Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

Structural Basis of Protection against H7N9 Influenza Virus by Human Anti-N9 Neuraminidase Antibodies.

Influenza virus neuraminidase (NA) is a major target for small-molecule antiviral drugs. Antibodies targeting the NA surface antigen could also inhibit virus entry and egress to provide host protection. However, our understanding of the nature and range of target epitopes is limited because of a lack of human antibody structures with influenza neuraminidase. Here, we describe crystal and cryogenic electron microscopy (cryo-EM) structures of NAs from human-infecting avian H7N9 viruses in complex with five human anti-N9 antibodies, systematically defining several antigenic sites and antibody epitope footprints. These antibodies either fully or partially block the NA active site or bind to epitopes distant from the active site while still showing neuraminidase inhibition. The inhibition of antibodies to NAs was further analyzed by glycan array and solution-based NA activity assays. Together, these structural studies provide insights into protection by anti-NA antibodies and templates for the development of NA-based influenza virus vaccines and therapeutics.

Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action.

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.

The 340-cavity in neuraminidase provides new opportunities for influenza drug development: A molecular dynamics simulation study.

Influenza neuraminidase (NA) is a pivotal target for viral infection control. However, the accumulating of mutations compromise the efficacy of NA inhibitors. Thus, it is critical to design new drugs targeted to different motifs of NA. Recently, a new motif called 340-cavity was discovered in NA subtypes close to the calcium binding site. The presence of calcium is known to influence NA activity and thermostability. Therefore, the 340-cavity is a putative ligand-binding site for affecting the normal function of NA. In this study, we performed molecular dynamics simulations of different NA subtypes to explore the mechanism of 340-loop formation. Ligand-binding site prediction and fragment library screening were also carried out to provide evidence for the 340-cavity as a druggable pocket. We found that residues G342 and P/R344 in the 340-loop determine the size of the 340-cavity, and the calcium ion plays an important role in maintaining the conformation of the 340-loop through contacts with G345 and Q347. In addition, the 340-cavity is predicted to be a ligand-binding site by metaPocket, and a sequence analysis method is proposed to predict the existence of the 340-cavity. Our study shows that the 340-cavity is not an occasional or atypical domain in NA subtypes, and it has potential to function as a new hotspot for influenza drug binding.

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR.

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2-3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-ß-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2-3- and α2-6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.

Cell-permeable probe for identification and imaging of sialidases.

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Combining molecular docking and molecular dynamics to predict the binding modes of flavonoid derivatives with the neuraminidase of the 2009 H1N1 influenza A virus.

Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R(2) = 0.75.

Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles.

Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.

On the lower susceptibility of oseltamivir to influenza neuraminidase subtype N1 than those in N2 and N9.

Aiming to understand, at the molecular level, why oseltamivir (OTV) cannot be used for inhibition of human influenza neuraminidase subtype N1 as effectively as for subtypes N2 and N9, molecular dynamics simulations were carried out for the three complexes, OTV-N1, OTV-N2, and OTV-N9. The three-dimensional OTV-N2 and OTV-N9 initial structures were represented by the x-ray structures, whereas that of OTV-N1, whose x-ray structure is not yet solved, was built up using the aligned sequence of H5N1 isolated from humans in Thailand with the x-ray structure of the N2-substrate as the template. In comparison to the OTV-N2 and OTV-N9 complexes, dramatic changes were observed in the OTV conformation in the OTV-N1 complex in which two of its bulky side chains, N-acethyl (-NHAc) and 1-ethylproxy group (-OCHEt2), were rotated to adjust the size to fit into the N1 catalytic site. This change leads directly to the rearrangements of the OTV's environment, which are i), distances to its neighbors, W-178 and E-227, are shorter whereas those to residues R-224, E-276, and E-292 are longer; ii), hydrogen bonds to the two nearest neighbors, R-224 and E-276, are still conserved in distance and number as well as percentage occupation; iii), the calculated ligand/enzyme binding free energies of -7.20, -13.44, and -13.29 kcal/mol agree with their inhibitory activities in terms of the experimental IC50 of 36.1-53.2 nM, 1.9-2.7 nM, and 9.5-17.7 nM for the OTV-N1, OTV-N2, and OTV-N9 complexes, respectively; and iv), hydrogen-bond breaking and creation between the OTV and neighborhood residues are accordingly in agreement with the ligand solvation/desolvation taking place in the catalytic site.

Structure of an influenza neuraminidase-diabody complex by electron cryomicroscopy and image analysis.

The structure of a complex between a bivalent diabody and its antigen, influenza neuraminidase, has been determined by electron cryomicroscopy of single particles and image analysis. A three-dimensional reconstruction has been interpreted in terms of high-resolution X-ray models of the component proteins. The complex consists of two neuraminidase tetramers cross-linked by four diabodies with 422 point symmetry. The structure and symmetry of the complex is determined uniquely by packing constraints consistent with the maximum possible number of diabody cross-links. Diabodies may provide a useful approach to the structure determination of small proteins by incorporating the proteins into large symmetric complexes followed by single-particle electron microscopy.

A single sequence change destabilizes the influenza virus neuraminidase tetramer.

A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-Neu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, which sits in a pocket in the bottom of the active site of the enzyme, and the 4-guanidinium moiety of the inhibitor to be lost. NA "heads" of the resistant enzyme produced only a few small crystals under conditions in which the wild-type enzyme readily formed large crystals. These small crystals were of sufficient quality to yield X-ray crystallographic data which confirmed the E119G change and demonstrated the presence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activity per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetramers, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crystals obtained were probably from the few tetramers remaining intact in the preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examination of the known X-ray crystallographic structure of the wild-type NA reveals a salt bridge between Glu119 and Arg156 of the same monomer. Arg156 is a conserved amino acid that is situated at the interface between monomers, and a salt link between this amino acid and Glu119 may contribute to the stability of enzyme tetramers. It is suggested that the E119G alteration in the 4-GuDANA-resistant NA leads to the abrogation of this interaction and thus to the instability of the NA tetramers.

Comparison of protein surfaces using a genetic algorithm.

A genetic algorithm (GA) is described which is used to compare the solvent-accessible surfaces of two proteins or fragments of proteins, represented by a dot surface calculated using the Connolly algorithm. The GA is used to move one surface relative to the other to locate the most similar surface region between the two. The matching process is enhanced by the use of the surface normals and shape terms provided by the Connolly program and also by a simple hydrogen-bonding descriptor and an additional shape descriptor. The algorithm has been tested in applications ranging from the comparison of small surface patches to the comparison of whole protein surfaces, and it has performed correctly in all cases. Examples of the matches are given and a quantitative analysis of the quality of the matches is performed. A number of possible future enhancements to the program are described which would allow the GA to be used for more complex surface comparisons.

Mutations in the cytoplasmic tail of influenza A virus neuraminidase affect incorporation into virions.

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.

Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoelectron microscopy of influenza A virus neuraminidase glycoprotein topography.

Using immunoelectron microscopy, the distribution of influenza A virus neuraminidase (NA) glycoproteins was examined, after performing immunoreactions to virions on the grid. With polyclonal antibody, the immunolabels of the glycoproteins were found to be homogeneously distributed, whereas with monoclonal antibody they were found to be distributed in clusters. After destruction of haemagglutinin (HA) but not of NA activity with a high concentration of trypsin, the remaining visible spikes were evenly distributed. This finding was consistent with the absence of immunolabelling with anti-HA antibody, and the homogeneous pattern of immunolabels with anti-NA polyclonal antibody, but not with the clustered labelling with the anti-NA monoclonal antibody. Thus, the immunolabelling image with anti-NA polyclonal antibody was considered to reflect the true one.

Spot-scan imaging of microcrystals of an influenza neuraminidase-antibody fragment complex.

Electron micrographs of two-dimensional microcrystals of a complex of an avian influenza virus neuraminidase and an antibody Fab fragment, termed 32/3, have been recorded using the spot-scan method of imaging. The crystals have a large unit cell (159.5 A x 159.5 A x 130.5 A) and a high solvent content (approximately 71% by volume) and are a challenging specimen for testing the spot-scan methodology. Crystalline order was preserved to beyond 4 A resolution as demonstrated by electron diffraction, using an embedding medium of a mixture of glucose and neutral potassium phosphotungstate. Using a Philips C400 computer control system interfaced to an EM420 electron microscope, and with the inclusion of additional software in the system, we have been able to record micrographs at low temperature with a relatively narrow (1500 A diameter) moving beam. There is evidence that the use of such a spot-scan beam reduces the effects of beam-induced specimen motion on the quality of micrographs. Conventional low-dose "flood-beam" images showed good isotropic optical diffraction in only 15% of cases whereas 30% of spot-scan images showed good diffraction. The best flood-beam images gave phases to only 15 A resolution after computer processing, whereas the best spot-scan images gave phases to 7 A resolution. Electron diffraction patterns were also recorded at low temperature, and the resulting diffraction amplitudes combined with phases from spot-scan images to yield a projection map of the structure. A 7 A resolution projection map of the complex is presented, and is compared with the projection map of the same avian influenza neuraminidase complexed with a different monoclonal Fab fragment, NC41, which has been solved to high resolution by X-ray diffraction.

Antigenic variation and the structure of influenza virus glycoproteins.

Influenza is impossible to control by vaccination because of variation in the two surface antigens of the virus, the haemagglutinin (HA) and neuraminidase (NA). This variation is caused by sequence changes in the genes coding these antigens and this review summarizes recent information on the structure of the HA and NA, the way in which these glycoproteins vary and the effects of the changes on the antigenic properties of the virus.