Neuraminidase from influenza virus

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.

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 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.)...

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... 

Sialidases are enzymes that catalyze the removal of terminal sialic acids from sialosides (56, 57). Human sialidases play pivotal roles in sialic acid metabolism (58). They relate to a number of disease states such as sialidosis (59-62) and cancer (63-65). Bacterial and viral play significant roles in the pathogenesis and pathology of bacterial and viral infections (66, 67). Viral sialidases such as neuraminidases of influenza virus catalyze the removal of sialic acid from the surface of infected host cells to release the newly formed progeny virus (68). 

There are several examples of parts of membrane proteins determined crystallographically. Here, a protein anchored in the membrane is clipped to provide just the hydrophilic domain for crystallisation. These studies were of cytochrome b5 (Mathews, Argos and Levine 1972), haemagglutinin (Wilson etal 1981) and neuraminidase (Varghese, Laver and Colman 1983) from influenza virus and the human class I histocompatibility antigen, HLA-A2 (Bjorkman et al 1987). 

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

Rafelson, M. E., Gold, S., and Priede, I., 1966, Neuraminidase (sialidase) from influenza virus. Methods Enzymol. 8 677-680. 

Ray, P. K., and Simmons, R. L., 1973, Differential release of sialic acid from normal and malignant cells by Vibrio cholerae neuraminidase or influenza virus neuraminidase. Cancer Res. 33 936-939. 

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. 

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... 

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... 

The influenza virus possesses a neuraminidase that plays a key role in elution of newly synthesized progeny from infected cells. If this process is inhibited, spread of the vims is markedly diminished. Inhibitors of this enzyme are now available for use in treating patients with influenza. 

Influenza (surface antigen) Allantoic fluid from embryonated hens eggs infected with Influenza viruses A and B 1 Inactivation and disruption 2 Separation of haemagglutinin and neuraminidase 3 Blending of haemagglutinins and neuraminidase of different serotypes Assay of haemagglutinin content by immunodiffusion Inoculation of embryonated hens eggs to exclude live virus... 

Immunopotentiating reconstituted influenza virosomes (IRTV) are spherical 150-nm sized particles consisting of a phospholipid bilayer in which influenza virus A/Singapore strain-derived hemagglutinin (HA) and neuraminidase (NA) are intercalated. As such, they resemble and mimic the influenza virus envelope. The difference from conventional liposome formulations lies in the inclusion of the viral envelope proteins HA and NA as well as viral phospholipids. Especially, the inclusion of influenza virus HA provides IRIV with delivery and immimogenic capacities. IRTV are licensed for human use as adjuvant in hepatitis A vaccination and as influenza subunit vaccine (1). 

Drug resistance - In clinical studies of naturally acquired infection with influenza virus, 1.3% of posttreatment isolates in adults and adolescents, and 8.6% in children from 1 to 12 years of age showed emergence of influenza variants with decreased neuraminidase susceptibility to oseltamivir carboxylate. 

Currently, two classes of drugs are available with antiviral activity against influenza viruses inhibitors of the ion channel activity of the M2 membrane protein, amantadine and rimantadine, and the neuraminidase inhibitors oseltamivir, and zanamivir. H5N1 viruses isolated from poultry and humans in Thailand and Viet Nam in 2004 invariably showed an amantadine-resistance indicating that amantadine treatment is not an option during the ongoing outb-treak in South-East Asia. 

Influenza virus resistant to oseltamivir has not been found in naturally acquired isolates but has been isolated from influenza patients who have undergone treatment with this drug. These resistant strains contain mutations in the active site of neuraminidase and are generally less virulent and infective than nonresistant virus. In vitro passage of influenza virus in the presence of oseltamivir carboxylate can produce mutations in hemagglutinin that decrease the overall dependence of viral replication on neuraminidase however, the clinical relevance of this resistance mechanism is unknown. 

Mechanism of Action An antiviral that appears to inhibit the influenza virus enzyme neuraminidase, which is essential for viral replication. Therapeutic Effect Prevents viral release from infected cells. 

The Design of Anti-Influenza Virus Drugs from the X-ray Molecular Structure of Influenza Virus Neuraminidase... 

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