Neuraminidase from viruses

A second example of up-and-down p sheets is the protein neuraminidase from influenza virus. Here the packing of the sheets is different from that in RBP. They do not form a simple barrel but instead six small sheets, each with four P strands, which are arranged like the blades of a six-bladed propeller. Loop regions between the p strands form the active site in the middle of one side of the propeller. Other similar structures are known with different numbers of the same motif arranged like propellers with different numbers of blades such as the G-proteins discussed in Chapter 13. 

Figure S.6 Schematic and topological diagrams of the folding motif in neuraminidase from influenza virus The motif is built up from four antiparallel P strands joined by hairpin loops, an up-and-down open P sheet.

Zhang J, Yu K, Zhu W, Jiang H (2006) Neuraminidase pharmacophore model derived from diverse classes of inhibitors. Bioorg Med Chem Lett 16 3009-3014 Ziircher T, Yates PJ, Daly J, Sahasrabudhe A, Walters M, Dash L, Tisdale M, McKimm-Breschkin JL (2006) Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro. J Antimicrob Chemother 58 723-732... 

Neuraminidases are enzymes present in viruses, bacteria, and parasites. They are implicated in serious diseases such as cholera, meningitis and pneumonia. Neuraminidase from influenza virus aids the transmission of the virus between cells and maintains viral infectivity. In different strains of influenza several amino acids are conserved, especially in the active site, giving rise to hopes of finding a single inhibitor (and so a drug) for all the neuraminidase enzymes from influenza strains. The crucial question is whether a covalent bond is formed between the enzyme and the reaction intermediate. 

Thomas et al. [90] investigated the reaction catalyzed by neuraminidase from influenza virus. QM/MM calculations were performed using AMI in the QM part and treating the MM region with either the CHARMM22 [97] or OPLS-AA force fields [98]. 

N. P. Arbatskii, A. O. Zheltova, D. V. Yurtov, V. A. Derevitskaya, and N. K. Kochetkov, Successive isolation of hemagglutinin and neuraminidase from influenza virus A/Krasnodar/ 101/59 (H2N2) using bromelain, Dokl. Akad. Nauk SSSR, 306 (1989) 1490-1493. 

Figure 1. Some protein structures solved by X-ray crystallography. The figure illustrates the different secondary structure assemblies. a-Helices are represented by spirals or cylinders -strands by arrows, (a) Hemerythrin, an all a-protein. (b) Superoxide dismutase, an all /7-protein, (c) Lysozyme, an a -i- /7 protein, (d) and (e) Two orthogonal views of the NAD binding domain of lactate dehydrogenase, an a/p protein, (f) Triose phosphate isomerase, an a/p structure, (g) A DNA binding protein, the CAP protein from E. coli. (h) Influenza virus haemaglutinin. (i) Influenza virus neuraminidase. From Blake and Johnson [12b], which also contains references to the original sources of these structures.

Sialidases (Neuraminidases). The three sialidase families (GH 33, 34 and 83) all have the same catalytic machinery, an aspartate, which appears to act as a proton donor, and a probable nucleophilic tyrosine, rather than a carboxylate, activated in all likelihood by a glutamate. GH 33 contains all transialidases as well as simple hydrolytic enzymes, whereas GH 34 and GH 83 contain only enzymes from viruses which are mammalian or avian pathogens. GH 33 and GH 34 act with retention of the anomeric configuration and it is currently assumed that GH 83 is similar. Obtaining crystal structures with mechanistically informative ligands bound is complicated by the facility with which sialidases dehydrate A -acetylneuraminic acid to its 2,3-dehydro derivative, DANA the process is most facile with GH 33 enzymes. The influenza sialidase in GH 34 was more amenable and not only bound the minor anomer of NANA, but bound it in the conformation. Structures of GH 83 sialidases are available only with uninformative ligands such as p-NANA bound. 

CHEMISTRY AND ANTIVIRAL ACTIVITY Zanamivir (4-guanidino-2,4-dideoxy-2, 3-dehydro-/V-acetyl neuraminic acid) is a siaMc acid analog that potently and specifically inhibits the neuraminidases of influenza A and B viruses. Depending on the strain, zanamivir competitively inhibits influenza neuraminidase activity but affects neuraminidases from other pathogens and mammalian sources only at much higher concentrations. Zanamivir inhibits in vitro replication of influenza A and B viruses, including amantadine- and rimantadine-resistant strains and several oseltamivir-resistant variants. 

Investigations of the neuraminidase from Sendai virus have shown that it is a cell-surface glycoprotein (mol. wt. 1.4 x 10 ) composed of subunits of molecular weight 7.5 x lO (rather than of mol. wt. 5.3 x lO or 1.14 x 10 , as suggested by others). ... 

A fluorogenic substrate for the assay of neuraminidase, 4-methylumbelliferone A-acetylneuraminic acid ketoside, has been synthesized. A detailed examination of the Km values for this substrate of neuraminidase from Clostridium perfringens and strains of A- and B-type influenza neuraminidases from chicken red blood cell influenza virus was conducted. 

Neuraminidase activity has been demonstrated in highly concentrated preparations of human para-influenza 1 virus (HA2 virus). The enzyme (pH optimum 5.0—5.4, temperature optimum 37—40 °C, Km 5 mM for iV-acetylneuraminlactose) was found to be thermolabile, and exhibited some characteristics similar to those of neuraminidases from other paramyxoviruses (Sendai, NDV, mumps, human para-influenza 2 virus). 

Streicher et al. have reported the application of a molecule (684) containing both the powerful influenza neuraminidase (NA) inhibitor phospha-oseltamivir and o-biotin, eonnected via an undecaethylene glycol spacer (Scheme 4). The compound (684) inhibited influenza virus neuraminidase (from the H3N2x31 virus) in the same range as oselta-mivir, with a slow off-rate, and produced a stable NA-coated surface when loaded onto streptavidin-coated biosensors. Thus molecule (684) has been found to be a potential candidate for the selective immobilisation of influenza virus in influenza diagnosis, vaccine choice, development or testing. ... 

Tsvetkova, I. V., and Lipkind, M. A., 1973, Studies on the role of myxovirus neuraminidase in virus-cell receptor interaction by means of direct determination of sialic acid split from cells. 3. One-step growth kinetics of accumulation of the sialic acid liberated from NDV-infected chick embryo cells. Arc/ . Gesamte Virusforsch. 42 125. 

Bachmayer, H., and Schmidt, G., 1972, Selective removal of neuraminidase from influenza Ag viruses, Med. Microbiol. Immunol. 158 91-94. 

Cuatrecasas, P., and Illiano, G., 1971a, Purification of neuraminidases from Vibrio cholerae, Clostridium perfringens, and influenza virus by affinity chromatography, Biochem. Biophys. Res. Commun. 44 178-184. 

Gregoriades, A., 1972, Isolation of neuraminidase from the WSN strain of influenza virus. Virology 49 333-336. 

The neuraminidase molecule is a homotetramer made up of four identical polypeptide chains, each of around 470 amino acids the exact number varies depending on the strain of the virus. If influenza virus is treated with the proteolytic enzyme pronase, the head of the neuraminidase, which is soluble, is cleaved off from the stalk projecting from the viral envelope. The soluble head, comprising four subunits of about 400 amino acids each, can be crystallized. 

Figure S.7 The subunit structure of the neuraminidase headpiece (residues 84-469) from influenza virus is built up from six similar, consecutive motifs of four up-and-down antiparallel fi strands (Figure 5.6). Each such motif has been called a propeller blade and the whole subunit stmcture a six-blade propeller. The motifs are connected by loop regions from p strand 4 in one motif to p strand 1 in the next motif. The schematic diagram (a) is viewed down an approximate sixfold axis that relates the centers of the motifs. Four such six-blade propeller subunits are present in each complete neuraminidase molecule (see Figure 5.8). In the topological diagram (b) the yellow loop that connects the N-terminal P strand to the first P strand of motif 1 is not to scale. In the folded structure it is about the same length as the other loops that connect the motifs. (Adapted from J. Varghese et al.. Nature 303 35-40, 1983.)...

The influenza virus inhibitors, zanamivir, and oseltamivir, act outside the cell after virus particles have been formed. The dtugs have been designed to fit into the active site of the viral envelope enzyme neuraminidase, which is required to cleave sialic acid off the surface of the producing cells. When its activity is blocked, new virus particles stay attached to the cell surface through binding of the virus protein hemagglutinin to sialic acid and are prevented from spreading to other cells. 

Neuraminidase inhibitors are the major class of drugs to treat or to prevent the infection with influenza viruses. Currently, two neuraminidase inhibitors are available, zanamivir and oseltamivir, which block the release of new influenza vims from infected host cells and thereby stop the spread of infection. The enzyme neuraminidase is a surface glycoprotein present on all influenza viruses. There are nine influenza neuraminidase sub-types known of which subtypes N1 and N2 appear to be the most important ones. Neuraminidase inhibitors are effective against all neuraminidase subtypes. The activity of the neuraminidase is required for the newly... 

Hurt AC, lanneUo P, Jachno K, Komadina N, Hampson AW, Barr IG, McKimm-Breschkin JL (2006) Neuraminidase inhibitor-resistant and -sensitive influenza B viruses isolated from an untreated human patient, Antimicrob Agents Chemother 50 1872-1874 Hurt AC, Selleck P, Komadina N, Shaw R, Brown L, Barr IG (2007) Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes. Antiviral Res 73 228-231... 

Palese P, Tobita K, Ueda M, Compans RW (1974b) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61 397 10 Pegg MS, von Itzstein M (1994) Slow-binding inhibition of sialidase from influenza virus, Biochem Mol Biol Int 32 851-858... 

Sheu TG, Deyde VM, Okomo-Adhiambo M, Garten RJ, Xu X, Bright RA, Butler EN, Wallis TR, Klimov AI, Gubareva LV (2008) Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008, Antimicrob Agents Chemother 52 3284-3292,... 

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