RESUMO
BACKGROUND: Haemagglutinin-neuraminidase (HN) is one of the membrane proteins of Newcastle disease virus (NDV) that plays a significant role during host viral infection. Therefore, antibodies against HN are vital for the host's ability to protect itself against NDV infection due to their critical functions in viral infection. As a result, HN has been a candidate protein in vaccine development against the Newcastle disease virus. METHODS: This report used the full-length sequence of the HN protein of NDV isolated in Iran (VIId subgenotype). We characterize and identify amino acid substitutions in comparison to other more prevalent NDV genotypes, VII subgenotypes and vaccine strains. Furthermore, bioinformatics tools were applied to determine the three-dimensional structure, molecular dynamics simulation and prediction of B-cell antigenic epitopes. RESULTS: The results showed that the antigenic regions of our isolate are quite comparable to the other VII subgenotypes of NDV isolated from different geographical places. Moreover, by employing the final 3D structure of our HN protein, the amino acid residues are proposed as a B-cell epitope by epitope prediction servers, which leads to the introduction of linear and conformational antigenic sites. CONCLUSIONS: Immunoinformatic vaccine design principles currently exhibit tremendous potential for developing a new generation of candidate vaccines quickly and economically to eradicate infectious viruses, including the NDV. In order to accomplish this, focus is directed on residues that might be considered antigenic.
Assuntos
Genótipo , Proteína HN , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Proteína HN/genética , Proteína HN/química , Sequência de Aminoácidos , Animais , Irã (Geográfico) , Sequência de Bases , Galinhas , Doenças das Aves Domésticas/virologia , Doença de Newcastle/virologiaRESUMO
Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteína HN , Vírus da Parainfluenza 3 Humana , Infecções por Respirovirus , Sigmodontinae , Proteínas Virais de Fusão , Animais , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/genética , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Proteína HN/imunologia , Proteína HN/química , Proteína HN/genética , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Modelos Animais de Doenças , Testes de Neutralização , Linfócitos B/imunologia , Modelos MolecularesRESUMO
Paramyxoviruses-including important pathogens like parainfluenza, measles, and Nipah viruses-use a receptor binding protein [hemagglutinin-neuraminidase (HN) for parainfluenza] and a fusion protein (F), acting in a complex, to enter cells. We use cryo-electron tomography to visualize the fusion complex of human parainfluenza virus 3 (HN/F) on the surface of authentic clinical viruses at a subnanometer resolution sufficient to answer mechanistic questions. An HN loop inserts in a pocket on F, showing how the fusion complex remains in a ready but quiescent state until activation. The globular HN heads are rotated with respect to each other: one downward to contact F, and the other upward to grapple cellular receptors, demonstrating how HN/F performs distinct steps before F activation. This depiction of viral fusion illuminates potentially druggable targets for paramyxoviruses and sheds light on fusion processes that underpin wide-ranging biological processes but have not been visualized in situ or at the present resolution.
Assuntos
Infecções por Paramyxoviridae , Proteínas Virais de Fusão , Humanos , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteína HN/química , Proteína HN/metabolismo , Receptores de Superfície Celular , Internalização do VírusRESUMO
Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 µM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.
Assuntos
Ácido N-Acetilneuramínico , Infecções por Paramyxoviridae , Humanos , Animais , Chlorocebus aethiops , Ácido N-Acetilneuramínico/farmacologia , Vírus da Doença de Newcastle , Antivirais/química , Neuraminidase , Hemaglutininas , Células Vero , Proteína HN/genética , Proteína HN/químicaRESUMO
Mumps virus is an infectious pathogen causing major health problems for humans such as encephalitis, orchitis, and parotitis. Therefore, designing an inhibitor for this virus is of great medical and public health importance. With this goal in mind, we investigate the affinity of different sialic acid-based compounds (ligands) against the hemagglutinin-neuraminidase (HN) protein of the mumps virus, using a combination of molecular dynamics (MD) simulations and quantum chemistry calculations. Our MD simulation results indicate that the ligands form stable complexes with the HN protein through a combination of electrostatic, van der Waals (vdW), and hydrogen bond (H-bond) interactions, which the electrostatic interactions play a more important role in the complexation process. Based on the obtained results from the structural analysis Arg381, Arg291, and Arg49 play a key role in the binding site interactions with the different ligands, in comparison with other residues. There are some candidates such as Neu5Acα2-6Galß1-4GlcNAcß, Neu5Acα2-3Galß1-3GlcNacß1-3Galß1-4Glc, and Neu5Acα2-6Galß1-4GlcNAcß1-3Galß1-4Glc that form more stable complexes with the HN than the α2-3-Sialyllactose confirmed by the calculated Gibbs binding energies (-39.65, -46.93, and -36.49 kcal.mol-1, respectively). To investigate the relationship between the molecular properties of the selected compounds and their affinity to the HN receptor, density functional theory dispersion corrected (DFT-D3) calculations were employed. According to our DFT-D3 results, neutral sialic acid-based compounds have lower reactivity to the mumps virus than the negativity charge structures. Moreover, by increasing the electronic chemical potential (µ) the vdW and H-bond interactions between drugs and the HN protein increase. In other words, by elevating the electron tendency of the selected ligands their affinity to the mumps virus increases. Our quantum chemistry calculations reveal that in addition to the structural features the molecular properties of the drugs can play important roles in their affinity and reactivity against the virus. The results of this study can provide useful details to design new compounds or improve their properties against the mumps virus.
Assuntos
Vírus da Caxumba , Ácido N-Acetilneuramínico , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Simulação de Dinâmica Molecular , Proteína HN/química , Ligantes , Proteínas Virais/metabolismoRESUMO
As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.
Assuntos
Proteína HN , Vírus da Doença de Newcastle , Animais , Proteína HN/química , Neuraminidase/genética , Neuraminidase/metabolismo , Hemaglutininas , Galinhas , Genótipo , MutaçãoRESUMO
Human parainfluenza viruses (HPIVs) are ( -)ssRNA viruses belonging to Paramyoviridaie family. They are one of the leading causes of mortality in infants and young children and can cause ailments like croup, bronchitis, and pneumonia. Currently, no antiviral medications or vaccines are available to effectively treat parainfluenza. This necessitates the search for a novel and effective treatment. Computer-aided drug design (CADD) methodology can be utilized to discover target-based inhibitors with high accuracy in less time. A library of 45 phytocompounds with immunomodulatory properties was prepared. Thereafter, molecular docking studies were conducted to characterize the binding behavior of ligand in the binding pocket of HPIV3 HN protein. The physicochemical properties for screened compounds were computed, and the top hits from docking studies were further analyzed and validated using molecular dynamics simulation studies using the Desmond module of Schrodinger Suite 2021-1, followed by MM/GBSA analysis. The compounds CID:72276 (1) and CID:107905 (2) emerged as lead compounds of our in silico investigation. Further in vitro studies will be required to prove the efficacy of lead compounds as inhibitors and to determine the exact mechanism of their inhibition. Computational studies predict three natural flavonoids to inhibit the HN protein of HPIV3.
Assuntos
Catequina , Infecções por Paramyxoviridae , Catequina/farmacologia , Catequina/uso terapêutico , Criança , Pré-Escolar , Proteína HN/química , Proteína HN/genética , Proteína HN/metabolismo , Hemaglutininas/farmacologia , Hemaglutininas/uso terapêutico , Humanos , Ligantes , Simulação de Acoplamento Molecular , Neuraminidase , Vírus da Parainfluenza 1 Humana/metabolismo , Vírus da Parainfluenza 3 Humana/genética , Infecções por Paramyxoviridae/tratamento farmacológico , Proteínas ViraisRESUMO
BACKGROUND: Human parainfluenza virus 3 (HPIV3) is a major respiratory pathogen that causes acute respiratory infections in infants and children. Since September 2021, an out-of-season HPIV3 rebound has been noted in Korea. The objective of this study was to analyze the molecular characteristics of the HPIV3 strains responsible for the outbreak in Seoul, South Korea. METHODS: A total of 61 HPIV3-positive nasopharyngeal swab specimens were collected between October and November 2021. Using 33 HPIV3-positive specimens, partial nucleotide sequences of the HPIV3 hemagglutinin-neuraminidase (HN) gene were aligned with previously published HN gene sequences for phylogenetic and genetic distance (p-distance) analyses. RESULTS: Phylogenetic tree revealed that all Seoul HPIV3 strains grouped within the phylogenetic subcluster C3. However, these strains formed a unique cluster that branched separately from the C3a lineage. This cluster showed 99% bootstrap support with a p-distance < 0.001. Genetic distances within the other C3 lineages ranged from 0.013 (C3a) to 0.023 (C3c). Deduced amino acid sequences of the HN gene revealed four protein substitutions in Seoul HPIV3 strains that have rarely been observed in other reference strains: A22T, K31N, G387S, and E514K. CONCLUSIONS: Phylogenetic analysis of Seoul HPIV3 strains revealed that the strain belonged to a separate cluster within subcluster C3. Genetic distances among strains within subcluster C3 suggest the emergence of a new genetic lineage. The emergence of a new genetic lineage could pose a potential risk of a new epidemic. Further monitoring of the circulating HPIV3 strains is needed to understand the importance of newly discovered mutations.
Assuntos
COVID-19 , Infecções por Paramyxoviridae , Criança , Proteína HN/química , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Lactente , Pandemias , Vírus da Parainfluenza 3 Humana/genética , Filogenia , SeulRESUMO
Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked ~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Parainfluenza 1 Humana/efeitos dos fármacos , Células A549 , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antivirais/química , Sítios de Ligação , Cães , Proteína HN/química , Proteína HN/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Vírus da Parainfluenza 1 Humana/fisiologia , Ligação Proteica , Internalização do Vírus , Replicação ViralRESUMO
BACKGROUND: The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization. METHODS: In this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmids and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test. RESULTS: Five IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328-342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242-256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope. CONCLUSION: The HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242-256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Proteína HN/imunologia , Epitopos Imunodominantes/imunologia , Vírus da Doença de Newcastle/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Galinhas , Sequência Conservada , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Proteína HN/química , Proteína HN/genética , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Camundongos , Modelos Moleculares , Testes de Neutralização , Vírus da Doença de Newcastle/genética , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Porcine rubulavirus (PRV), which belongs to the family Paramyxoviridae, causes blue eye disease in pigs, characterized by encephalitis and reproductive failure in newborn and adult pigs, respectively. There is no effective treatment against PRV and no information on the effectiveness of the available vaccines. Continuous outbreaks have occurred in Mexico since the early 1980s, which have caused serious economic losses to pig producers. Vaccination can be used to control this disease. Searching for effective antigen candidates against PRV, we first sequenced the PAC1 F protein, then we used various immunoinformatics tools to predict antigenic determinants of B-cells and T-cells against the two glycoproteins of the virus (HN and F proteins). Finally, we used AutoDock Vina to determine the binding energies. We obtained the F gene sequence of a PRV strain collected in the early 1990s in Mexico and compared its amino acid profile with previous and more recent strains, obtaining an identity similarity of 97.78 to 99.26%. For the F proteins, seven linear B-cell epitopes, six conformational B-cell epitopes and twenty-nine T-cell MHC class I epitopes were predicted. For the HN proteins, sixteen linear B-cell epitopes, seven conformational B-cell epitopes and thirty-four T-cell MHC class I epitopes were predicted. The ATRSETDYY and AAYTTTTCF epitopes of the HN protein might be important for neutralizing the viral infection. We determined the in silico binding energy between the predicted epitopes on the F and HN proteins and swine MHC-I molecules. The binding energy of these epitopes ranged from -5.8 to -7.8 kcal/mol. The present study aimed to assess the use of HN and F proteins as antigens, either as recombinant proteins or as a series of peptides that could activate different responses of the immune system. This may help identify relevant immunogens, saving time and costs in the development of new vaccines or diagnostic tools.
Assuntos
Epitopos/química , Proteína HN/imunologia , Rubulavirus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Chlorocebus aethiops , Biologia Computacional/métodos , Epitopos/imunologia , Proteína HN/química , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Suínos , Células Vero , Proteínas Virais de Fusão/químicaRESUMO
Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions.
Assuntos
Proteína HN/química , Proteína HN/metabolismo , Vírus da Caxumba/fisiologia , Caxumba/virologia , Polissacarídeos/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Cristalografia por Raios X , Células HEK293 , Proteína HN/isolamento & purificação , Humanos , N-Acetilglucosaminiltransferases/deficiência , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Tropismo Viral , Internalização do VírusRESUMO
Human respirovirus 3 (HRV3) is a major causative agent of acute respiratory infections in humans. HRV3 can manifest as a recurrent infection, although exactly how is not known. In the present study, we conducted detailed molecular evolutionary analyses of the major antigen-coding hemagglutinin-neuraminidase (HN) gene of this virus detected/isolated in various countries. We performed analyses of time-scaled evolution, similarity, selective pressure, phylodynamics, and conformational epitope prediction by mapping to HN protein models. In this way, we estimated that a common ancestor of the HN gene of HRV3 and bovine respirovirus 3 diverged around 1815 and formed many lineages in the phylogenetic tree. The evolutionary rates of the HN gene were 1.1 × 10-3 substitutions/site/year, although the majority of these substitutions were synonymous. Some positive and many negative selection sites were predicted in the HN protein. Phylodynamic fluctuations of the gene were observed, and these were different in each lineage. Furthermore, most of the predicted B cell epitopes did not correspond to the neutralization-related mouse monoclonal antibody binding sites. The lack of a link between the conformational epitopes and neutralization sites may explain the naturally occurring HRV3 reinfection.
Assuntos
Evolução Molecular , Proteína HN/genética , Vírus da Parainfluenza 3 Humana/genética , Filogenia , Teorema de Bayes , Biologia Computacional , Mapeamento de Epitopos , Epitopos/genética , Proteína HN/química , Humanos , Cadeias de Markov , Método de Monte CarloRESUMO
The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed ß-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.
Assuntos
Proteína HN/química , Infecções por Paramyxoviridae/virologia , Paramyxoviridae/fisiologia , Proteínas Virais/química , Internalização do Vírus , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Ácido N-Acetilneuramínico/metabolismo , Paramyxoviridae/química , Paramyxoviridae/genética , Infecções por Paramyxoviridae/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ligação ViralRESUMO
Complexity in understanding allosteric stimulation of the hemagglutinin-neuraminidase (HN) protein of paramyxoviruses by host sialic acids (SIAs) stems from (1) unavailability of structure in its SIA-bound state and (2) the observation that this process is temperature sensitive. To consider simultaneously SIA's effect on structure and thermal fluctuations, we use molecular dynamics and simulate the dimeric form of the Newcastle disease virus HN. We find that SIA induces only minor structural changes in individual monomers, yet it reorients dimer interface by 10°. Interface reorientation is accompanied by constriction of SIA binding groove and enhanced fluctuations of interfacial residues that disrupt hydrophobic interactions and favor creation of new salt bridges. Supervised machine learning analysis of non-equilibrium data reveals that the allosteric signal is not formed from a directed sequence of these events. Altogether, we propose a detailed model of the initial events of allosteric stimulation, consistent with experiments on engineered mutations.
Assuntos
Proteína HN/química , Proteína HN/metabolismo , Vírus da Doença de Newcastle/metabolismo , Ácidos Siálicos/farmacologia , Regulação Alostérica , Sítios de Ligação , Cristalografia por Raios X , Proteína HN/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Vírus da Doença de Newcastle/química , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica , Aprendizado de Máquina SupervisionadoRESUMO
Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid as a receptor. However, given the MuV tropism toward glandular tissues and the central nervous system, an additional glycan motif(s) may also serve as a receptor. Here, we performed a large-scale glycan array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN proteins bound to newly identified glycan motifs, sialyl LewisX (SLeX) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLeX and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLeX and GM2-glycan have several unique interactions compared with those of 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLeX at the second and third carbohydrates from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLeX, or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The α2,3-sialylated oligosaccharides, such as SLeX and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism.IMPORTANCE Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl LewisX and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV.
Assuntos
Proteína HN/metabolismo , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Vírus da Caxumba/fisiologia , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Tropismo Viral , Ligação Viral , Cristalografia por Raios X , Proteína HN/química , Antígenos do Grupo Sanguíneo de Lewis/química , Análise em Microsséries , Ligação Proteica , Conformação Proteica , Ácidos Siálicos/químicaRESUMO
Human parainfluenza virus type 1 (hPIV1) has two spike glycoproteins, the hemagglutinin-neuraminidase (HN) glycoprotein as a receptor-binding protein and the fusion (F) glycoprotein as a membrane-fusion protein. The F glycoprotein mediates both membrane fusion between the virus and cell and membrane fusion between cells, called syncytium formation. Wild-type C35 strain (WT) of hPIV1 shows little syncytium formation of infected cells during virus growth. In the present study, we isolated a variant virus (Vr) from the WT that showed enhanced syncytium formation of infected cells by using our previously established hPIV1 plaque formation assay. Vr formed a larger focus and showed increased virus growth compared with WT. Sequence analysis of the spike glycoprotein genes showed that the Vr had a single amino acid substitution of Ile to Val at position 131 in the fusion peptide region of the F glycoprotein without any substitutions of the HN glycoprotein. The Vr F glycoprotein showed enhanced syncytium formation in F and HN glycoprotein-expressing cells. Additionally, expression of the Vr F glycoprotein increased the focus area of the WT-infected cells. The single amino acid substitution at position 131 in the F glycoprotein of hPIV1 gives hPIV1 abilities to enhance syncytium formation and increase cell-to-cell spread. The present study supports the possibility that hPIV1 acquires increased virus growth in vitro from promotion of direct cell-to-cell transmission by syncytium formation.
Assuntos
Vírus da Parainfluenza 1 Humana/fisiologia , Proteínas Virais de Fusão/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Gigantes , Proteína HN/química , Proteína HN/fisiologia , Humanos , Macaca mulatta , Valina/química , Proteínas Virais de Fusão/química , Replicação ViralRESUMO
Human parainfluenza virus (hPIV) infections are a major cause of respiratory tract illnesses in children, with currently no available vaccine or drug treatment. The surface glycoprotein haemagglutinin-neuraminidase (HN) of hPIV has a central role in the viral life cycle, including neuraminic acid-recognising receptor binding activity (early stage) and receptor-destroying activity (late stage), which makes it an ideal target for antiviral drug disovery. In this study, we showed that targeting the catalytic mechanism of hPIV-1 HN by a 2α,3ß-difluoro derivative of the known hPIV-1 inhibitor, BCX 2798, produced more potent inhibition of the neuraminidase function which is reflected by a stronger inhibition of viral replication. The difluorosialic acid-based inhibitor efficiently blocked the neuraminidase activity of HN for a prolonged period of time relative to its unsaturated neuraminic acid (Neu2en) analogue, BCX 2798 and produced a more efficient inhibition of the HN neuraminidase activity as well as in vitro viral replication. This prolonged inhibition of the hPIV-1 HN protein suggests covalent binding of the inhibitor to a key catalytic amino acid, making this compound a new lead for a novel class of more potent hPIV-1 mechanism-based inhibitors.
Assuntos
Inibidores Enzimáticos/química , Proteína HN/química , Vírus da Parainfluenza 1 Humana/enzimologia , Antivirais/química , Antivirais/farmacologia , Azidas/química , Azidas/farmacologia , Biocatálise , Inibidores Enzimáticos/farmacologia , Proteína HN/genética , Proteína HN/metabolismo , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Humanos , Vírus da Parainfluenza 1 Humana/efeitos dos fármacos , Vírus da Parainfluenza 1 Humana/genética , Infecções por Respirovirus/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Human parainfluenza viruses cause acute respiratory tract infections and disease predominantly in young children and immunocompromised individuals. Currently, there are no vaccines to prevent hPIV infections, nor licensed anti-hPIV drugs. There is therefore a need for specific antiviral therapies to decrease the morbidity and mortality associated with hPIV diseases. Haemagglutinin-neuraminidase (HN) is one of two hPIV surface proteins with critical roles in host receptor recognition, binding and cleavage; it has been explored as a key drug development target for the past few decades with variable success. Recent advancements in computational modelling and the availability of the X-ray crystal structure of hPIV3 HN have improved our understanding of the structural and mechanistic features of HN. This review explores structural features of the HN protein that are being exploited for structure-guided inhibitor design. We describe past and present hPIV HN inhibition strategies based on sialic acid scaffolds, together with other novel approaches that decrease hPIV infectivity. Although many HN inhibitors have been developed and evaluated as anti-hPIV agents, currently only a host-directed therapy (DAS181) has succeeded in phase II clinical drug trials. Hence, the review concludes with future considerations for targeting the specific function(s) of hPIV HN and suggestions for antiviral drug design.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteína HN , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidase/antagonistas & inibidores , Infecções por Paramyxoviridae/tratamento farmacológico , Antivirais/síntese química , Antivirais/farmacologia , Criança , Pré-Escolar , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Farmacorresistência Viral/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Genoma Viral , Proteína HN/química , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Hospedeiro Imunocomprometido , Ácido N-Acetilneuramínico/síntese química , Ácido N-Acetilneuramínico/farmacologia , Vírus da Parainfluenza 1 Humana/efeitos dos fármacos , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/genética , Infecções por Paramyxoviridae/patologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
The main objective of the study was to evaluate neuraminidase inhibiting (NI) antibodies against A/H1N1pdm09 influenza viruses in the community as a whole and after infection. We evaluated NI serum antibodies against A/California/07/09(H1N1)pdm and A/South Africa/3626/2013(H1N1)pdm in 134 blood donors of different ages using enzyme-linked lectin assay and in 15 paired sera from convalescents with laboratory confirmed influenza. The neuraminidase (NA) proteins of both A/H1N1pdm09 viruses had minimal genetic divergence, but demonstrated different enzymatic and antigenic properties. 5.2% of individuals had NI antibody titers ≥1:20 against A/South Africa/3626/2013(H1N1)pdm compared to 53% of those who were positive to A/California/07/2009(H1N1)pdm NA. 2% of individuals had detectable NI titers against A/South Africa/3626/13(H1N1)pdm and 47.3% were positive to A/California/07/2009(H1N1)pdm NA among participants negative to hemagglutinin (HA) of A/H1N1pdm09 but positive to seasonal A/H1N1. The lowest NI antibody levels to both A/H1N1pdm09 viruses were detected in individuals born between 1956 and 1968. Our data suggest that NI antibodies against A/South Africa/3626/13 (H1N1)pdm found in the blood donors could have resulted from direct infection with a new antigenic A/H1N1pdm09 variant rather than from cross-reaction as a result of contact with previously circulating seasonal A/H1N1 variants. The immune responses against HA and NA were formed simultaneously right after natural infection with A/H1N1pdm09. NI antibodies correlated with virus-neutralizing antibodies when acquired shortly after influenza infection. A group of middle-aged patients with the lowest level of anti-NA antibodies against A/California/07/2009 (H1N1)pdm was identified, indicating the highest-priority vaccination against A/H1N1pdm09 viruses.