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Structure and Receptor Specificity of an Avian Flu Antigen Print

 

To date, the H5N1 avian influenza viruses, which are currently circulating in domestic and wild birds on three continents, have only a limited ability to infect humans. However, with continued outbreaks of the virus in poultry and wild birds, the potential for the emergence of a human-adapted H5 virus, either by reassortment (the mixing of genetic material from similar viruses) or mutation, is seen as a major threat to public health worldwide. Of the three influenza pandemics of the last century, the 1957 (H2N2) and 1968 (H3N2) pandemics involved avian–human reassortments. The origin of the 1918 influenza virus (H1N1), which killed about 50 million people worldwide, is unknown. Although a number of viral factors can determine the host range restriction and pathogenicity of influenza A viruses, recent evidence suggests that hemagglutinin (HA), the principal antigen on the viral surface, is a critical factor for efficient human-to-human transmission. Researchers have now solved the HA structure to 2.9 Å at the ALS and analyzed its specificity to human receptors.

A Few Bird Flu Facts

The H5N1 avian influenza virus, commonly called “bird flu,” is a highly contagious and deadly pathogen in poultry. Since late 2003, H5N1 has reached epizootic levels in domestic fowl in a number of Asian countries, including China, Vietnam, Thailand, Korea, Indonesia, Japan, and Cambodia, and has now spread to wild bird populations. More recently, the H5N1 virus has spread to bird populations across much of Europe and into Africa. Although its spread to the human population has so far been limited, it has a high mortality rate, accounting for 191 deaths out of 317 documented severe cases (as of June 29, 2007, according to the World Health Organization). In this work, Stevens et al. analyze the structure of the hemagglutinin (HA) from a highly pathogenic Vietnamese H5N1 influenza virus. HA, the principal antigen on the viral surface, is the primary target for neutralizing antibodies and is responsible for viral binding to host receptors, enabling the virus to enter the host cell. As such, the HA is an important target for both drug and vaccine development.

Ribbon diagram of the Viet04 trimer. The three monomers are colored blue, green, and gray. The receptor binding site on one monomer is highlighted.

The HA homotrimer is responsible for viral binding to host receptors containing glycans (complex sugar chains) terminated by sialic acids; avian viruses preferentially bind to sugar receptors with sialic acid in an α2-3 linkage, whereas human-adapted viruses prefer sialic acids in an α2-6 linkage. The researchers utilized a number of technologies to study the structure–function relationship of an HA from a highly pathogenic H5N1 influenza virus, A/Vietnam/1203/2004 (Viet04). Functional HA trimers were produced in a baculovirus expression system, eliminating the difficulty and hazard of extracting the HA from live influenza viruses. Three hundred and eighty-four crystallization conditions were tested using less than 6 µL of protein material. The most promising condition based on crystal quality was translated to sitting-drop conditions, and subsequent optimization of these conditions yielded diffraction-quality crystals. The Viet04 HA structure was then solved by molecular replacement to 2.95-Å resolution from data collected at ALS Beamline 8.2.2.

The resulting Viet04 HA trimer structure is very similar to other avian, human, and swine HAs, with a globular head containing the receptor binding domain and a vestigial esterase domain, and a membrane proximal domain with its distinctive, central alpha-helical stalk and HA1/HA2 cleavage site. Comparison of human, avian, and swine HA structures revealed that the Viet04 HA is more closely related to human 1918 H1 HA than to the other HA structures, including a related 1997 duck H5 HA (A/Duck/Singapore/3/1997).

The same HA protein can also be used to analyze HA receptor specificity using a recently described technique involving a glycan microarray—a glass slide imprinted with hundreds of different glycan chains to systematically analyze their binding properties. Glycan microarray analysis of Viet04 HA revealed a preference for binding to avian α2-3 sialic acid receptors. The introduction of mutations that can convert H1 serotype HAs to human α2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, the introduction of mutations at positions 226 and 228, which are known to convert avian H2 and H3 HAs to human receptor specificity, permitted binding to natural human biantennary α2-6 glycans.

Top: Glycan microarray analysis of the wild-type Viet04 reveals avian preference. Bottom: Mutations at positions 226 and 228 result in a reduction in preference for avian receptor analogs and an increase in human biantennary glycans.

Thus, these mutations on the H5 HA not only reduced avidity to avian sialosides, they increased specificity for human α2-6–linked biantennary N-linked glycans, which could serve as receptors on lung epithelial cells. These combined effects could allow the Viet04 virus to escape entrapment by mucins in the lungs and increase binding to susceptible human epithelial cells. These mutations therefore provide only one possible route by which H5 viruses could gain a foothold in the human population, but other mutations are likely required to facilitate the complete switch in receptor specificity that appears to be critical for human-to-human transmission.

 


 

Research conducted by J. Stevens, O. Blixt, J.C. Paulson, and I.A. Wilson (The Scripps Research Institute); T.M. Tumpey (Centers for Disease Control and Prevention); and J.K. Taubenberger (Armed Forces Institute of Pathology).

Research funding: National Institutes of Health and Skaggs Institute for Chemical Biology. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (BES).

Publication about this research: J. Stevens, O. Blixt, T.M. Tumpey, J.K. Taubenberger, J.C. Paulson, and I.A. Wilson, "Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus," Science 312, 404 (2006).