Virus Neuraminidase Research Articles

Glycosylation and Crowded Membrane Effects on Influenza Neuraminidase Stability and Dynamics.

All protein simulations are conducted with varying degrees of simplification, oftentimes with unknown ramifications about how these simplifications affect the interpretability of the results. In this work, we investigated how protein glycosylation and lateral crowding effects modulate an array of properties characterizing the stability and dynamics of influenza neuraminidase. We constructed three systems: (1) glycosylated neuraminidase in a whole virion (i.e., crowded membrane) environment, (2) glycosylated neuraminidase in its own lipid bilayer, and (3) unglycosylated neuraminidase in its own lipid bilayer. We saw that glycans tend to stabilize the protein structure and reduce its conformational flexibility while restricting the solvent movement. Conversely, a crowded membrane environment encouraged exploration of the free energy landscape and a large-scale conformational change, while making the protein structure more compact. Understanding these effects informs what factors one must consider in attempting to recapture the desired level of physical accuracy.

Quadrivalent neuraminidase RNA particle vaccine protects pigs against homologous and heterologous strains of swine influenza virus infection

Influenza A virus in swine (IAV-S) continues to cause significant negative impact to both sows and growing pigs. The viral hemagglutinin (HA) and neuraminidase (NA) genes continue to evolve with HA diversifying at a faster rate than NA. Depending on country, whole inactivated virus (WIV) commercial and autogenous vaccines, as well as veterinary prescription vaccines targeting HA, are currently available. The use of these vaccines is focused on reducing virus and clinical signs in sows and to provide HA-specific maternally derived antibodies (MDA) to their suckling pigs. The deficiency in this strategy is that HA-MDA does not persist long enough to protect pigs through their growing phase from infection, and HA-MDA can interfere with effective pig immunization. This study evaluated the immunogenicity and efficacy of an adjuvanted, quadrivalent RNA Particle vaccine (Sequivity NA), currently licensed as Sequivity® IAV-S NA. This vaccine was formulated based on four NA antigens representing the major NA clades of IAV subtypes H1N1, H1N2 and H3N2 circulating in swine herds in the United States. In a series of trials, pigs were vaccinated twice, at three days and three weeks of age (WOA), followed by challenge with either homologous or heterologous IAV strains at 8 or 15 WOA. The Sequivity NA vaccine induced robust serum NA inhibition (NI) antibody and protected against IAV-S strains with homologous and heterologous NA to that of the vaccine. The magnitude and duration of nasal shedding was reduced in vaccinated-pigs challenged with either homologous or heterologous virus within the same NA clade. This NA-based RNA Particle vaccine avoids the known impact of HA-MDA on pig vaccination and provides a new tool to successfully reduce IAV-induced disease in the pig population.

The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus.

The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

Microsecond dynamics of H10N7 influenza neuraminidase reveals the plasticity of loop regions and drug resistance due to the R292K mutation.

At the beginning of the last century, multiple pandemics caused by influenza (flu) viruses severely impacted public health. Despite the development of vaccinations and antiviral medications to prevent and control impending flu outbreaks, unforeseen novel strains and continuously evolving old strains continue to represent a serious threat to human life. Therefore, the recently identified H10N7, for which not much data is available for rational structure-based drug design, needs to be further explored. Here, we investigated the structural dynamics of neuraminidase N7 upon binding of inhibitors, and the drug resistance mechanisms against the oseltamivir (OTV) and laninamivir (LNV) antivirals due to the crucial R292K mutation on the N7 using the computational microscope, molecular dynamics (MD) simulations. In this study, each system underwent long 2 × 1 μs MD simulations to answer the conformational changes and drug resistance mechanisms. These long time-scale dynamics simulations and free energy landscapes demonstrated that the mutant systems showed a high degree of conformational variation compared to their wildtype (WT) counterparts, and the LNV-bound mutant exhibited an extended 150-loop conformation. Further, the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculation and MM/GBSA free energy decomposition were used to characterize the binding of OTV and LNV with WT, and R292K mutated N7, revealing the R292K mutation as drug-resistant, facilitated by a decline in binding interaction and a reduction in the dehydration penalty. Due to the broader binding pocket cavity of the smaller K292 mutant residue relative to the wildtype, the drug carboxylate to K292 hydrogen bonding was lost, and the area surrounding the K292 residue was more accessible to water molecules. This implies that drug resistance could be reduced by strengthening the hydrogen bond contacts between N7 inhibitors and altered N7, creating inhibitors that can form a hydrogen bond to the mutant K292, or preserving the closed cavity conformations.

Deep mutational scanning of influenza A virus neuraminidase facilitates the identification of drug resistance mutations in vivo.

NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA's mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

Pharmacoinformatics and Breed-Based De Novo Hybridization Studies to Develop New Neuraminidase Inhibitors as Potential Anti-Influenza Agents.

Influenza represents a profoundly transmissible viral ailment primarily afflicting the respiratory system. Neuraminidase inhibitors constitute a class of antiviral therapeutics employed in the management of influenza. These inhibitors impede the liberation of the viral neuraminidase protein, thereby impeding viral dissemination from the infected cell to host cells. As such, neuraminidase has emerged as a pivotal target for mitigating influenza and its associated complications. Here, we apply a de novo hybridization approach based on a breed-centric methodology to elucidate novel neuraminidase inhibitors. The breed technique amalgamates established ligand frameworks with the shared target, neuraminidase, resulting in innovative inhibitor constructs. Molecular docking analysis revealed that the seven synthesized breed molecules (designated Breeds 1-7) formed more robust complexes with the neuraminidase receptor than conventional clinical neuraminidase inhibitors such as zanamivir, oseltamivir, and peramivir. Pharmacokinetic evaluations of the seven breed molecules (Breeds 1-7) demonstrated favorable bioavailability and optimal permeability, all falling within the specified parameters for human application. Molecular dynamics simulations spanning 100 nanoseconds corroborated the stability of these breed molecules within the active site of neuraminidase, shedding light on their structural dynamics. Binding energy assessments, which were conducted through MM-PBSA analysis, substantiated the enduring complexes formed by the seven types of molecules and the neuraminidase receptor. Last, the investigation employed a reaction-based enumeration technique to ascertain the synthetic pathways for the synthesis of the seven breed molecules.

New insights into the neuraminidase-mediated hemagglutination activity of influenza A(H3N2) viruses

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48–86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

Structural diversity and biological activities of indole-diterpenoids from Penicillium janthinellum by co-culture with Paecilomyces formosus

Co-culturing the marine-derived fungi Penicillium janthinellium with Paecilomyces formosus led to the isolation of nine new indole-diterpenes, janthinellumines A–I (1–9), along with twelve known analogues (10–21). The chemical structures including their absolute configurations of them were assigned by the analysis of extensive spectroscopic data and calculated ECD and VCD methods. These indole-diterpenoids displayed extensive biological activities, including anti-influenza A virus, protein tyrosine phosphatase (PTP) inhibitory, and anti-Vibrio activities. Among them, the anti-influenza mechanism of compounds 1, 2, and 7 was further investigated using neuraminidase inhibitory assay, molecular docking, and reverse genetics methods, suggesting that 1, 2, and 7 could interact with Arg371 of the viral neuraminidase. The structure–activity relationship (SAR) of PTPs inhibitory activity for indole-diterpene derivatives (1, 2, 4, 5, 9–16, and 19–21) was also summarized.

Different Outcomes of Chicken Infection with UK-Origin H5N1-2020 and H5N8-2020 High-Pathogenicity Avian Influenza Viruses (Clade 2.3.4.4b).

Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) of the "goose/Guangdong" lineage have caused a series of European epizootics since 2014. During autumn/winter 2020-2021, several H5Nx subtypes were detected in the UK, with H5N8 being the dominant subtype in wild birds and poultry. Despite the greater subtype diversity (due to viral neuraminidase gene reassortment) reported in wild birds, only H5N8 and H5N1 subtypes caused clade 2.3.4.4 UK HPAIV poultry outbreaks during this period. The direct inoculation of layer chickens showed that H5N8-2020 was more infectious than H5N1-2020, which supported the European H5N8 dominance during that season. However, the mean death time was longer for H5N8-2020 (3.42 days) than for H5N1-2020 (2.17 days). Transmission from directly infected to naive in-contact chickens was inefficient for both subtypes. Histological lesions, the tissue dissemination of viral antigen, and nucleic acid were more extensive and abundant and accumulated more rapidly for H5N1-2020 compared with H5N8-2020. Although inefficient, H5N1-2020 transmission was faster, with its greater virulence indicating that this subtype posed a major concern, as subsequently shown during H5N1 dominance of the clade 2.3.4.4 epizootic since autumn 2021. An evaluation of these in vivo viral characteristics is key to understanding the continuing poultry threats posed by clade 2.3.4.4 H5Nx HPAIVs.

A mosaic influenza virus-like particles vaccine provides broad humoral and cellular immune responses against influenza A viruses

The development of a universal influenza vaccine to elicit broad immune responses is essential in reducing disease burden and pandemic impact. In this study, the mosaic vaccine design strategy and genetic algorithms were utilized to optimize the seasonal influenza A virus (H1N1, H3N2) hemagglutinin (HA) and neuraminidase (NA) antigens, which also contain most potential T-cell epitopes. These mosaic immunogens were then expressed as virus-like particles (VLPs) using the baculovirus expression system. The immunogenicity and protection effectiveness of the mosaic VLPs were compared to the commercial quadrivalent inactivated influenza vaccine (QIV) in the mice model. Strong cross-reactive antibody responses were observed in mice following two doses of vaccination with the mosaic VLPs, with HI titers higher than 40 in 15 of 16 tested strains as opposed to limited cross HI antibody levels with QIV vaccination. After a single vaccination, mice also show a stronger level of cross-reactive antibody responses than the QIV. The QIV vaccinations only elicited NI antibodies to a small number of vaccine strains, and not even strong NI antibodies to its corresponding vaccine components. In contrast, the mosaic VLPs caused robust NI antibodies to all tested seasonal influenza virus vaccine strains. Here, we demonstrated the mosaic vaccines induces stronger cross-reactive antibodies and robust more T-cell responses compared to the QIV. The mosaic VLPs also provided protection against challenges with ancestral influenza A viruses of both H1 and H3 subtypes. These findings indicated that the mosaic VLPs were a promising strategy for developing a broad influenza vaccine in future.

High-resolution neuraminidase inhibition profiling of Arnebia euchroma (Royle) I.M. Johnst. based on HR-MS and target isolation: An example study of anti-infectious constituents in traditional Chinese medicine

Ethnopharmacological relevanceTraditional Chinese Medicines (TCMs) are an important source to discover new anti-infectious drugs. Neuraminidases (NAs) not only play a key role on human health, but also are promising targets for anti-infectious drugs. Arnebia euchroma which is a widely used traditional Chinese medicine with the effect of cooling blood and detoxifying showed potential inhibitory activities on both bacterial NA and virus NA, suggesting that the material basis of A. euchroma deserves in-depth study. Aim of the studyTo investigate the anti-infectious constituents of A. euchroma based on NA inhibition. Material and methodsA HPLC-DAD system incorporated an auto-sampler was used for micro-fractionation. A nanoliter liquid handler and a high sensitive multimode plate reader system were used for high throughput NA inhibition screening. Thus a high-resolution NA inhibition profiling platform was constructed. The structures of potential active components in A. euchroma obtained by the high-resolution bioassay profiling were identified by DAD and MS in parallel. Then, a target and rapid isolation of NAIs from A. euchroma was achieved under the guidance of the spectrum-effect relationship obtained above. Finally, the isolated compounds were elucidated by extensive spectroscopic methods and their bioactivities were validated by in vitro assay and molecular docking. Results16 potential active ingredients in A. euchroma were isolated and identified, including a new mero-monoterpenoid. The in vitro bioassay results revealed that 12 out of the 16 isolated compounds showed potent inhibitory activities on bacterial NA (IC50s = 1–6 μM) and five of them exhibited potent anti-microbial activities on methicillin-resistant Staphylococccus aureus (MRSA) with MICs in the range of 0.5–4 μg/mL. Furthermore, some isolated compounds showed equal or even better inhibitory activities on oseltamivir resistant viral NA than oseltamivir sensitive NA. The mechanism study in silicon revealed that these natural compounds possessed absolutely different binding modes on the bacterial and viral NAs. ConclusionsOur study gave a clear spectrum-effect relationship of A. euchroma, providing a scientific evidence for future study of the multi-components synergistic effect of TCMs.

Worth the Weight: Sub-Pocket EXplorer (SubPEx), a Weighted Ensemble Method to Enhance Binding-Pocket Conformational Sampling.

Structure-based virtual screening (VS) is an effective method for identifying potential small-molecule ligands, but traditional VS approaches consider only a single binding-pocket conformation. Consequently, they struggle to identify ligands that bind to alternate conformations. Ensemble docking helps address this issue by incorporating multiple conformations into the docking process, but it depends on methods that can thoroughly explore pocket flexibility. We here introduce Sub-Pocket EXplorer (SubPEx), an approach that uses weighted ensemble (WE) path sampling to accelerate binding-pocket sampling. As proof of principle, we apply SubPEx to three proteins relevant to drug discovery: heat shock protein 90, influenza neuraminidase, and yeast hexokinase 2. SubPEx is available free of charge without registration under the terms of the open-source MIT license: http://durrantlab.com/subpex/.

Novel mono- and multivalent N-acetylneuraminic acid glycoclusters as potential broad-spectrum entry inhibitors for influenza and coronavirus infection

N-acetylneuraminic acid (Neu5Ac) is a glycan receptor of viruses spread in many eukaryotic cells. The present work aimed to design, synthesis and biological evaluation of a panel of Neu5Ac derivatives based on a cyclodextrin (CD) scaffold for targeting influenza and coronavirus membrane proteins. The multivalent Neu5Ac glycoclusters efficiently inhibited chicken erythrocyte agglutination induced by intact influenza virus in a Neu5Ac density–dependent fashion. Compared with inhibition by Neu5Ac, the multivalent inhibitor with 21 Neu5Ac residues on the primary face of the β-CD scaffold afforded 1788-fold higher binding affinity inhibition for influenza virus hemagglutinin with a dissociation constant (KD) of 3.87 × 10−7 M. It showed moderate binding affinity to influenza virus neuraminidase, but with only about one-thirtieth the potency of that with the HA protein. It also exhibited strong binding affinity to the spike protein of three human coronaviruses (severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2), with KD values in the low micromolar range, which is about 10-time weaker than that of HA. Therefore, these multivalent sialylated CD derivatives have possible therapeutic application as broad-spectrum antiviral entry inhibitors for many viruses by targeting the Neu5Ac of host cells.

Evaluation of berberine inhibitory effects on influenza neuraminidase enzyme: A molecular dynamics study

Evaluation of berberine inhibitory effects on influenza neuraminidase enzyme: A molecular dynamics study

Medicinal plants with antiviral effect: A review

Several phytochemicals exhibited high level of antiviral activity. Medicinal plant possessed antiviral activity via many mechanisms included inhibition of viral replication, inhibition of the assembly of intracellular infectious virus particles, inhibition of viral infectivity, inhibition of RNA polymerase, DNA polymerase, viral neuraminidase, protease, reverse transcriptase and viral protein expression and many other mechanisms. The current review discuss the medicinal plants with antiviral activity with their mechanisms of action.

Enzymatic evaluation of chromenone derivatives from Flemingia philippinensis as viral neuraminidase (H3N2) inhibitors

The chromenones (1–6) from F. philippinensis displayed significant inhibition against the viral neuraminidase of H3N2. All the target chromenones including the new compound 5 have a triketone skeleton consisting of 2-acetyl-1,3-cyclohexanedione, which provides a being stable structure as exocyclic keto and enol form itself. The most potent inhibitor was identified as chromenone 1 (IC50 = 4.6 µM), but a subtle change in the functionality markedly affected the inhibitory potency (IC50 = 4.6 ∼ 28.5 µM) and mode. In particular, chromenones (1 and 4) with the exocyclic enol form exhibited the competitive mode, whereas the keto forms (2, 3, 5, and 6) were noncompetitive. The competitive inhibitor, chromenone 1, was proved to interact in a reversible simple slow-binding mode, according to the respective parameters: Kiapp = 2.38 µM, k3 = 0.0013 µM−1min−1, and k4 = 0.0031 min−1. The binding interactions of the competitive (1 and 4) and noncompetitive (2 and 3) inhibitors at the catalytic and allosteric sites, respectively, were predicted by the molecular docking method.

Consequences of asymmetric mutational pressure for the dynamic of linear B-cell epitopes repertoire of influenza a virus neuraminidase rearrangement

Full-length nucleotide sequences of avian influenza A virus neuraminidase coding region (20,631 sequences) were analyzed and compared with those isolated from viruses infecting human and swine (63,750 sequences). If in fourfold degenerate sites there is asymmetric A-bias that may be more or less asymmetric depending on the type of neuraminidase and the host, than in twofold degenerate sites from third codon positions there is a strong asymmetric U-bias in coding regions of N4, N5, and N8 isolated from viruses infecting birds, as well as in those of N1 and N2 isolated from viruses infecting human, swine, and birds, while in coding regions of N9 isolated from birds, there is surprisingly strong C-bias, and in sequences of N3, N6, and N7 the usage of C is quite close to the level of U. Revealed stabilization of both U and C in twofold degenerate sites is the evidence of frequent changes in mutational pressure direction. Asymmetric mutational pressure was one of the sources of amino acid replacements that resulted in an equal percentage of sites with appeared and disappeared linear B-cell epitopes in N1, N2, N4, and N5 (33.62–35.33% vs. 32.41–36.45%, respectively), and controlled by the immune pressure it resulted in a stronger tendency to disappear for B-cell epitopes of N3, N6, N7, N8, and N9 of avian viruses (8.74–28.77% vs. 28.96–38.89%). The lack of correlation between nucleotide usages in fourfold and twofold degenerate sites for three nucleotides, except U, is a strong evidence of mutational pressure theory.

Design, Synthesis, Inhibitory Activity, and SAR Studies of Hydrophobic Phenylacetic Acid Derivatives as Neuraminidase Inhibitors

Introduction: A series of hydrophobic phenylacetic acid derivatives introducing an aromatic lipophilic side chain at C-3 amino and an acetyl or isopropionyl at C-4 amino were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus. Methods: All compounds were synthesized in good yields starting from commercially available 2-(4- aminophenyl) acetic acid using a suitable synthetic strategy. Results: These compounds showed potent inhibitory activity against influenza A NA. Several compounds with alkylated amino group showed moderate NA subtype selectivity, among which compound (7i) displayed the best. Conclusion: Compound (7i) activity against H5N1 was more than 10 times better than H9N2, and could be used as lead compounds in the future.

An armed anti-immunoglobulin light chain nanobody protects mice against influenza A and B infections.

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.