylated spike protein-targeting IgG. Our results also question an existing assumption that controllers of HIV exhibit gp120-targeting IgG that are less fucosylated than those of progressors. Additionally, we confirm that afucosylated IgG is associated with membrane-associated antigens for COVID-19 and HIV, and present new evidence indicating that this relationship is specific to the host cell membrane. Finally, we use the model to identify redundant assay measurements and subsets of information-rich measurements from which Fc properties can be inferred. In total, our modeling approach provides a quantitative framework for the reasoning typically applied in these studies, improving the ability to draw mechanistic conclusions from these data.

Exploring the influence of anionic lipids in the host cell membrane on viral fusion.

Membrane fusion is an essential component of the viral lifecycle that allows the delivery of the genetic information of the virus into the host cell. Specialized viral glycoproteins exist on the surface of mature virions where they facilitate fusion through significant conformational changes, ultimately bringing opposing membranes into proximity until they eventually coalesce. This process can be positively influenced by a number of specific cellular factors such as pH, enzymatic cleavage, divalent ions, and the composition of the host cell membrane. In this review, we have summarized how anionic lipids have come to be involved in viral fusion and how the endosomal resident anionic lipid BMP has become increasingly implicated as an important cofactor for those viruses that fuse via the endocytic pathway.

Sequence of the SARS-CoV-2 Spike Transmembrane Domain Encodes Conformational Dynamics.

The homotrimeric SARS-CoV-2 spike protein enables viral infection by undergoing a large conformational transition, which facilitates the fusion of the viral envelope with the host cell membrane. The spike protein is anchored to the SARS-CoV-2 envelope by its transmembrane domain (TMD), composed of three TM helices, each contributed by one of the protomers of spike. Although the TMD is known to be important for viral fusion, whether it is a passive anchor of the spike or actively promotes fusion remains unknown. Specifically, it is unclear if the TMD and its dynamics facilitate the prefusion to postfusion conformational transition of the spike. Here, we computationally study the dynamics and self-assembly of the SARS-CoV-2 spike TMD in homogeneous POPC and cholesterol containing membranes. Atomistic simulations of a long TM helix-containing protomer segment show that the membrane-embedded segment bobs, tilts and gains and loses helicity, locally thinning the membrane. Coarse-grained multimerization simulations using representative TM helix structures from the atomistic simulations exhibit diverse trimer populations whose architecture depends on the structure of the TM helix protomer. While a symmetric conformation reflects the symmetry of the resting spike, an asymmetric TMD conformation could promote membrane fusion through the stabilization of a fusion intermediate. Together, our simulations demonstrate that the sequence and length of the SARS-CoV-2 spike TM segment make it inherently dynamic, that trimerization does not abrogate these dynamics and that the various observed TMD conformations may enable viral fusion.

An Advanced Sensing Approach to Biological Toxins with Localized Surface Plasmon Resonance Spectroscopy Based on Their Unique Protein Quaternary Structures.

Botulinum neurotoxins (BoNTs), ricin, and many other biological toxins are called AB toxins possessing heterogeneous A and B subunits. We propose herein a quick and safe sensing approach to AB toxins based on their unique quaternary structures. The proposed approach utilizes IgG antibodies against their A-subunits in combination with those human cell-membrane glycolipids that act as the natural ligands of B-subunits. In practice, an IgG antibody against the A-subunit of a target toxin is selected from commercially available sources and immobilized on the surface of Au nanoparticles to constitute a multivalent IgG/Au nanoconjugate. The derived IgG/Au conjugate is used in the pretreatment process of test samples for deactivating biological toxins in the form of a ternary toxin/antibody/Au complex. This process is implemented in advance to reduce the risk of handling biological toxins in laboratory work. On the other hand, the human glycolipid is immobilized on a tiny glass plate and used as a biosensor chip. The biosensor chip is set in the chamber of a flow sensing system using localized surface plasmon resonance (LSPR) spectrometry available in portable size at relatively low cost. In principle, the LSPR sensing system enables us to perform a rapid and selective detection for different kinds of biological toxins if the human glycolipid is correctly selected and installed in the sensing system. In the present LSPR sensing approach, a target AB toxin may have been deactivated during the pretreatment process. The test sample containing the deactivated AB toxin becomes a real target to be analyzed by the sensing system. In the present, we describe the concept of employing the commercially available IgG antibody in the pretreatment process followed by a typical procedure for converting it into the multivalent antibody/Au nanoconjugate and its preliminary applications in the LSPR detection of a ricin homologue (RCA120) and BoNTs in different serotypes. The tested LSPR sensing approach has worked very well for the ricin homologue and certain serotypes of botulinum neurotoxins like BoNT/A, indicating that the prior deactivation process at their A-domains causes no significant damage to the function of their B-domains with respect to determining the host cell-membrane glycolipid. The experimental results also indicated that LSPR responses from these pretreated AB toxins are significantly amplified. That is obviously thanks to the presence of Au nanoparticles in the multivalent IgG/Au nanoconjugate. We suggest in conclusion that the proposed LSPR sensing approach will provide us with a safe and useful tool for the study of biological AB toxins based on their unique quaternary protein structures.

Epsilon Toxin from Clostridium perfringens Induces the Generation of Extracellular Vesicles in HeLa Cells Overexpressing Myelin and Lymphocyte Protein.

Epsilon toxin (ETX) from Clostridium perfringens is a pore-forming toxin (PFT) that crosses the blood-brain barrier and binds to myelin structures. In in vitro assays, ETX causes oligodendrocyte impairment, subsequently leading to demyelination. In fact, ETX has been associated with triggering multiple sclerosis. Myelin and lymphocyte protein (MAL) is widely considered to be the receptor for ETX as its presence is crucial for the effects of ETX on the plasma membrane of host cells that involve pore formation, resulting in cell death. To overcome the pores formed by PFTs, some host cells produce extracellular vesicles (EVs) to reduce the amount of pores inserted into the plasma membrane. The formation of EVs has not been studied for ETX in host cells. Here, we generated a highly sensitive clone from HeLa cells overexpressing the MAL-GFP protein in the plasma membrane. We observed that ETX induces the formation of EVs. Moreover, the MAL protein and ETX oligomers are found in these EVs, which are a very useful tool to decipher and study the mode of action of ETX and characterize the mechanisms involved in the binding of ETX to its receptor.

Very broad distribution of β sheet registries of the HIV gp41 fusion peptide supports mutational robustness for fusion and infection

HIV, like other membrane-enveloped viruses, has protein spikes that include a fusion peptide (Fp) segment that binds the host cell membrane and plays a critical role in fusion (joining) viral and cell membranes. The HIV Fp is the ~23 N-terminal residues of the gp41 spike protein. Fp adopts intermolecular antiparallel β sheet structure when lipid fraction cholesterol ≈0.3, which is comparable to host cells. Rotational-echo double-resonance NMR was applied to probe the registries (alignments) of adjacent Fp molecules in membrane-bound sheets. The data were fitted to determine quantitative populations, f(t)'s, of individual antiparallel registries indexed by t, the number of residues in the registry. Both wild-type (WT) and fusion-defective V2E Fp sheets have broad but very different registry distributions, each with at least eight populated registries with f(t) > 0.02, and [Formula: see text] and ⟨t⟩V2E = 18.5. The broad WT distribution likely improves mutational robustness for HIV, as Fp is a neutralization epitope of the immune system, and Fp mutations are required for immune evasion during chronic HIV infection. V2E fusion is reduced because longer Fp sheets increase separation between initially apposed membranes. The f(t)WT were well-fitted to free energies that were sums of contributions from sheet length, aligned leucines, and sidechain membrane insertion. The f(t)V2E's were similarly well-fitted except there wasn't the insertion contribution. Relative to V2E, WT fusion is enhanced by deeper membrane insertion of Fp with accompanying greater dislocation of neighboring lipids. This study provides a rare quantitative determination of broad molecular structural distributions by experiment.

Research note: The critical role of the interaction between Eimeria tenella invasion protein RON2 and host receptor annexin A2 in mediating parasite invasion.

Avian coccidiosis, caused by protozoan parasites of the genus Eimeria, is a globally prevalent and highly pathogenic disease that poses a serious threat to the poultry industry, resulting in significant economic losses. However, the mechanism by which Eimeria species invade host cells remains unclear. Previous studies have identified rhoptry neck protein 2 (RON2) from Eimeria tenella as a critical factor in host cell invasion, but a comprehensive understanding of the role of EtRON2 in host cell invasion and its relationship with E. tenella invasion is lacking. To address this gap, this study focused on the secreted protein EtRON2 from E. tenella and its interaction with host cell receptors. The receptor interacting with the EtRON2 protein was identified through a GST pull-down assay, followed by mass spectrometry, and the interaction was further validated through subcellular co-localization analysis. Furthermore, sporozoites and host cells were treated with a specific antibody targeting the EtRON2/annexin A2 interaction, and the invasion rate of E. tenella was assessed using RT-qPCR to analyze the effect of inhibiting this interaction on host cell invasion by E. tenella. Annexin A2 protein, located on the surface of the host cell membrane, was screened, and the results of the sporozoite invasion assay revealed that inhibiting the interaction between these two proteins reduced F-actin aggregation. Understanding the interaction between the EtRON2 protein and its receptor during parasite invasion could help elucidate the function of the EtRON2 receptor and provide a theoretical foundation for further studies on the invasion mechanisms of E. tenella and the prevention and control of avian coccidiosis.

Characterization and Fluctuations of an Ivermectin Binding Site at the Lipid Raft Interface of the N-Terminal Domain (NTD) of the Spike Protein of SARS-CoV-2 Variants.

Most studies on the docking of ivermectin on the spike protein of SARS-CoV-2 concern the receptor binding domain (RBD) and, more precisely, the RBD interface recognized by the ACE2 receptor. The N-terminal domain (NTD), which controls the initial attachment of the virus to lipid raft gangliosides, has not received the attention it deserves. In this study, we combined molecular modeling and physicochemical approaches to analyze the mode of interaction of ivermectin with the interface of the NTD-facing lipid rafts of the host cell membrane. We characterize a binding area that presents point mutations and deletions in successive SARS-CoV-2 variants from the initial strain to omicron KP.3 circulating in many countries in 2024. We show that ivermectin has exceptional flexibility, allowing the drug to bind to the spike protein of all variants tested. The energy of interaction is specific to each variant, allowing a classification according to their affinity for ivermectin in the following ascending order: Omicron KP.3 < Delta < Omicron BA.5 < Alpha < Wuhan (B.1) < Omicron BA.1. The binding site of ivermectin is subject to important variations of the NTD, including the Y144 deletion. It overlaps with the ganglioside binding domain of the NTD, as demonstrated by docking and physicochemical studies. These results suggest a new mechanism of antiviral action for ivermectin based on competitive inhibition for initial virus attachment to lipid rafts. The current KP.3 variant is still recognized by ivermectin, although with an affinity slightly lower than the Wuhan strain.

Monkeypox virus A29L protein as the target for specific diagnosis and serological analysis

The unexpected monkeypox (Mpox) outbreak has been reported in many non-endemic countries and regions since May 2022. The mutant strains of Mpox virus (MPXV) were found with higher infectivity and greater capability for sustained human-to-human transmission, posing a significant public health threat. MPXV A29L, a protein homolog of vaccinia virus (VACV) A27L, plays an important role in viral attachment to host cell membranes. Therefore, MPXV A29L is considered the diagnostic target and the potential vaccine candidate for eliciting neutralizing antibodies and protective immune responses. In response to the escalating Mpox outbreak, three monoclonal antibodies (mAbs) (2-9B, 3-8G, and 2-5H) targeting the different domains of MPXV A29L have been developed in the study. Among them, 2-5H is highly specific for MPXV A29L without exhibiting cross-reactivity with VACV A27L. The antibody pairing composed of 2-5H and 3-8G has been developed as the lateral flow immunochromatographic assay for specific detection of MPXV A29L. However, these three mAbs were unable to inhibit A29L binding to heparin column or prevent MPXV infection in the neutralization test assays. The results of the serological assays using the truncated A29L fragments as the antigens showed that the Mpox patient sera contained significantly lower levels of antibodies targeting the N-terminal 1–34 residues of A29L, suggesting that the N-terminal portion of A29L is less immunogenic upon natural infection.Key points• MAbs 2-9B, 3-8G, and 2-5H neither interrupted A29L binding to heparin nor neutralized MPXV.• The LFIA composed of 3-8G and 2-5H can specifically distinguish MPXV A29L from VACV A27L.• Mpox patient sera contained lower levels of antibodies targeting the N-terminal portion of A29L.

Antimicrobial Peptides: A Promising Alternative to Conventional Antimicrobials for Combating Polymicrobial Biofilms.

Polymicrobial biofilms adhere to surfaces and enhance pathogen resistance to conventional treatments, significantly contributing to chronic infections in the respiratory tract, oral cavity, chronic wounds, and on medical devices. This review examines antimicrobial peptides (AMPs) as a promising alternative to traditional antibiotics for treating biofilm-associated infections. AMPs, which can be produced as part of the innate immune response or synthesized therapeutically, have broad-spectrum antimicrobial activity, often disrupting microbial cell membranes and causing cell death. Many specifically target negatively charged bacterial membranes, unlike host cell membranes. Research shows AMPs effectively inhibit and disrupt polymicrobial biofilms and can enhance conventional antibiotics' efficacy. Preclinical and clinical research is advancing, with animal studies and clinical trials showing promise against multidrug-resistant bacteria and fungi. Numerous patents indicate increasing interest in AMPs. However, challenges such as peptide stability, potential cytotoxicity, and high production costs must be addressed. Ongoing research focuses on optimizing AMP structures, enhancing stability, and developing cost-effective production methods. In summary, AMPs offer a novel approach to combating biofilm-associated infections, with their unique mechanisms and synergistic potential with existing antibiotics positioning them as promising candidates for future treatments.