Influenza hemagglutinin H3
Influenza hemagglutinin (H3 serotype) was the first glycoprotein structure to be solved at atomic resolution, by Ian Wilson, John Skehel and Don Wiley in 1981. The collaboration between the Skehel and Wiley labs provided great insight into hemagglutinin function, and it remains the prototype for understanding receptor recognition, antigenic variation, and the extraordinary conformational changes associated with target membrane insertion and ultimately fusion of viral with cell membrane to allow the viral genome to enter the cell and replicate.
In the 1980s, the Paulson lab made the seminal discovery that human and avian viruses with the H3 serotype have different receptor specificities; that human viruses bind to Neu5Acα2-6Gal, while avian viruses bind Neu5Acα2-3Gal. In two very elegant experiments, they were able to switch these specificities by applying selective pressure, and showed that a single amino acid change (L226Q) was all that was required for early H3N2 viruses to switch between human and avian specificities. These results showed how easy it can be for avian viruses to cross the species barrier into humans. Seasonal influenza viruses with the H3 serotype continue to circulate in the human population, and subtleties in their receptor specificities appear to be playing a role in how clinical isolates can be recovered in laboratory hosts. CFG investigators are using tools, such as the glycam microarray, provided by the CFG to analyze the detailed receptor specificity of the circulating H3N2 as well as other influenza viruses and their interaction with laboratory hosts to better understand this phenomenon, which has direct consequences on production of vaccines.
Although the influenza H3 hemagglutinin has been chosen as the paradigm, since so much is known, there are 16 subtypes of influenza HA (H1-H16), defined by lack of antigenic cross-reactivity. There is typically only about 20% amino acid sequence identity between HAs of different subtypes. There are interesting and important differences in how easily a particular strain within the subtype can change its binding specificity between avian-like and human-like receptors, leading to the failure so far of H5N1 to be established in the human population, whereas swine-origin H1N1 showed high transmissibility between humans from the time it was first isolated.
To understand the transmission of influenza viruses and how new pandemics begin, it will be important to study a variety of HA subtypes and strains. but for other subtypes the precise rules may differ. Hoever, much progress had been made in the CFG with participating investiigators of understanding the receptor specificity and transmissability of H1 and H2 subypes. Fortunately, the H5N1 avian virus has still not acquired the ability to transmit between humans, as the rules seem more complex compared to H1, H2 and H3, despite at least 15 years of opportunity. The CFG has facilitated considerable advances in our knowledge of the role of sialic acid binding in influenza host specificity and tropism for the upper or lower respiratory tract, and these studies need to be continued until we understand how influenza viruses enter the human population to cause each new pandemic, and the role of receptor specificity in pathogenicity.
CFG Participating Investigators contributing to the understanding of this paradigm
CFG Participating Investigators (PIs) contributing to the understanding of H3 include: Gillian Air, Rafi Ahmed, Nicolai Bovin, Ruben Donis, Chwan-Chuen King, Vladimir Lugovtsev, Christopher Olsen, Peter Palese, James Paulson, Andrew Pekosz, Daniel Perez, Peter P.J.M. Rottier, Charles Russell, Ram Sasisekharan, Dorothy Scott, David Smith, James Stevens, Stephen Mark Tompkins, Reinhard Vlasak, Qinghua Wang, Ian Wilson
Progress toward understanding this GBP paradigm
Ligands for H3 hemagglutinin are sialylated glycans. The H3 hemagglutinin of human viruses (subtype H3N2) binds to N-acetylneuraminic acid linked α2-6 to galactose, sometimes N-acetylgalactosamine. Glycan array analyses indicate that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. Recent human H3 HAs have shown variation in their specificity of binding downstream sugars.. Occasionally human H3N2 HAs bind to 2-3 linked sialic acids, such as sialyl-Lewis x 
Cellular expression of GBP and ligands
HA is expressed on the surface of influenza virus infected cells before being budded out into progeny virions. H3N2 viruses infect the respiratory tract of humans and birds; in birds they may also infect the gut epithelia. Using MAA and SNA lectins, the upper respiratory tract appears to have more 2-6 linked sialic acid while 2-3 sialic acid appears more abundant in the lungs, but relationship between the different specificities of H3N2 HAs and the cell types infected remains unclear . H3N2 viruses infect very few continuous cell lines. Madin-Darby canine kidney cells are most commonly used. Non-permissive cell lines may take up virus efficiently, replicate RNA and express HA on the cell surface but do not bud new virus particles .
Biosynthesis of ligands
Sialylated glycoproteins or glycolipids recognized by human influenza hemagglutinin H3 are synthesized by host cells. The H3 hemagglutinin shows considerable diversity in binding but with rare exceptions the sialic acid is attached 2-6 to the next sugar on structures that are mostly typical of N-linked glycans on proteins. The enzymes required for biosynthesis of the type 2 poly N-acetyllactosamine chains and modification with sialic acid or with sialic acid and fucose, have been defined ([poly N-acetyllactosamine extension biosynthesis]). The sialyltransferases that generate ligands for most H3 subtype hemagglutinins are ST6Gal1, ST6GalII, ST6GalNAc1, ST6GalNAcII, ST6GalNAcIV.
The crystal structure of H3 HA was determined by Wilson, Wiley & Skehel in 1981. This has served as a model for more recent HA structure determinations such as H1 HA .
The image of the HA trimer was made with PyMol (Delano Scientific) from PDB file 5HMG. The three subunits are colored green, blue and magenta. For each, the darker shade is the HA1 polypeptide and the lighter shade is HA2.
Biological roles of GBP-ligand interaction
Sialylated glycans on the surface of cells lining the respiratory tract serve to capture virus to initiate infection. Glycan array analyses have confirmed that human influenza viruses such as those carrying the H3 HA bind only to structures with NeuAcα2-6 and avian isolates bind only to structures containing NeuAcα2-3. The role of this GBP-glycan interaction in initiating endocytosis is still unclear, but in the low pH of the endosomal compartment, the HA undergoes a large conformational change  that brings about fusion of the viral membrane with the cell membrane so that viral nucleocapsids are released, enter the nucleus and initiate viral transcription and replication.
CFG resources used in investigations
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for "hemagglutinin".
Virologists have used lectin binding to try to determine where the influenza virus receptors specific for human or avian HAs are located in the human respiratory tract, with mixed results . A complete profile of human trachea as well as lung is needed.
There are no glycogene array results with the H3 HA, but related paradigm H1 HA has been used by Dr Linda Sherman to assess the role of protein glycosylation in the decision between deletion vs. anergy in immune tolerance. The antigen used was a peptide of A/PR/8/34 (H1N1) HA, 518-IYSTVASSL-526. CFG Request #1155
Knockout mouse lines
Unfortunately the mouse is a very poor model of influenza infection. Some viruses with H3 HA infect mice quite readily, but do not cause a human-like disease. This means that studies of infection and transmission of H3N2 influenza viruses in SiaT knockout mice are difficult to translate to the human disease. However, studies were done using a mouse-adapted virus 
The majority of PI-initiated requests for CFG resources to study influenza have been requests for analysis of receptor specificity on the glycan array (click here for example), and the remainder have been requests for compounds to conduct in vitro assays in investigators' laboratories. In addition, the CFG glycan array library has been used print custom sialic acid glycan arrays for the U.S. Centers for Disease Control (CDC) for analysis of the receptor specificity of emerging viruses, with data deposited to the CFG database. Glycan Array analyses of H3 HAs have been run for the following PI's:
Compans (Resource Request #1781; A/Aichi/1/68, A/Udorn/72 and A/Wyoming/3/03), Steinhauer (#1777; A/Aichi/68 and mutants), Olsen (#1796, A/swine/Mn/593/99 and A/swine/Ontario/130/97), Rottier (#1797, A/Finland), Air (#1660, 1380, 1033, 948, 175; A/Oklahoma/483/2008, A/OK/309/06, A/Oklahoma/323/2003, A/OK/370/05, A/OK/369/05, A/OK/1992/05, A/Wyoming/3/03, A/Philippines/82), Chen (#1468; A/Victoria/75), Donis (#138; A/canine/Florida/2004, A/equine/MA/2003), Paulson (#451; duck/Ukraine/63, A/Moscow/10/99)
Influenza virus HAs of other serotype H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 and type B. Type A subtypes H1, H2, H5, H6, H7, and H9 are all being actively investigated by CFG investigators for their potential to jump to humans and type B for its failure to spread in non-human species. CFG data for many of these subtypes are available in the CFG database search results for "hemagglutinin."
- ↑ Gulati S, Smith DF, Air GM. Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses. Virology J 2009;6(22)
- ↑ Stevens, J., Chen, L.-M., Carney, P.J., Garten, R., Foust, A., Le, J., Pokorny, B.A., Manojkumar, R., Silverman, J., Devis, R., Rhea, K., Xu, X., Bucher, D.J., Paulson, J.C., Paulson, J., Cox, N.J., Klimov, A., Donis, R.O., 2010. Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs. Journal of Virology 84, 8287-8299.
- ↑ NICHOLLS, J., CHAN, R., Russell, R., Air, G., PEIRIS, J., 2008. Evolving complexities of influenza virus and its receptors. Trends in Microbiology 16, 149-157.
- ↑ Kumari K, Gulati S, Smith DF, Gulati U, Cummings RD, Air GM. Receptor binding specificity of recent human H3N2 influenza viruses. Virol J 2007;4(42):1-12.
- ↑ Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science 2010 Apr 16;328(5976):357-60.
- ↑ Bullough et al “Structure of influenza hemagglutinin at the pH of membrane fusion”. Nature 371: 37-43 (1994)
- ↑ Nicholls JM, Chan RW, Russell RJ, Air GM, Peiris JS. Evolving complexities of influenza virus and its receptors. Trends Microbiol 2008 2008 Apr;16(4):149-57.
- ↑ Glaser L, Conenello G, Paulson J, Palese P. Effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase. Virus Res. 2007 Jun;126(1-2):9-18.
- ↑ Stevens, J., Blixt, O., Chen, L. M., Donis, R. O., Paulson, J. C., and Wilson, I. A. (2008). Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381(5), 1382-94.
- ↑ Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol 355(5), 1143-55.
- ↑ Stevens, J., Blixt, O., Paulson, J. C., and Wilson, I. A. (2006). Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat Rev Microbiol 4(11), 857-64.
- ↑ Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C., and Wilson, I. A. (2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312(5772), 404-10.
The CFG is grateful to the following PIs for their contributions to this wiki page: Gillian Air, James Paulson, Ian Wilson