Synthesis and Antiviral Activity of Camphene Derivatives against Different Types of Viruses

To date, the ‘one bug-one drug’ approach to antiviral drug development cannot effectively respond to the constant threat posed by an increasing diversity of viruses causing outbreaks of viral infections that turn out to be pathogenic for humans. Evidently, there is an urgent need for new strategies to develop efficient antiviral agents with broad-spectrum activities. In this paper, we identified camphene derivatives that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses, including influenza virus A/PR/8/34 (H1N1), Ebola virus (EBOV), and the Hantaan virus. The lead-compound 2a, with pyrrolidine cycle in its structure, displayed antiviral activity against influenza virus (IC₅₀ = 45.3 µM), Ebola pseudotype viruses (IC₅₀ = 0.12 µM), and authentic EBOV (IC₅₀ = 18.3 µM), as well as against pseudoviruses with Hantaan virus Gn-Gc glycoprotein (IC₅₀ = 9.1 µM). The results of antiviral activity studies using pseudotype viruses and molecular modeling suggest that surface proteins of the viruses required for the fusion process between viral and cellular membranes are the likely target of compound 2a. The key structural fragments responsible for efficient binding are the bicyclic natural framework and the nitrogen atom. These data encourage us to conduct further investigations using bicyclic monoterpenoids as a scaffold for the rational design of membrane-fusion targeting inhibitors.     

Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase

The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.     

液相色谱-质谱联用分析流感病毒感染引起宿主蛋白类泛素化修饰

干扰素刺激基因15编码蛋白质(Interferon stimulated gene 15 kDa protein, ISG15)是最早被鉴定的类泛素分子蛋白质,在病毒感染和免疫调节等方面具有重要作用。本研究利用免疫沉淀技术将被类泛素 ISG15修饰的蛋白富集纯化,采用液相色谱-质谱联用技术对流感病毒感染 A549宿主细胞过程中产生的类泛素 ISG15修饰蛋白进行了分析。实验结果表明,在流感病毒感染的实验组 A549细胞中,鉴定到了22种来源于宿主细胞的ISG15修饰的蛋白,包括类泛素蛋白 ISG15、细胞周期蛋白-T1、热休克蛋白71、钙调素结合蛋白、真核翻译起始因子等,以及1种来源于流感病毒的非结构蛋白 NS1。在鉴定的22种宿主蛋白中,有6种蛋白在未感染病毒的对照组 A549细胞中也得到鉴定,包括膜联蛋白 A1、果糖二磷酸醛缩酶 A、线粒体三磷酸腺苷合成酶亚基 g、烯醇化酶、肌动蛋白、微管蛋白。生物信息学分析表明,流感病毒感染引起的 ISG15修饰的宿主蛋白分别归属于9个不同的蛋白分类,包括细胞骨架蛋白、分子伴侣蛋白、酶调节剂、核酸结合蛋白、激酶类、转移酶类、转录因子、氧化还原酶类以及结构蛋白。本研究为大规模分析鉴定 ISG15修饰蛋白提供了一种特异、有效的研究方法。     

禽流感病毒HA蛋白与人受体的分子对接

血凝素(hemagglutinin,HA)是位于禽流感病毒表面的糖蛋白。在病毒感染过程中,HA与禽类宿主细胞表面受体结合,介导病毒膜与宿主核内体膜的融合,在传染过程中发挥关键作用。自然界中的禽流感病毒处于不断演化之中,其HA的禽受体结合位点常常发生氨基酸变异。因此,当HA变异体与人受体结合能力较强时,禽流感病毒往往会发生跨种传播而感染人。为预防禽流感的跨种传播,人们迫切需要发展大规模快速检测或预测HA变异体与人受体结合亲和力的方法,以评估各种新发禽流感病毒的跨种传播能力,提前筛选出有潜在危险的病毒株。针对此问题,本研究以H7N9亚型的HA蛋白H7为研究对象,发展了一种运用分子对接的计算方法,预测HA变异体与人受体的结合亲和力。该方法的计算结果表明,H7与人受体的结合亲和力普遍弱于有较强传染人能力的H1,说明H7N9亚型病毒的跨种传播能力普遍较弱;但是,计算分析也揭示,部分新发的H7N9毒株的HA有强的人受体结合亲和力,提示在自然演化过程中,H7N9病毒有可能演化出具有较强的感染人能力的新毒株,这与2013年禽流感疫情的实际发生情况相一致。因此,本文所发展的计算方法可用于快速预测新发禽流感病毒HA与人受体的结合亲和力,为新发禽流感病毒的跨种传播风险评估提供理论依据。     

Ultrastructural Damages to H1N1 Influenza Virus Caused by Vapor Essential Oils

Influenza viruses are transmitted from human to human via airborne droplets and can be transferred through contaminated environmental surfaces. Some works have demonstrated the efficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined the liquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viral cytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs and their vapor phase by the head-space gas chromatography–mass spectrometry technique, observing that the major component found in each liquid EO is the same one of the corresponding vapor phases, with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrity of virus particles was checked by negative staining transmission electron microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes. We speculated that the most abundant components of the vapor EOs might directly interfere with influenza virus envelope structures or mask viral structures important for early steps of viral infection.     

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand–receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC₅₀ values in low picomolar concentrations and also blocks the virus entry into MDCK‐II cells.     

Origins of the reassortant 2009 pandemic influenza virus through proteotyping with mass spectrometry

The application of a proteotyping approach employing high resolution mass spectrometry based is shown to be able to determine the gene origin of all major viral proteins in a triple reassortant pandemic 2009 influenza strain. Key to this approach is the identification of unique swine‐host‐specific signature and indicator peptides that are characteristic of influenza viruses circulating in North American and Eurasian swine herds in the years prior to the 2009 influenza pandemic. These swine‐and human pandemic‐specific signatures enable the origins of viral proteins in a clinical virus specimen to be determined and such strains to be rapidly and directly differentiated from other co‐circulating seasonal influenza viruses from the same period. The proteotyping strategy offers advantages over traditional RT‐PCR‐based approaches that are currently the mainstay of influenza surveillance at the molecular level. Copyright © 2014 John Wiley & Sons, Ltd.     

流感病毒糖蛋白糖链的作用和功能研究

流感病毒是近几年的研究热点之一.糖链在流感病毒生活周期中发挥重要作用,例如宿主细胞表面的唾液酸化糖链是病毒侵染细胞时的特异性受体,宿主决定的病毒糖蛋白糖链结构影响病毒的宿主范围和毒力.本文从糖组学角度综述糖链在甲型流感病毒生活周期中的重要作用,着重阐述病毒血凝素糖基化的影响因素及其对病毒宿主范围、毒力的影响和在病毒演化...     

Discovery of potential M2 channel inhibitors based on the amantadine scaffold via virtual screening and pharmacophore modeling

The M2 channel protein on the influenza A virus membrane has become the main target of the anti-flu drugs amantadine and rimantadine. The structure of the M2 channel proteins of the H3N2 (PDB code 2RLF) and 2009-H1N1 (Genbank accession number GQ385383) viruses may help researchers to solve the drug-resistant problem of these two adamantane-based drugs and develop more powerful new drugs against influenza A virus. In the present study, we searched for new M2 channel inhibitors through a combination of different computational methodologies, including virtual screening with docking and pharmacophore modeling. Virtual screening was performed to calculate the free energies of binding between receptor M2 channel proteins and 200 new designed ligands. After that, pharmacophore analysis was used to identify the important M2 protein-inhibitor interactions and common features of top binding compounds with M2 channel proteins. Finally, the two most potential compounds were determined as novel leads to inhibit M2 channel proteins in both H3N2 and 2009-H1N1 influenza A virus.     

Effect of the β-propiolactone treatment on the adsorption and fusion of influenza A/Brisbane/59/2007 and A/New Caledonia/20/1999 virus H1N1 on a dimyristoylphosphatidylcholine/ganglioside GM3 mixed phospholipids monolayer at the air-water interface

The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosis into the endosome. In the presence of the native virus the pH decrease caused a reduction in domain size, whereas lipid layer thickness and surface pressure were increased. These observations are consistent with a fusion of the viral membrane with the lipid monolayer. Importantly, this fusion was not observed with adsorbed inactivated virus, which indicates that BPL treatment inhibits the first step of virus-membrane fusion. Our data also indicate that BPL chemically modifies hemagglutinin, which mediates the interaction with GM3.     

Binding interaction analysis of the active site and its inhibitors for neuraminidase (N1 subtype) of human influenza virus by the integration of molecular docking, FMO calculation and 3D-QSAR CoMFA modeling

Recently, the worldwide spread of A/H5N1 avian influenza with high virulence has highlighted the potential threat of human influenza pandemic. Tamiflu and Relenza are currently the only two anti-influenza drugs targeting the neuraminidase (NA) enzyme of human influenza virus. Reports of the emergence of drug resistance further make the development of new potent anti-influenza inhibitors a priority. The X-ray crystallographic study of A/H5N1 avian influenza NA subtypes (Russell, R. J. Nature 2006, 443, 45-49) has demonstrated that there exist two genetically distinct groups, group-1 (N1, N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9), whose conformations are substantially different. The detailed comparison of their active sites has established, heretofore, the most accurate and solid molecular basis of structure and mechanism for the development of new anti-influenza drugs. In the present study, a three-dimensional structure of N1 subtype of human influenza type A virus (N1hA) has been generated by homology modeling using the X-ray crystallographic structure of N1 subtype of avian influenza virus (N1aA) as the template. Binding interaction analysis between the active site and its inhibitors has been performed by combining ab initio fragment molecular orbital (FMO) calculations and three-dimensional quantitative structure-activity relationship with comparative molecular field analysis (3D-QSAR CoMFA) modeling. Integrated with docking-based 3D-QSAR CoMFA modeling, molecular surface property (electrostatic and steric) mapping and FMO pair interaction analysis, a set of new receptor-ligand binding models and bioaffinity predictive models for rational design and virtual screening of more potent inhibitors of N1hA are established. In addition, the flexibility of the loop-150 of N1hA and N1aA has been examined by a series of molecular dynamics simulations.     

A two-dimensional electrophoresis map of Chinese hamster ovary cell proteins based on fluorescence staining

We report on the development of a detailed two-dimensional electrophoresis map of Chinese hamster ovary (CHO) cell proteins based on fluorescence staining and tandem time-of-flight (TOF/TOF)-mass spectrometry. We observed a 71% success rate in the identification of proteins even though the CHO genome is not sequenced. The map consists of 224 protein identifications present in 274 two-dimensional gel electrophoresis (2-DE) gel spots. We have also initiated a study of the phosphoproteome using a commercially available phosphoprotein-specific fluorescent stain. Using this stain, we observe 672 phosphorylated proteins, including many proteins known to be phosphorylated, which is 36% of the proteins we visualized with a total protein stain and consistent with expectations.     

Studying Spatial Distributions of Influenza Hemagglutinin on the Plasma Membrane of Fibroblasts: A Work in Progress

The major envelope protein of influenza virus, hemagglutinin (HA), mediates the fusion of virus to cell for infection, and can mediate cell-cell fusion. It has been studied as a “raft” protein, as it is found in detergent-resistant membranes (DRM) and trafficks apically in polarized epithelia. Moreover, the viral envelope of influenza itself is rich in sphingomyelin and cholesterol. Using both immunogold electron microscopy and fluorescence resonance energy transfer (FRET) microscopy, we are examining the distribution of HA on the surface of fibroblasts expressing wild-type HA.     

Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.     

Selective proteome-wide detection of hydrophobic integral membrane proteins using a novel fluorescence-based staining technology

Integral proteins containing two or more alpha-helical membrane-spanning domains are underrepresented in two-dimensional gels. While sodium dodecyl sulfate (SDS)-polyacrylamide gels separate these proteins, staining profiles are usually dominated by high-abundance hydrophilic proteins in the specimen. A fluorescence-based stain is presented that selectively highlights integral proteins containing two or more alpha-helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5-10 ng of bacteriorhodopsin, a seven-helix transmembrane protein. Stained proteins are detected using a laser scanner or charge-coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.     

N-glycan analysis by CGE-LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production

Glycoproteins, such as monoclonal antibodies as well as recombinant and viral proteins produced in mammalian cell culture play an important role in manufacturing of many biopharmaceuticals. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation affects important properties of the corresponding proteins, including bioactivity and antigenicity. This study describes the establishment of a method for analyzing N-glycosylation patterns of mammalian cell culture-derived influenza A virus glycoproteins used in vaccine manufacturing. It comprises virus purification directly from cell culture supernatant, protein isolation, deglycosylation, and clean-up steps as well as "fingerprint" analysis of N-glycan pools by CGE-LIF, using a capillary DNA-sequencer. Reproducibility studies of CGE-LIF, virus purification, and sample preparation have been performed. For demonstrating its applicability, the method was exemplarily used for monitoring batch-to-batch reproducibility in vaccine production, with respect to the glycosylation pattern of the membrane protein hemagglutinin of influenza A/PR/8/34 (H1N1) virus. This method allows characterization of variations in protein glycosylation patterns, directly by N-glycan "fingerprint" alignment.     

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.     

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Rapid separation and identification of the subtypes of swine and equine influenza A viruses by electromigration techniques with UV and fluorometric detection

Influenza A is viral disease, which is a cause of yearly epidemics and, potentially, pandemics. The conventional techniques used today are equipment-demanding, time-consuming and laborious. Recently, we have confirmed that the capillary isoelectric focusing is a suitable fast alternative for the verifying of virus purity. In the wide pH gradient of pH range 2.0-7.5 the isoelectric points for subtypes of equine (H3N8) and swine (H1N2) influenza A viruses were determined approximately as 6.6 and 6.5, respectively. In this contribution we have verified these findings using different isolates of different viral subtypes of swine influenza, H1N1, H1N2, and of equine influenza, H3N8, H7N7, which were separated by capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) in the narrow pH gradient pH range from 6.0 to 7.0. It was found that the isoelectric points of different isolates and subtypes of equine and swine influenza are almost independent of their origin. The electromigration velocities of subtypes of equine or swine influenza viruses were dependent on the antigenic subtypes of their surface glycoproteins. The detection sensitivity of the influenza viruses labeled by the fluorescent non-ionogenic tenside based on poly(ethylene glycol)pyrenebutanoate for fluorometric detection was increased and down to ten labeled viruses were detected. The isoelectric points of the native and labeled equine and swine influenza A viruses and their subtypes do not differ. According to our experiments these methods appear to be useful for the fast preliminary differentiation of influenza viruses in future.     

Rapid Identification of the Receptor‐Binding Specificity of Influenza A Viruses by Fluorogenic Glycofoldamers

The re‐emergence of influenza raises a global concern that viral pandemics can unpredictably occur. However, effective approaches that can probe the infection risk of influenza viruses for humans are rare. In this work, we develop a glycofoldamer that can rapidly identify the glycan‐receptor specificity of influenza viruses in a high‐throughput manner. The coupling of glycan receptors that can be recognized by hemagglutinin (a surface protein on the virion capsid of influenza) to a fluorogenic‐dye foldamer produces the glycofoldamers with minimal fluorescence in aqueous solution. After interaction with human‐infecting virus strains for only five minutes, the fluorescence intensity of the glycofoldamer is remarkably enhanced with a blue‐shifted emission peak. The probes have also proven effective for the rapid identification of 1) the human‐ or bird‐infecting properties of influenza viruses in a high‐throughput manner and 2) the receptor‐specificity switch of a virus strain by mutations.