1. Biological Role of Neuraminic Acid in Avian Influenza Virus Infection Maitree Suttajit School of Mewdical Sciences, Univ. Phayao Email: maitree.suttajit@gmail.com AGCG 2013 October 14-18 1

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 2

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 3

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

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 5

6. Sialic Acid as a Terminal Sugar in Glycoconjugates 6

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 9

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

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 11

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. 12

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

14. Influenza virus 14 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: sialic acid in cell- surfaceglycoprotein 15

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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 17

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

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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 20

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

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

23. Influenza Infection 23

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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 acid on 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 26

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 27

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 28

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 29

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) 30

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32. SAα2,3-galSAα2,6-gal HA top view HA side view

33. 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  2-3Gal  2-6Gal  2-3Gal  2-6Gal  2-3Gal  2-6Gal 16 HA subtypes 9 NA subtypes

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 35

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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 40

41. Neuraminidase inhibitors Hemagglutinin (HA) sticks to cellular sialic acid Neuraminidase (NA) degrades cellular sialic acid 1.NA hydrolyzes sialic acid from the cell surface and frees new virus particles to the outside of infected cell. 2. This released new viruses can infect other cells and spread infection. 3.NA inhibitors prevent such cleavage of virus from being released, thereby limiting the spread of infection.  HA NA->

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

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 46 Suttajit M 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 Maitree Suttajit and Richard J. Winzler Maitree SuttajitRichard 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 Vibrio cholerae, 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. 47

48. H5N1Viral Hemagglutination Inhibition 48 HA positive HA negative CM= Collocalia mucin

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, Maitree Suttajit, Janice Beidler and 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 Collocalia mucoid 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. 49

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 Maitree Suttajit, 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. 50

51. NANA 51

52. Conclusions Neuraminic acid or sialic acid is the the IFV receptor H5N1 is endemic in certain countries in S.E. Asia The virus is present in wild birds and it may be a source of transmission to poultry The virus has shown the ability to change and infect new species More research prevention will be needed to control the viral problem 52

53. 53 Thanks for your attention Phayao Lake, northern Thailand

54. NANA modification in FSH Effect of Modification of N-Acetylneuraminic Acid on the Biological Activity of Human and Ovine Follicle-stimulating Hormone Maitree Suttajit, Leo E. Reichert Jr. and Richard J. Winzler Maitree SuttajitLeo E. Reichert Jr.Richard J. Winzler + Department of Biochemistry, Faculty of Medicine, Chiengmai University, Chiengmai, Thailand + From the Department of Biochemistry, Division of Basic Health Sciences, Emory University, Atlanta, Georgia 30322 ; Department of Chemistry, Florida State University, Tallahassee, Florida 32306 Abstract Ovine and human follicle-stimulating hormone (FSH) were modified by periodate oxidation followed by borohydride reduction, yielding FSH-containing analogues of N-acetylneuraminic acid (NANA) shortened by 1 or 2 carbon atoms. The FSH activity of the modified preparations was assayed by the human chorionic gonadotropin augmentation assay. The treatment decreased the biological activity of FSH somewhat, but, even when no unmodified NANA remained, the potency was still about half that of untreated FSH. Treatment of either native or modified FSH with neuraminidase resulted in the release of all NANA and NANA-8 and some NANA-7, and the loss of hormonal activity. It appears, therefore, that an intact 3-carbon polyhydroxy side chain in NANA is not required for full FSH activity. 54

55. Differences in Species Susceptibility All the H5N1 viruses tested are highly pathogenic for chickens-killing rapidly (1-2 days by I.V. route) Differences in domestic duck pathogenicity – Historically HPAI viruses can infect but do not kill ducks (including Asian H5N1) – Starting in 2002 some H5N1 viruses from Hong Kong were highly pathogenic for ducks – Some recent viruses may cause high mortality in ducks Other species – Little work done with other species-Hong Kong 97 viruses was generally lethal only for gallinaceous birds 55