Virus neuraminidase

The authors would like to acknowledge Dr. This work was supported by the Australian Government National Health and Medical Research Council grant and program grant National Center for Biotechnology Information , U. Front Microbiol. Published online Jan Julie L. Gilbertson , 1 Sanja Trifkovic , 1 , 2 Lorena E.

Brown , 1 and Jennifer L. McKimm-Breschkin 1. Brad P. Lorena E. Jennifer L. Author information Article notes Copyright and License information Disclaimer. McAuley, ua. This article was submitted to Virology, a section of the journal Frontiers in Microbiology.

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This article has been cited by other articles in PMC. Abstract With the constant threat of emergence of a novel influenza virus pandemic, there must be continued evaluation of the molecular mechanisms that contribute to virulence. Introduction Influenza viruses are members of the family Orthomyxoviridae , a group of enveloped viruses containing a segmented negative-sense single-stranded RNA genome.

Open in a separate window. Figure 1. Transmembrane Domain The N-terminal hydrophobic transmembrane domain, which attaches the NA to the viral envelope Bos et al. Stalk The stalk domains of NAs of different IAV subtypes share some structural features, but the number and sequence of amino acid residues can vary considerably Blok and Air, Figure 2.

Head Domain Peptide maps from crystallized NA catalytic heads were first detailed in Laver, Figure 3. Figure 4. Structural Relationships between NA Subtypes Phylogenic mapping, which included comparisons of genetic and structural relationships between NAs from different viruses not including the recently discovered bat viruses revealed that IAV NAs fall into two distinct groups, regardless of their serotype identification i.

NA Functional Roles in Replication Virus Entry NA activity and cleavage of sialic acids have long been thought to enable movement of the virion through mucus Burnet, Virus Internalization NAIs were found to reduce infection efficiency of cell lines without inhibiting virus binding or fusion activity, supporting a role for the NA during the viral entry process Ohuchi et al.

Catalytic Activity By far, the most characterized function of NA is its action as a sialidase enzyme, enabling release of new virion progeny by enzymatically cleaving sialic acids from cell surface receptors and from carbohydrate side chains on nascent virions Gottschalk, ; Palese et al. HA:NA Balance With respect to the ability for IAV to circumnavigate the mucosal environment and successfully infect underlying epithelial cells, the HA and NA need to have complementary receptor and ligand-binding specificity.

Figure 5. Conclusion Rather than just a sialidase that facilitates virus release from infected cells, the NA is a complicated multifunctional protein with an important role at many stages of the infectious process.

Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments The authors would like to acknowledge Dr. Footnotes Funding. References Abed Y. Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds. Influenza neuraminidase. Influenza Other Respir. Viruses 6 , — Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein.

Mechanism of cavity formation in influenza neuraminidase. Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res. Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding. Transport of viral proteins to the apical membranes and interaction of matrix protein with glycoproteins in the assembly of influenza viruses.

Analysis of the transmembrane domain of influenza virus neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft association. Sialic acid is incorporated into influenza hemagglutinin glycoproteins in the absence of viral neuraminidase. The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity.

Virology , 10— Changes in the length of the neuraminidase stalk region impact H7N9 virulence in mice. The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to 4-guanidino-Neu5Ac2en. Virology , 95— Biochemistry 21 , — The short stalk length of highly pathogenic avian influenza H5N1 virus neuraminidase limits transmission of pandemic H1N1 virus in ferrets. NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation.

The 2. EMBO J. Mucins and mucoids in relation to influenza virus action; inhibition of virus haemagglutination by glandular mucins. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia.

Science , — Biologic importance of neuraminidase stalk length in influenza A virus. The global spread of drug-resistant influenza. Interface 9 , — PLoS One 8 :e Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell , — e Influenza virus sialidase: effect of calcium on steady-state kinetic parameters. Acta , 65— Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase.

Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature , 41— Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity. Global epidemiology of influenza: past and present. Assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains. Mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of H7N9 influenza A virus.

Substrate binding by the 2 nd sialic acid-binding site of influenza A virus N1 neuraminidase contributes to enzymatic activity. Microsecond molecular dynamics simulations of influenza neuraminidase suggest a mechanism for the increased virulence of stalk-deletion mutants.

B , — Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs. An amino acid deletion in an influenza neuraminidase. Virology , — Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein. Differential transport of influenza A neuraminidase signal anchor peptides to the plasma membrane.

FEBS Lett. Vaccine 24 , — A single EK mutation far from the active site of influenza B virus neuraminidase contributes to reduced susceptibility to multiple neuraminidase-inhibitor drugs. What adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor?

Biochemistry 80 , — The cytoplasmic tail of the neuraminidase protein of influenza A virus does not play an important role in the packaging of this protein into viral envelopes.

Mouse saliva inhibits transit of influenza virus to the lower respiratory tract by efficiently blocking influenza virus neuraminidase activity. The influenza virus neuraminidase. Nature , — Consequences of resistance: in vitro fitness, in vivo infectivity, and transmissibility of oseltamivir-resistant influenza A viruses.

Viruses 7 Suppl. Human H3N2 influenza viruses isolated from to show varying preference for receptor substructures with no apparent consequences for disease or spread. PLoS Pathog. Influenza virus pleiomorphy characterized by cryoelectron tomography. Epidemiological risk factors for animal influenza A viruses overcoming species barriers. EcoHealth 14 , — Length variations in the NA stalk of an H7N1 influenza virus have opposite effects on viral excretion in chickens and ducks.

A mutant influenza virus that uses an N1 neuraminidase as the receptor-binding protein. Community transmission of oseltamivir-resistant A H1N1 pdm09 influenza. Optimizing antiviral therapy for influenza: understanding the evidence.

Expert Rev. Salivary blockade protects the lower respiratory tract of mice from Lethal influenza virus infection. Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape. Variation in the divalent cation requirements of influenza a virus N2 neuraminidases. Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense.

Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity. Science , 71— Amino acid residues contributing to the substrate specificity of the influenza A virus neuraminidase. Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains.

A secondary sialic acid binding site on influenza virus neuraminidase: fact or fiction? Endocytosis of influenza viruses. Microbes Infect. Virology 86 , 78— Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy. Proteins 78 , — Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism. Biochemistry 26 , — The pandemic H1N1 neuraminidase N1 lacks the cavity in its active site.

A amino-acid deletion in the neuraminidase stalk and a five-amino-acid deletion in the NS1 protein both contribute to the pathogenicity of H5N1 avian influenza viruses in mallard ducks. PLoS One 9 :e Airway mucus: its components and function. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed.

Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Keywords: influenza vaccine; neuraminidase; vaccine design. Abstract Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Publication types Research Support, Non-U. Firstly, it is considered that it helps the virus approach the target cells by cleavage of sialic acids from respiratory tract mucins [ 26 ]. Secondly, it may take part in the fusion of viral and cell membranes [ 27 ].

Thirdly, it facilitates budding of new virions by preventing their aggregation, caused by the interaction of the HA of the first virus with the sialylated glycans of the second one [ 27 ].

In addition, there is data suggesting that NA amplifies HA haemagglutinating activity by cleavage of the terminal neuraminic acid residues of the oligosaccharides surrounding the receptor-binding site of HA [ 28 ]. One of the most interesting features of the influenza virus is the coexistence of two proteins whose functions are to some extent contradictory, namely: haemagglutinin, which has a receptor-binding function; and neuraminidase, which has a receptor destroying function.

Studies of the viruses resistant to NA inhibitors, artificial viral reassortants which have HA and NA of different origins , and virus particles designed by means of reverse genetics, which lack NA or HA activity, show that the NA and HA of the influenza virus act in concert and their evolution proceeds interdependently [ 29 - 35 ].

Also, it raises a question as to their oligosaccharide specificity, because Neu5Ac-terminated oligosaccharide chains in viral hosts are quite diverse. The method based on the use of this substrate was proposed [ 36 ] first as an alternative to colorimetric or radioactive methods. After cleavage of the neuraminic acid, MU-Neu5Ac forms a fluorophore which is activated by light at a wavelength of nm, and its fluorescence maximum is achieved at pH High fluorescence intensity fold higher than for MUNeu5Ac is useful in studies of low-activity neuraminidases [ 37 ].

The main disadvantage of this method is the short lifetime of the product of chemiluminescent hydrolysis, which has to be recorded within 5 minutes. The amount of free neuraminic acid is usually determined after cleavage [ 39 ]; the most convenient procedure for assay of Neu5Ac allows for conducting measurements in the presence of the sialylated substrate [ 40 ].

An alternative procedure is based on assay of the second product of the hydrolysis, the desialylated glycoprotein, with the help of lectin for example, Peanut agglutinin , which is specific for the unmasked terminal galactose [ 41 , 42 ].

The substrate specificity of NA is its ability to discriminate between sialic acids for example, Neu5Ac and Neu5Gc and linkage type with the next residue , or , as well as the ability to identify internal regions of the oligosaccharide chain. In particular, the following structures have been used for the determination of NA substrate specificity:.

Methods based on the use of those substrates achieve only one of the listed goals; in particular, they allow to study specificity at the level of SiaGal or SiaGal. More broad specificity can be studied with the use of an analytical procedure which employs a number of synthetic substrates.

In [ 42 ], a panel of three oligosaccharides was used: 3'SiaLac, 6'SiaLac and 6'SiaLacNAc, in the form of polyacrylamide conjugates; and neuraminidase activity was measured by lectin, specific for galactose residues, which appear as the result of NA action see above. A new simple and sensitive method for NA specificity determination has been developed recently [ 48 ]. Stability, relative hydrophility, electroneutrality, small size, and ability to use standard fluorescent filter for detection are the advantages of this label.

The method is based on a quantitative separation of the electroneutral product of the reaction and the negatively charged substrate, separation is performed either on a microcartrige with an anion-exchange sorbent or microplates, the semipermeable bottom of which consists of an anion-exchange material. For greater reliability one may quantify the amount of the reaction substrate, along with the quantity of the reaction product.

The high sensitivity of the method makes it possible to work with low substrate concentrations mol , as well as with low virus concentrations. Studies of desialylation kinetics, in particular the reaction velocity and its dependence on substrate and enzyme concentration, is important for understanding the reaction mechanism, as well as for the choice of the correct concentration range. In turn, the correct range allows to study desialylation specificity in cases when the NA quantity in the test sample is unknown [ 49 ].

It is worth mentioning that only this approach allows to study many aspects of NA substrate specificity see above , namely to study the influence of the sialic acid type, the type of linkage between the sialic acid and the next sugar, and the influence of the inner glycan sugars.

As already mentioned above, high molecular weight substrates, along with low molecular weight substrates, can be used for studying NA activity and specificity. Low molecular weight substrates allow to study the reaction mechanism and desialylation kinetics without the complications of multivalent interactions NA is a tetramer and the possible influence of HA, which interacts with multivalent conjugate 3 — 5 orders of magnitude better than with the monomeric one [ 50 ].

High molecular weight substrates appear to be a more accurate model for studying natural interactions; that is when there is a necessity to account the NA tetrameric organization, the clustering of NA molecules on the cell surface, and the involvement of the second surface glycoprotein, HA, which is present on the viral surface in larger amount. Investigation of the evolution of the influenza virus NA substrate specificity for viruses isolated from humans, and its comparison with the substrate specificity of influenza virus NAs isolated from different hosts, such as ducks and pigs, is of great importance.

The first undertaking could shed light on the question of the unique character of pandemic strains, while the second could help detect in advance the properties of the enzyme which facilitate the crossing of the interspecies barrier.

Hydrolytic activity towards 6'SiaLac was identified only for viruses isolated in and further, and starting from isolates an increase of activity towards this substrate was registered [ 46 ].

It has been shown recently that N2 influenza viruses are highly active towards 3'SiaLac, while their activity towards 6'SiaLac varies from extremely low avian and early human isolates to high swine and latter human isolates. It has been shown that NA activity towards 6'SiaLac depends also on the host type and, for human viruses, on the year of isolation [ 45 ]. For N1 strains isolated in the s [ 43 , 44 ], it was shown that their neuraminidase equally recognizes 3'SiaLac and 6'SiaLac.

Data on the substrate specificity of N1 and the N2 NAs of several duck, swine and human influenza virus isolates were obtained with the use of BODIPY-labeled synthetic oligosaccharides [ 48 - 51 ]. In summary, the nature of the host cell line used for virus accumulation influences NA substrate specificity [ 42 ]. The reason for this effect remains unknown. It is difficult to compare the results of substrate specificity studies conducted by different authors due to the use of both different influenza virus strains and different substrates in varying concentrations.

It is also worth mentioning that studies of the influenza virus with the simultaneous use of high- and low-molecular weight substrates of a defined structure have yet to be conducted. Despite the limited amount of data published to date, it is already possible to discuss some features. Firstly, the NA substrate specificity of human isolates differs from that of avian isolates.

Secondly, the oligosaccharide specificity of the NA of viruses which circulate in different hosts birds, pigs, humans notably differs, at least for the characteristic "ratio of the hydrolysis velocity of oligosaccharides towards oligosaccharides. Data on NA functioning would be incomplete without taking into account another surface protein of the influenza virus, haemagglutinin.

There is only a limited number of publications describing the simultaneous study of HA and NA substrate specificity, and there is virtually no research where the dependence of HA and NA oligosaccharide specificity on one hand and virus infectivity on another hand have been studied.

The state-of-the-art analytical procedures for NA introduced in the current review are now up to par with the more advanced analytical methods of HA analysis, which always developed faster; therefore, it is quite easy to predict that one of the main trends in influenza virus studies in the future would be joint studies of HA and NA specificity. National Center for Biotechnology Information , U.

Journal List Acta Naturae v. Acta Naturae. Shtyrya , 1 L. Mochalova , 1 and N. Author information Copyright and License information Disclaimer. Corresponding author. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC.

Abstract The structure of the influenza virus neuraminidases, the spatial organization of their active site, the mechanism of carbohydrate chains desialylation by neuraminidase, and its role in the influenza virus function at different stages of the viral infectious cycle are considered in this review. Head The enzyme active site and calcium binding domain, which stabilizes the enzyme structure at low pH values, are situated in the head of NA [ 2 ; 8 ].

Open in a separate window. Glycosylation Carbohydrate chains are attached to Asn residues located in the different regions of the NA's head. Disulfide bonds There are eight conservative disulfide bonds in the NA structure, and one additional bond in the N2, N8, and N9 subtypes. Active site structure The Neu5Ac binding site is located above the first strands of the third and the fourth motifs in a big loop on the NA surface. Scheme 1. Methods for determination of NA substrate specificity The substrate specificity of NA is its ability to discriminate between sialic acids for example, Neu5Ac and Neu5Gc and linkage type with the next residue , or , as well as the ability to identify internal regions of the oligosaccharide chain.

Functional features of some influenza virus neuraminidases As already mentioned above, high molecular weight substrates, along with low molecular weight substrates, can be used for studying NA activity and specificity. Varghese J. Colman P. NA enzyme and antigen. In: Sasaki Y, editor. The influenza viruses. New York: Plenum Publishing Corporation; Russell R.

The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Harris A. Influenza virus pleiomorphy characterized by cryoelectron tomography.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases. Bossart-Whitaker P. Threedimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. Janakiraman M.