Biological Role of Neuraminic Acid in Avian Influenza Virus Infection

1. Biological Role of Neuraminic Acid in Avian Influenza Virus Infection MaitreeSuttajit School of Mewdical Sciences, Univ. Phayao Email: maitree.suttajit@gmail.com

2. Topics: • Neuraminic acid and neuraminidase (NA) • Influenza virus (IFV): Types and infection • Hemagglutinin (H) and its role in IFV infection • NA inhibitors and prevention of IFV infection

3. Topic 1: • Neuraminic acid and neuraminidase (NA) • Influenza virus (IFV) : Types and infection • Hemagglutinin (H) and its role in IFV infection • NA inhibitors and prevention of IFV infection

4. Neuraminic Acid or Sialic Acid 5-(acetylamino)-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosonic acid

5. N-Acetylneuraminic acid(Neu5Ac or NANA or Sialic Acid) • Predominantly found in mammalian cells. • Found in complex glycans on mucins and glycoproteins , glycolipids, such as gangliosides, a crucial component of neuronal membranes. • Can be cleaved from glyoproteins/glycolipids by neuraminidase

6. Sialic Acid as a Terminal Sugar in Glycoconjugates

7. Neuramindase (NA) An enzyme cleaves sialic acid sugar moiety from selected glycoproteins(GP) and glycolipids(GL). The cleavage of GP or GL by NA promotes the release of progeny virus from influenza viral - infected cells.

8. The neuraminidase enzyme • Neuraminidase (NA) is attached to the viral surface by a single hydrophobic sequence of 29 amino acids • Its active site is located in a deep pocket and the 18 amino acids . • NA is critical to the infective process, particularly including preventing viral aggregation or binding to hemaglutinin or inactivation by respiratory mucous. • NA is essential for proper liberation (shedding) of the new virus. • Both NA and hemaglutanin (HA) act as antigens for flu vaccines. • NA can be easily mutated. There are two main types corresponding to influenza A and B. • Due to the frequency with which influenza A mutates these proteins, new flu vaccines are required each year.

9. Protein-Bound Glycans Are Targets For Many Pathogens and Toxins: Mucins are at the “Front Lines” Acting as Decoys

10. A. Neuraminidase Activity B. Neuraminidase Inhibitor Anne Moscona. Neuraminidase Inhibitors for Influenza 2005;353:1363-73

11. Topic 2: • Neuraminic acid and neuraminidase (NA) • Influenza virus (IFV): Types and infection • Hemagglutinin (H) and its role in IFV infection • NA inhibitors and prevention of IFV infection

12. Influenza virus • Influenza types: A, B and C • Is a large enveloped virus, about 110nm in diameter, with hemagglutinin (HA) and neuraminidase (NA) projections protruding through the glycoprotein membrane, and containing a segmented, single-stranded RNA. • A and B types cause respiratory infection . • Both types, A & B, have high frequency of HA or NA mutation and cause periodic epidemics of influenza worldwide. • Influenza C is less common and causes mild in upper respiratory illness than types A and B due to its lack of neuraminidase .

13. Influenza virus • It has an envelope • Spike is composed of transmembraneproteins:Haemagglutinin (HA), Neuraminidase (NA) • HA binds with sialic acid (N-acetyl neuraminic acid) • NA is anesterase which cleaves sialic acid from glycoproteins

14. Influenza virus http://www.ucsd.tv/evolutionmatters/lesson3/study.shtml

15. Haemagglutinin spike • Transmembrane glycoprotein • Trimeric molecule; fibrous stem region, globular domain (highly conserved amino acids, receptor binding domain) • Binding receptor: sialicacid in cell- surfaceglycoprotein

17. Influenza Virus • Size of Virion Is 90-100 nm • Surrounded By Plasma Membrane Of Host • 2 glycoproteins on surface • Hemagglutin (HA) and Neuraminidase (NA) • HA is responsible for viral attachment to sialic acid found on glycoproteins • NA is responsible for detaching from sialic acid (budding) • Matrix Protein Beneath Lipid Bi-layer • Nucleocapsid contains 8 ssRNA • Types of Influenza Virus Are Bases On Protein Matrix Composition and nucleoprotein composition • Type A, B, C • Type A is responsible for major pandemics in humans • Antigenic variation in HA (13 variants) and NA (9 variants) determines subtype • Ex. H1N1

18. Influenza Subtypes • 16 Hemagglutinin subtypes • 9 Neuraminidase subtypes • 2 Nonstructural subtypes • Can occur in any combination • Useful for epidemiology

20. Topic 3: • Neuraminic acid and neuraminidase (NA) • Influenza virus (IFV): Types and infection • Hemagglutinin (H) and its role in IFV infection • NA inhibitors and prevention of IFV infection

21. N-Acetylneuraminic acid (Sialic acid) is the influenza virus receptor (hemmagutinin) • Allowing attachment to mucous cells via viral hemagglutinin (HA) • Involve in preventing infections of mucosal cells in mouth, nose, GI, respiratory tract.

22. Influenza A Virus :Negative sense RNA , Single stranded Segmented 16 Hemagglutinin subtypes 9 Neuraminidase Subtypes

23. Influenza Infection

26. Influenza Hemagglutinin (HA) • HA is a glycoprotein found on the surface of the influenza viruses . • It is responsible for binding the virus to cells with sialic acidon the membranes, such as cells in the upper respiratory tract or erythrocytes • It is also responsible for the fusion of the viruses envelope membrane with the endosome membrane at acidic pH

27. What Defines a Subtype? • Neutralizing antibody produced against one virus will neutralize all other viruses of the same subtype • A different subtype is defined when neutralizing antibody produced for one subtype will not neutralize viruses from other subtypes • Subtypes are defined by antigenic characteristics of the virus • Virus isolates will occasionally cross react with more than one reference antibodies • Hemagglutination inhibition tests provide a simple way to measure subtype differences

28. Hemagglutinin (HA) Protein • Protein is cleaved into HA1 and HA2 subunits by host proteases • Cleavage of HA is necessary for virus to be infectious (necessary to release fusion domain) • HA has receptor binding site (receptor = sialic acid) • Fusion domain becomes active when pH is lowered in endosome

29. H5 Hemagglutinin Cleavage Site • For H5 LPAI waterfowl viruses, the consensus cleavage site sequence is Arg Glu Thr Arg/ Gly • Most H5 HPAI viruses have additional basic amino acids at cleavage site • Mexico 1995 Arg Lys Arg Lys Thr Arg/ Gly • Hong Kong 1997 Arg Glu Arg Arg Arg Lys Lys Arg/Gly • The loss of a glycosylation site was also important in the emergence of HPAI in Pennsylvania in 1983 • LPAI PA/83 Lys Lys Lys Arg/ Gly + glycosylation at 11-13 • HPAI PA/83 Lys Lys Lys Arg/ Gly - glycosylation at 11-13

30. H5N1 Asian “Bird Flu” • The HPAI H5N1 Asian lineage was first detected in China in 1996 with the Goose/Guangdong/1/96 isolate • This isolate had a unique multi-basic aa cleavage site and was highly pathogenic for chickens • 1997 Hong Kong poultry and human H5N1 viruses had same H5 gene but different internal genes • 1999 Hong Kong goose viruses were most similar to Guangdong/96 virus • 2001 Korean quarantine station isolate (from China) 4 genes like Guangdong/96 including HA and four unique genes • 2001 Hong Kong H5N1 viruses with 5 distinct combinations of genes observed (same HA)

32. HA side view HA top view SAα2,3-gal SAα2,6-gal

33. The patterns of the viral receptors in different hosts restricts infection with and replication of influenza A viruses

34. Type A influenza cannot be eradicated 16 HA subtypes 9 NA subtypes a2-6Gal a2-3Gal a2-3Gal a2-6Gal a2-3Gal a2-6Gal

35. H5N1 Epizootic • The virus started spreading more widely at the end of 2003 • Has spread to at least 40 different countries, including European and African countries • Virus is changing in its ability to cause disease in ducks and wild birds • There are H5N1 viruses with different biological properties

40. Topic 4: • Neuraminic acid and neuraminidase (NA) • Influenza virus (IFV): Types and infection • Hemagglutinin (H) and its role in IFV infection • NA inhibitors and prevention of IFV infection

41. Neuraminidase inhibitors NA hydrolyzes sialic acid from the cell surface and frees new virus particles to the outside of infected cell. This released new viruses can infect other cells and spread infection. NA inhibitors prevent such cleavage of virus from being released, thereby limiting the spread of infection. HA NA-> Hemagglutinin(HA) sticks to cellular sialic acid Neuraminidase (NA) degrades cellular sialic acid

42. A. Neuraminidase Activity B. Neuraminidase Inhibitor Anne Moscona. Neuraminidase Inhibitors for Influenza 2005;353:1363-73

43. Antiviral medications 2 Classes of Medications Available • Adamantanes • Amantadine, Rimantadine • Activity only against influenza A viruses • Neuraminidase inhibitors • Oseltamivir, Zanamivir • Activity against influenza A and influenza B viruses

44. Neuraminidase inhibitors Shorten flu duration 1-2 days if started within 48 h of onset. Zanamivir

45. Neuraminidase inhibitors cont. Oseltamivir (Tamiflu ®  Roche) (UW Formulary) Oseltamivir

46. Periodate Oxidation of NANA • SuttajitM and Winzler R.J. (1971) Effect of Modification of N-Acetylneuraminic Acid on the Binding of Glycoproteins to Influenza Virus and on Susceptibility to Cleavage by Neuraminidase. J. Biol. Chem., 246,(10), 3398-3404

47. N-Acetylneuraminic Acid modification and influenza virus binding • J. Biol. Chem., 1971, 246:3398-3404. • Effect of Modification of N-Acetylneuraminic Acid on the Binding of Glycoproteins to Influenza Virus and on Susceptibility to Cleavage by Neuraminidase • MaitreeSuttajit and Richard J. Winzler • + Department of Chemistry, Florida State University, Tallahassee, Florida 32306 • Abstract • Glycoprotein substrates for neuraminidase and glycoprotein inhibitors of the agglutination of erythrocytes by influenza virus were subjected to oxidation by very low concentrations of periodate under conditions which selectively oxidize N-acetylneuraminic acid (NANA), to the 8carbon and 7-carbon aldehydes. Molar ratios of periodate to NANA were varied from 0.2 to 4.5. Reduction of the oxidized glycoproteins with sodium borohydride yielded glycoproteins containing the 8- and 7-carbon analogues of NANA as well as the parent 9-carbon compound in amounts depending on the quantity of periodate employed. These were quantitated in the mixture by means of gas-liquid chromatography. Under the conditions used, periodate oxidation of other sugars in the glycoproteins could not be detected. • The capacity of neuraminidases from Vibriocholerae, Clostridium perfringens, and PR-8 influenza virus to cleave N-acetylneuraminic acid or its 8-carbon (NANA-8) or 7-carbon (NANA-7) analogues from native and modified orosomucoid and from NANA-, NANA-8-, and NANA-7-N-acetylgalactosaminitol were studied. NANA-8 was hydrolyzed from the glycoproteins and the reduced disaccharide at an initial rate about 30% of that for NANA. The 7-carbon analogue of NANA was not cleaved from the reduced disaccharide, but was slowly cleaved from modified orosomucoid at an initial rate of 5 to 10% of that for NANA. It was concluded that the optimum activity of neuraminidase requires the intact structure of the polyhydroxy side chain of NANA. • When the molar ratio of periodate to NANA exceeded 1.5, all of the NANA was converted to the 8- and 7-carbon analogues, and the capacity of the glycoproteins to inhibit agglutination of human erythrocytes by PR-8 influenza virus was reduced by 95 to 100%. It is concluded that carbon atoms 7, 8, and 9 in the polyhydroxy side chain of NANA acid in glycoproteins are involved in their binding to influenza virus.

48. H5N1Viral Hemagglutination Inhibition HA positive HA negative CM= Collocaliamucin

49. N-Acetylneuraminic Acid Analogues • J. Biol. Chem., 1971, 246:803-809. • I. PREPARATION OF THE 8-CARBON AND 7-CARBON COMPOUNDS • Robert L. McLean, MaitreeSuttajit, Janice Beidlerand Richard J. Winzler • + Author Affiliations: Department of Biochemistry, Schools of Medicine, Dentistry, and Pharmacy, State University of New York at Buffalo, Buffalo, New York 14214 • Abstract • The 8-carbon and the 7-carbon analogues of N-acetyl-neuraminic acid were necessary in order to study some of the structural requirements of N-acetylneuraminic acid in its role as substrate for certain enzymes. The 8-carbon analogue of N-acetylneuraminic acid was synthesized by the alkaline condensation of 2-acetamido-2-deoxy-d-lyxose and di-tert-butyloxaloacetate. This same 8-carbon compound and the 7-carbon analogue of N-acetylneuraminic acid were isolated after acid hydrolysis of Collocaliamucoid which had been modified by periodate oxidation followed by borohydride reduction. The two analogues could also be detected when periodate oxidation and borohydride reduction were applied to crystalline β-methoxy-N-acetylneuraminic acid methyl ester.

50. N-Acetylneuraminic Acid Analogues • J. Biol. Chem., 1971, 246:803-809. • II. THE ACTION OF N-ACETYLNEURAMINIC ACID ALDOLASE ON 8-CARBON AND 7-CARBON ANALOGUES • MaitreeSuttajit, Carol Urban and Robert L. McLean • Department of Biochemistry, Schools of Medicine, Dentistry, and Pharmacy, State University of New York at Buffalo, Buffalo, New York 14214 • Abstract • The action of N-acetylneuraminic acid aldolase on 7- and 8-carbon analogues of N-acetylneuraminic acid has been studied. The 8-carbon analogue was slowly and reversibly cleaved by the aldolase to yield 2-acetamido-2-deoxy-d-lyxose and pyruvic acid. The 7-carbon analogue was not cleaved by the enzyme. The 8-carbon analogue competed with N-acetylneuraminic acid for the active site of the enzyme, but the 7-carbon analogue exhibited no inhibitory effect even when present at a 10-fold greater concentration than N-acetylneuraminic acid.