Structure of the influenza virus with reference

This site is surrounded by twelve flexible loops, which go upwards from that axis [ 6 ]. Functional a. Recent X-ray studies of neuraminidases from the first phylogenic group have shown that, in comparison with neuraminidases from the second phylogenic group, they have a slightly different structure of the polypeptide chain around the enzyme active centre. In particular, there is a cavity in close proximity to the active site, which is formed by a change in the dimensional orientation of "loop The NA's reaction mechanism Scheme 1 was proposed based on the results of structural studies of the crystallized protein [ 7 ].

Mechanism of substrate desialylation by influenza virus neuraminidase according to [ 7 ], [ 15 ], and [ 16 ]. After the introduction of the Neu5Ac residue into the active centre, Neu5Ac conformation changes from chair to half-chair, i. The molecule of aglycone leaves the enzyme active site with glycosidic oxygen, protonated by the solvent.

Multiple contacts between the intermediate product and the a. Neu5Ac2en, in which the C2 atom is in sp2-form, mimics the intermediate reaction product in planar conformation [ 6 ]. At this stage of the reaction, the neuraminic acid residue is covalently bound to the hydroxyl group of Tyr, which is characteristic of all exosialidases [ 15 , 17 ]. Hydroxylation of the oxocarbonium ion with the solvent and product leaving the enzyme active site in the form of Neu5Ac are the limiting stages of the catalytic reaction.

It is worth mentioning that there are no significant changes in the coordinates of the NA active site during the reaction [ 18 ]. The presence of invariant residues in the active site, the similarity of the structural organization, and the architecture of complexes with Neu5Ac and with Neu5Ac2en allow to assume that the mechanism of NA functioning for the A and B influenza viruses is identical [ 6 ].

The structure of the neuraminidase active site is strictly conservative not only between subtypes, but also between the types of the enzyme, which points to the importance of all its components and the evolutionary stabilized functioning of this system.

This observation has allowed to design an NA inhibitor for the influenza virus which mimics the transition state of the hydrolysis reaction, and Neu5Ac2en Fig. The success of this drug has initiated a number of studies aimed at designing new NA inhibitors. The main structural elements of the new class of inhibitors without the oxygen atom in the cycle are cyclohexane and cyclopentane.

One of those structures is the 3S,4R,5R aminoacetamido 1-ethylpropoxy cyclohexenecarboxylic acid oseltamivir or Tamiflu Fig. The structure of this molecule is adjusted to coordinates of the amino acids, which interact with the glycerol chain of Neu5Ac2en [ 20 ]. Successful use of this drug has stimulated the development of new NA inhibitors with hydrophobic groups [ 21 ]. Besides, a NA inhibitor on the base of a cyclopentane structure has been developed; it has all the functionally important parts of zanamivir carboxyl, acetamide, C4-hydroxyl which fit into the NA active centre.

BCX preamivir Fig. At present, preamivir analogs are at the development stage. Zanamivir and oseltamivir are already used as drug products, whereas BCX has entered the last phase of clinical trials.

Until recently, it was considered that active uncontrolled use of zanamivir and oseltamivir would not have a significant influence on the development of resistance in influenza virus strains.

That is, even if resistant strains emerge, they would not be able to replicate in the absence of the inhibitor [ 24 ]. The mutation HisTyr had been spotted in studies of resistance in vitro and in vivo, as well as in clinical isolates [ 26 ]. This leaves us with hope that the strain of the influenza virus that will cause the next pandemic might be susceptible to this NA inhibitor. There is data indicating that NA is relevant at different stages of infection. 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.

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USA 97 , — Universal primer set for the full-length amplification of all influenza A viruses. Tannock, G. Download references. We thank G. Brownlee and J. Kenyon for helpful discussions, J. Kenyon, J.

Aw, and Y. Wan for sharing protocols, J. Robertson for making the 1M7 reagent, J. This work was supported by a Wellcome Trust studentship no. ID to L. Bernadeta Dadonaite, Michael L. You can also search for this author in PubMed Google Scholar. Correspondence to Lorena E. Brown , Ervin Fodor or David L.

Reprints and Permissions. Dadonaite, B. The structure of the influenza A virus genome. Nat Microbiol 4, — Download citation. Received : 13 February Accepted : 12 June Published : 22 July Issue Date : November Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.

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