M2 Protein Research Articles (Page 1)

The protective role of Astragaloside IV in adriamycin-induced renal injury: A focus on macrophage polarization and PPARγ activation.

Human metapneumovirus (HMPV) was first discovered in the Netherlands in 2001 and is now considered one of the most important contributors to viral respiratory diseases. It is often asymptomatic in healthy adults but can cause serious illness among immunocompromised or older patients. In response to the infection, the viral immune evasion mechanism remains a key approach for evading the immune response. In hMPV, the M2-2 protein interacts with the hMAVS protein to evade the immune response. It is essential to understand how the mechanism takes place for designing potential therapeutic agents. Thus, herein, we provide structural mechanisms of the interaction between M2-2 and MAVS through biomolecular interactions, in silico alanine scanning, and classical simulation approaches (repeated). We selected the HADDOCK-generated complex from the docking results, leaving the others from ZDOCK, Cluspro, and PyDOCK. Using alanine scanning, 18 interface residues were identified consensually, among which 8 residues, P29A, E30A, M31A, W33A, E37A, Q39A, E40A, and K48A, significantly affected the binding and were selected for the subsequent analysis. The docking results of these alanine mutants reported a significant reduction in the HADDOCK score, electrostatic energies, and vdW forces. Moreover, the stability of these mutations has been significantly compromised during simulation, while the total binding free energy also corroborates with the docking scores. From the detailed hydrogen-bond analysis, the interactions were significantly reduced in the mutants' complexes compared to the wild type, suggesting that alanine substitutions weaken the M2-1 and MAVS interaction by disrupting its finely tuned interaction network, highlighting potential vulnerabilities in its binding mechanism. The dissociation constant (Kd) results further validated discrepancies in the binding strength caused by the alanine substitutions. This study provides insights into the immune evasion mechanism of the hMPV virus and provides a basis for therapeutic development.

ObjectiveThe World Health Organization (WHO) grading based on histopathology cannot always accurately predict tumor behavior of meningiomas. To overcome the limitations of the WHO grading, the study aims to propose a novel oxidative stress-based molecular classification for WHO grade 2/3 meningiomas.MethodsDifferentially expressed oxidative stress-related genes were analyzed between 86 WHO grade 1 (low grade) meningiomas and 99 grade 2/3 (high grade) meningiomas. An oxidative stress-based molecular classification was developed in high-grade meningiomas through consensus clustering analysis. Immune microenvironment features, responses to immunotherapy and chemotherapy, and targeted drugs were evaluated. Three machine learning models: logistic regression, support vector machine, and random forest, were built for differentiating the classification. Key oxidative stress-related genes were verified in human meningeal cells (HMC) and two meningioma cells (CH-157MN and IOMM-Lee) via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. After knockdown of Forkhead Box M1 (FOXM1) or Prion Protein (PRNP), cell growth, migration, and reactive oxygen species (ROS) levels were measured through cell counting kit-8 (CCK-8), transwell, and immunofluorescence, respectively.ResultsWe classified high-grade meningiomas into two oxidative stress-based clusters, termed cluster 1 and cluster 2. Cluster 1 exhibited higher infiltrations of immune and stromal cells and higher expression of classic immune checkpoints: Cluster of Differentiation 86 (CD86), Programmed Cell Death 1 (PDCD1), and Leukocyte-Associated Immunoglobulin-Like Receptor 1 (LAIR1), indicating that cluster 1 meningiomas might respond to immunotherapy. Drug sensitivity was heterogeneous between the two clusters. Three classifiers were established, which could accurately differentiate this molecular classification. FOXM1 and PRNP were experimentally evidenced to be highly expressed in meningioma cells, and their knockdown hindered cell growth and migration and triggered ROS accumulation.ConclusionIn summary, our findings established a novel oxidative stress-based molecular classification and identified potential treatment vulnerabilities in high-grade meningiomas, which might assist personalized clinical management.

Background and aimsRespiratory syncytial virus (RSV) is a major respiratory pathogen afflicting both infants and the elderly. Although three RSV vaccines have been approved for adults over the age of 60 or pregnant individuals, there are ongoing efforts to develop novel vaccines against RSV infection. This study was designed to develop and evaluate virus-like particles (VLPs) as potential RSV subunit vaccine candidates, with the goal of balancing immunogenicity, protective efficacy, and safety.MethodsTwo types of VLPs were constructed using a recombinant baculovirus (rBV)-insect cell expression system: GECD-VLPs (containing the extracellular domain [GECD] of RSV G protein) and GECD/M282-90-VLPs (containing GECD fused with the CTL epitope M282-90 of M2 protein). BALB/c mice were vaccinated with these VLPs, and immune responses were assessed via RSV-specific IgG and neutralizing antibody titers, cytokine profiles (IFN-γ, IL-2, TNF-α, IL-10, IL-4, IL-5), and lung T-cell subsets (CD25+FoxP3+ Treg and Th17 cells). Protective efficacy against RSV infection and immunopathology was further evaluated post-challenge.ResultsVaccination with both VLPs induced robust RSV-specific IgG and neutralizing antibodies, conferring defense against RSV infection. Compared with the UV-RSV control group, both GECD/M282-90-VLPs and GECD-VLPs groups exhibited significantly increased Th1-type cytokine levels and decreased Th2-type cytokine concentrations (P<0.05, P<0.001). Importantly, compared to GECD-VLPs, GECD/M282-90-VLPs further significantly upregulated the expression of Th1-type cytokines (IFN-γ, IL-2) and regulatory cytokine IL-10, while significantly downregulating Th2-type cytokine IL-4 (all P<0.05). Post-RSV challenge, mice vaccinated with GECD/M282-90-VLPs exhibited a substantially increased proportion of CD25+FoxP3+ Treg cells and a decreased percentage of Th17 cells in the lungs. Notably, GECD/M282-90-VLP vaccination prevented RSV-induced immunopathology.DiscussionOur findings demonstrate that vaccination with GECD/M282-90-VLPs elicited a balanced immune response and conferred protection against RSV infection without immunopathology. These data demonstrate that the GECD/M282-90-VLPs are a potential RSV subunit vaccine candidate.

The M2 proton channel within the Influenza A virus constitutes an essential element for viral replication, with its functionality depending on the protonation states of four histidine residues located within the channel. This study meticulously investigates the impact of polarizability on the channel’s gating dynamics across various protonation states, employing both polarizable and non-polarizable models for water, protein, and membrane. Through a comprehensive analysis, we elucidate the nuanced role of polarizability in the channel’s operational mechanisms, differentiating between the solvent and protein polarizable effects. This investigation not only enriches our understanding of the M2 channel’s biophysical behavior but also highlights the significance of polarizability.

The aggregation of α-synuclein (αSyn) is a central feature of Parkinson’s disease (PD) and other synucleinopathies. The efficient clearance of αSyn depends largely on the autophagy–lysosomal pathway. Emerging genetic evidence highlights the role of pleckstrin homology and RUN domain-containing M1 protein (PLEKHM1), a critical regulator of autophagosome–lysosome fusion, in the pathogenesis of multiple neurodegenerative diseases. This study investigates the possible effects of increased PLEKHM1 expression on αSyn pathology and neurodegeneration in mice. We utilized a mouse model of PD that is based on A53T-αSyn overexpression, achieved by the stereotactic injection of recombinant adeno-associated viral vectors (rAAV) into the substantia nigra. Additionally, this study explores the effect of PLEKHM1 overexpression on the autophagy–lysosomal pathway under physiological conditions, using transgenic autophagy reporter mice. PLEKHM1 overexpression facilitated the αSyn-induced degeneration of dopaminergic somata in the substantia nigra and degeneration of dopaminergic axon terminals in the striatum. In concert with αSyn expression, PLEKHM1 also potentiated microglial activation. The extent of αSyn pathology, as reported by staining for phosphorylated αSyn, was not affected by PLEKHM1. Using RFP-EGFP-LC3 autophagy reporter mice, rAAV-mediated PLEKHM1 overexpression reduced lysosomal and autolysosomal area, increased LAMP1-LC3 colocalization, and decreased the autolysosome-to-autophagosome ratio. Concurrently, PLEKHM1 overexpression in both genotypes caused p62 accumulation, accompanied by reduced overlap with lysosomal and autophagosomal markers but increased colocalization with autolysosomal markers, indicating impaired cargo degradation during late-stage autophagy. Taken together, elevated PLEKHM1 levels exacerbate neurodegeneration in αSyn-overexpressing mice, possibly by impairing autophagic flux. Now, with in vivo evidence complementing genetic data, alterations in PLEKHM1 expression appear to compromise autophagy, potentially enhancing neuronal vulnerability to secondary insults like αSyn pathology.

To explore the cerebrospinal fluid (CSF) proteome in systemic lupus erythematosus (SLE) and the associations between the CSF proteomic patterns and clinical manifestations. CSF samples from 29 female outpatients with SLE were analyzed with label-free liquid chromatography tandem mass spectrometry. Inclusion and CSF collection were conducted irrespective of clinical manifestations and disease duration. Proteomic data were used for sample clustering and analyzed for clinical variance. Proteins were clustered using Weighted Gene Co-expression Correlation Network Analysis. Modules were biologically characterized and analyzed for correlation to the clinical dataset. Three patient clusters were identified. Cluster 1 was characterized by the highest frequency of nephritis, depression, and cognitive dysfunction. Cluster 2 showed the highest frequency of alopecia and Sjogren disease antigen A-antibodies (anti-SSA) and a low frequency of cognitive impairment. Cluster 3 had a higher frequency of autonomic neuropathy and headache. Six protein modules were identified (module 1 [M1]-M6). Modules were characterized by nervous tissue proteins (M1), central nervous system (CNS) lipoproteins (M2), macrophage proteins (M3), plasma proteins (M4), Ig (M5), and intracellular metabolic proteins (M6). M1 and M2 proteins were most abundant in cluster 1 and correlated with nephritis, depression, and cognitive impairment. Increased abundance of M4 and M5 proteins were most distinct in cluster 2 and inversely correlated to cognitive impairment and brain atrophy. Patients clustered by their CSF proteomic pattern had different disease phenotypes. Nephritis and neuronal damage defined the group with higher levels of neuronal proteins in CSF, which may suggest shared pathogenetic pathways in SLE affecting the kidney and CNS.

Post-translational modifications, SUMOylation, and Ubiquitination are critical in protein activities and half-life regulations in physiological and pathological processes, such as cancers, immune responses, and virus infections. SUMOylation requires an activating enzyme E1, conjugating enzyme E2, and E3 ligase to catalyze the attachment of SUMO peptide to substrates. Using synthetic biology techniques, we reconstituted the SUMOylation cascade in bacterial cells, aiding future research to be more efficient. We determine the activities of the SUMOylation enzymes expressed in the polycistronic SUMO construct using the quantitative FRET assay developed in our lab for CyPet-SUMO1 conjugation to substrate YPet fused-influenza A virus (IAV) M1 protein. In this project, we screened the CyPet protein expression of Polycistronic SUMO in 13 E.Coli strains to determine optimal bacteria for the assay. From the best performing strain, BL21 (DE3) PlysS, we performed double transformation of PolycistronicSUMO and IAV M1 constructs into bacterial cell culture for FRET-based SUMOylation conjugation assay. We also applied this system for SUMOylation inhibitor testing. In summary, using qFRET technology, we developed a novel synthetic biology approach for the SUMOylation cascade in bacterial cells. The synthetic biology approach for polycistronic SUMO cascade is a novel strategy to reconstitute the cascade reaction in bacterial cells.

The major bacterial pathogen Streptococcus pyogenes (Group A Streptococcus, or Strep A) recruits the negative regulator of the alternative complement pathway factor H (FH) to its surface. Antigenically sequence variable regions of several Strep A M proteins, including M5 and M6, bind FH but have no obvious sequence homology. A second Strep A surface-localized protein, FbaA, binds FH through a purported coiled-coil region, suggesting mimicry of the well-known coiled coil of M proteins. We determined the structures of fragments of M5 protein, M6 protein, and FbaA complexed with FH domains 6 and 7 (FH(6–7)). M5 and M6 proteins formed dimeric α-helical coiled coils, as expected, while FbaA instead consisted of a monomeric three-helix bundle preceded by a loop. FH(6–7) accommodated different binding modes in these three proteins, with very few common interacting amino acids. Based on contributions to binding, distinct FH-binding sequence patterns were constructed for M5 and M6 proteins, enabling identification of FH-binding sequences in M or M-like Enn proteins in 32 strains of differing M types. While FbaA was allelically sequence variable, its critical FH-binding amino acids were absolutely conserved in 95 strains of differing M types. Together, FH-binding sequences were identified in about half of the known 250 Strep A strains, with the majority due to FbaA. Our structural and functional elucidation of the mechanism of FH recruitment is applicable to precise investigation of its role in Strep A virulence.

Glioblastoma is the most common type of brain cancer, with a prognosis that is unfortunately poor. Despite considerable progress in the field, the intricate molecular basis of this cancer remains elusive. The aim of this study was to identify genetic indicators of glioblastoma and reveal the processes behind its development. The advent and integration of supercomputing technology have led to a significant advancement in gene expression analysis platforms. Microarray analysis has gained recognition for its pivotal role in oncology, crucial for the molecular categorization of tumors, diagnosis, prognosis, stratification of patients, forecasting tumor responses, and pinpointing new targets for drug discovery. Numerous databases dedicated to cancer research, including the Gene Expression Omnibus (GEO) database, have been established. Identifying differentially expressed genes (DEGs) and key genes deepens our understanding of the initiation of glioblastoma, potentially unveiling novel markers for diagnosis and prognosis, as well as targets for the treatment of glioblastoma. This research sought to discover genes implicated in the development and progression of glioblastoma by analyzing microarray datasets GSE13276, GSE14805, and GSE109857 from the GEO database. DEGs were identified, and a function enrichment analysis was performed. Additionally, a protein-protein interaction network (PPI) was constructed, followed by module analysis using the tools STRING and Cytoscape. The analysis yielded 88 DEGs, consisting of 66 upregulated and 22 downregulated genes. These genes' functions and pathways primarily involved microtubule activity, mitotic cytokinesis, cerebral cortex development, localization of proteins to the kinetochore, and the condensation of chromosomes during mitosis. A group of 27 pivotal genes was pinpointed, with biological process analysis indicating significant enrichment in activities, such as division of the nucleus during mitosis, cell division, maintaining cohesion between sister chromatids, segregation of sister chromatids during mitosis, and cytokinesis. The survival analysis indicated that certain genes, including PCNA clamp-associated factor (PCLAF), ribonucleoside-diphosphate reductase subunit M2 (RRM2), nucleolar and spindle-associated protein 1 (NUSAP1), and kinesin family member 23 (KIF23), could be instrumental in the development, invasion, or recurrence of glioblastoma. The identification of DEGs and key genes in this study advances our comprehension of the molecular pathways that contribute to the oncogenesis and progression of glioblastoma. This research provides valuable insights into potential diagnostic and therapeutic targets for glioblastoma.

Introduction. The rapid evolution of highly pathogenic avian influenza (HPAI) viruses through antigenic drift and reassortment can lead to enhanced replication efficiency and cross-species transmission to mammals, as evidenced by recent outbreaks in various animal populations. Identifying mammalian pathogenicity markers in circulating HPAI viruses is crucial for evaluating their pathogenic potential and ability to cross species barriers. The aim. This study analyzed genomic sequences of highly pathogenic H5 avian influenza virus (AIV) isolates collected in the Russian Federation between 2018 and 2022. Materials and methods. We utilized original complete genome sequencing data alongside with nucleotide sequences of H5 AIV isolates and strains available in public databases. Results. Analysis revealed a predominance of viruses with replication complexes adapted to avian cells. Examination of viral hemagglutinin amino acid sequences showed that most strains maintained receptor-binding sites of avian origin, with enhanced affinity for SAα-2,3-Gal receptors present in avian epithelial cells. However, we identified several mammalian virulence factors that have emerged and spread within the avian influenza virus population, including full-length active PB1-F2 protein, a 5-amino-acid insertion in the NS1 protein, and specific amino acid substitutions in the M1 protein. Conclusion. The presence of mammalian pathogenicity factors in the avian influenza virus population may facilitate successful cross-species transmission through suppression of specific immune responses, followed by adaptation of viral hemagglutinin to mammalian cell receptors through antigenic drift and natural selection. The observed elimination of certain adaptive mutations from the avian influenza virus population validates the effectiveness of stamping-out policies and vaccination restrictions in industrial poultry farming as important measures to mitigate the zoonotic potential of avian influenza.

The M1 protein of Group A Streptococcus (GAS) is a critical virulence factor contributing to a wide range of diseases, from common pharyngitis to life-threatening invasive infections and autoimmune sequelae. Its role in immune evasion, inflammation, and tissue invasion makes it an attractive target for drug and vaccine development. Addressing M1-mediated pathogenesis is crucial to reducing the impact of GAS infections, particularly in vulnerable populations like children. This research study proposes the dry lab approach that leverages computational methods to provide valuable pre-analysis insights to optimise the wet lab experiments. This approach aims to reduce the experimental costs and time by predicting key outcomes in silico, guiding researchers in selecting the most promising candidates for further validation in the wet lab. The affinity of drug-protein interaction is central to developing effective treatments against M1 protein-associated GAS infections. High-affinity drugs can inhibit M1 functions, reduce immune-mediated damage, and ensure specificity and safety. By targeting M1 with high precision, these drugs can significantly mitigate the global burden of GAS diseases. This research study shows that using neural networks to predict drug-protein affinity to know the pros and cons of the proposed method that can significantly accelerate and enhance the development of therapeutics targeting the M1 protein of Group A Streptococcus (GAS).

Glycosaminoglycans (GAGs) are enriched in the cutaneous extracellular matrix and have important roles in bacterial colonisation. Group A Streptococcus (GAS) can be categorised by emm patterning and M-family protein expression. M proteins of GAS are major adhesins with lectin-binding properties. This study aimed to provide a comprehensive specificity and affinity profile of phylogenetically diverse M proteins to a range of sulfated host GAGs and to investigate the physiological relevance of these interactions. Chondroitin sulfate preferentially associated with M proteins of A-C pattern strains, with binding localised to the central variable region of M1 protein. Dermatan sulfate was shown to associate with M proteins of all pattern type strains, with recognition involving multiple sites on M proteins. Heparin and heparan sulfate exclusively interacted with M proteins of A-C and D pattern strains. Multiple sites of M proteins were involved in heparin recognition, as indicated by surface plasmon resonance and site-directed mutagenesis of the heparin-binding XBXBX motif in the hypervariable-central region of M53 protein. In contrast, binding of heparan sulfate was localised to the non-repeat region between the B2 repeat and C1 repeat of M53 proteins. 5448 (M1-expressing GAS, A-C pattern) was shown to bind chondroitin sulfate, dermatan sulfate and heparin in an M protein-dependent manner. Furthermore, recruitment of chondroitin sulfate or dermatan sulfate by M1 proteins, but not heparin, was shown to increase GAS adherence to human HaCaT keratinocytes. This study increases our understanding of the molecular mechanisms underlying GAS adhesion, with key implications for bacterial colonisation and persistence of infection.

M1 protein is a major virulence determinant of group A Streptococcus (GAS). During infection, M1 is cleaved from the cell surface by host and bacterial proteases resulting in soluble M1 at the site of infection. M1 forms a supramolecular complex with host fibrinogen. We hypothesize that this supramolecular complex affects the formation of fibrin clots. Fibrin formation is an essential part of innate immunity, sealing off infections to limit bacteria spreading. The effects of recombinant M1 (rM1) were assessed in fibrin clots made from whole blood, plasma, or purified fibrinogen incubated in thrombin by a semiautomated coagulation analyzer, permeation studies, confocal microscopy, and scanning electron microscopy. Clotting and lysis profiles (with plasminogen activators and plasminogen) were investigated using microtiter plate assays and kinetically with rotational thromboelastography. Factor XIII crosslinking was quantified using commercial kits and sodium dodecyl sulfate-polyacrylamide gel electrophoresis densitometry analysis. This study demonstrated that rM1-bound (0.47-60 μg/mL) fibrinogen produced clots with remarkably different structures and properties compared with clots without rM1. Inclusion of rM1 formed heterogeneous clots with irregular fiber bundles and compacted fibrin. Formation of the protective fibrin film was disrupted by rM1. Furthermore, mechanical strength of fibrin clots was reduced, and the fibrin networks were more porous with increased fluid permeability. Fibrin clots formed using whole blood incorporating rM1 were more susceptible to lysis by plasmin. GAS strains of M1 type are often associated with invasive infections; the impact of M1 on fibrin structure could contribute to the severity of GAS infection by compromising the fibrin barrier that limits bacterial proliferation and migration.

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 106.5 EID50 showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated.

The influenza A virus (IAV) is an extremely contagious virus responsible for both seasonal flu and global pandemics. Cumin (Cuminum cyminum L., family Apiaceae) is a spice widely used in numerous Asian nations. The cumin fruit, commonly termed ‘cumin seed’, has been used in traditional medicine for the treatment of several ailments; however, its effect on IAV is not completely understood. This study investigated the effect of cumin fruit hot-water extract (CWE) on IAV infection. Madin-Darby canine kidney (MDCK) cells were infected with IAV (H1N1) and used for in vitro experiments. Pre-infection treatment of the target cells with CWE suppressed M1 protein expression in a dose-dependent manner, whereas post-infection treatment had no such effect. CWE at concentrations of 12.5 µg/mL or higher also inhibited IAV-induced haemagglutination and clathrin-dependent endocytosis. Even, a plaque formation assay was also conducted to confirm the efficacy of CWE on virus replication. The assay results showed that CWE significantly reduced IAV replication. However, the expression of type I interferon (IFN) and IFN-stimulated antiviral protein genes was not affected by CWE in the virus-infected cells. Furthermore, the presence of cuminaldehyde in CWE was examined using high-performance liquid chromatography. Cuminaldehyde was not detected in the CWE used in this study. Moreover, cells that were pre-treated with a cuminaldehyde standard did not show any inhibition of IAV infection. The current in vitro study showed that CWE inhibited IAV infection without harming host cells. Thus, CWE may be used to prevent IAV infections by limiting viral attachment and absorption.

Aromatic residues play important roles in protein structure and function, but are difficult to study at atomic resolution by NMR because of their low spectral sensitivity and resolution. The M2 proton channels of influenza A and B viruses use a histidine for proton selection and a tryptophan for gating. High-resolution structures and dynamics of His37 and Trp41 side chains in AM2 have provided detailed insights into the proton conduction mechanism of AM2. However, the side chain structures of the corresponding His19 and Trp23 in BM2 have not been established. Here, we directly determine the side chain conformations of His19 and Trp23 using 13C-15N and 13C-19F distance measurements. Interestingly, we find that His19 adopts a distribution of χ1 torsion angles: the major conformer places the imidazole ring in a tilted and partly transverse orientation from the channel axis, while a minor population orients the imidazole ring parallel to the channel axis, similar to His37 in AM2. Trp23 adopts χ1 and χ2 angles similar to those of Trp41 in AM2, but the indole ring orientation inside the pore differs moderately from that of Trp41 due to backbone conformational differences. Finally, a membrane-surface histidine in BM2, His27, is dynamic, consistent with its distinct function from His19. These results provide new insights into the structural basis for the different proton conduction behaviors of influenza AM2 and BM2 and illustrate how interactions among aromatic residues influence the structure and function of membrane proteins.

The influenza A M2 protein is an acid-activated proton channel and an established pharmaceutical target for antiflu drugs. Here, we studied the conductance domain of the tetrameric M2 channel (construct 18-60) using proton-detected solid-state NMR under native-like conditions in lipid bilayers. We obtained results at different pH values relevant to the virus life cycle: pH 7.8 (nonconducting, closed), pH 6.0 (opening), and pH 4.5 (conducting, fully open). In the closed state at pH 7.8, we detected two sets of resonances of the functionally important side chain of H37. Employing quantum mechanics/molecular mechanics (QM/MM) simulations, we assigned them to hydrogen-bonded and free H37 side chains occurring in varying ratios in the tetrameric arrangement. Additionally, some backbone signals also appear twice, suggesting conformational heterogeneity. The arrangement appears rather rigid, explaining the nonconducting nature of the channel. Lowering the pH to 6.0 leads to increased dynamics of the side chains, as manifested by their disappearance in CP based solid-state NMR spectra. This dynamic arrangement, which results from additional protonation of the four H37 side chains, allows for the efficient transport of protons through the channel. Finally, at pH 4.5, the conformational heterogeneity observed at higher pH values disappears completely, and a unique set of highly resolved resonances becomes visible. This suggests a well-defined acid-activated state of the M2 channel. Notably, in this state, the signals of the His37 side chains are absent due to dynamics, as well as the signals of the amphipathic helix (residues 45-52). This study provides strong evidence to a model of proton conduction through M2 which relies on dynamic vs rigid H37 side chains and furthermore lays the basis for an atomic structure of the acid-activated state of M2.

The eight-segmented RNA genome of influenza A virus (IAV) is transcribed and spliced into 10 major viral mRNAs in the nucleus of infected cells. Both transcription and splicing are facilitated by the host RNA polymerase II (Pol II) machinery via interactions between the viral ribonucleoprotein (vRNP) complex and various host factors. In this study, we demonstrate that IAV vRNPs recruit species-specific heterogeneous nuclear ribonucleoprotein M (hnRNPM) to support their replication in human and avian cells through distinct mechanisms. In A549 cells, human hnRNPM specifically facilitates the efficient transcription of HA, NA, M, and NS segments of WSN virus in a gene coding sequence-dependent manner. In contrast, in DF-1 cells, chicken hnRNPM restricts excessive splicing of M segment mRNA to ensure proper M2 protein production. Notably, human hnRNPM, with 34 additional amino acids compared with its chicken counterpart, fails to inhibit the M2 expression in DF-1 cells, whereas both human and chicken hnRNPM regulate WSN virus replication similarly in A549 cells. These findings highlight the host-specific roles of M2 levels in IAV replication and reveal how IAV co-opts host factors through virus genome sequence-dependent and host species-specific mechanisms, underscoring its high flexibility and adaptability during cross-species transmission.IMPORTANCEThe transcription and splicing of IAV genome in the nucleus of infected cells are precisely regulated to produce optimal amounts of viral proteins, ensuring efficient virus replication. In this study, we discovered that human hnRNPM regulates the IAV segment-specific differential transcription in a coding sequence-dependent manner in human cells. In contrast, chicken hnRNPM specifically inhibits M2 mRNA splicing to maintain proper M2 protein levels in avian cells. These species-specific regulatory mechanisms highlight the distinct replication strategies employed by IAV in human versus avian cells and underscore the complexity of cross-species transmission.

The influenza A virus (IAV) is a major cause of recurrent seasonal epidemics and global pandemics, posing a significant threat to public health. Although lycorine has demonstrated broad-spectrum antiviral activity, its specific mechanisms of action against IAV remain incompletely understood. In this study, we characterized the potent inhibitory effects of lycorine on seasonal and drug-resistant IAV subtypes (H1N1/H3N2) as well as the influenza B virus, showing its ability to suppress viral mRNA, viral titers, and M2 protein expression across multiple cell lines. Time-of-addition and time-course assays revealed that lycorine exerts multiphasic interference, and the critical late stage of the IAV life cycle aroused our interest to study this further. Mechanistically, we discovered that lycorine specifically interferes with the de novo synthesis of nucleoporin Nup93, thereby disrupting the nuclear export of viral nucleoprotein (NP). These findings not only establish lycorine as a promising broad-spectrum anti-influenza candidate but also provide new insights for developing host-targeted antiviral strategies.