An avian influenza H5N1 virus that binds to a human-type receptor

Abstract

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.

FIG. 1.

(a) Abolition of hemagglutination titers in SAα2,3Gal-specific sialidase-treated red blood cells compared to those in untreated cells indicated SAα2,3Gal monospecificity for H5N1 viruses except for Th676 and the L129V/A134V reverse genetic virus, the titers of which were only modestly affected, suggesting an enhanced binding to SAα2,6Gal. (b) Electropherogram of sequencing reaction of the Th676 virus shows a T-to-G mutation changing the amino acid from leucine to valine at position 129 and a C-to-T mutation changing the amino acid from alanine to valine at position 134 (arrowheads).

FIG. 2.

Direct binding assay using sialylglycopolymers. Human H1N1 virus (A/Thailand/Siriraj-12/06) showed receptor preference for SAα2,6Gal (a), whereas the reverse genetic virus with Kan-1 wild-type HA showed receptor preference for SAα2,3Gal (b). The reverse genetic virus carrying HA with the L129V mutation showed a receptor preference similar to that of the wild-type virus (c), whereas the virus carrying HA with L129V/A134V mutations showed receptor preference for both SAα2,3Gal and SAα2,6Gal (d). Diamonds represent absorbencies of SAα2,3Gal binding, and squares represent absorbencies of SAα2,6Gal binding.

FIG. 3.

The two glycoside backbone conformations, SAα2,6Gal and SAα2,3Gal, cut from the X-ray cocrystals under PDB accession numbers 1JSI and 1JSIN (4) with different values for torsion (Φ) angles that were used as the cis or trans conformation characteristics (a). For a human H1 virus (PDB accession number 1RVZ), SAα2,6Gal showed an average Φ angle of 63°, indicating a cis conformation in the molecular dynamic simulation (b). The amount of time that each receptor analog spent having a particular Φ angle for SAα2,3Gal (top) and SAα2,6Gal (bottom) in the binding sites of the template H5 (A/Duck/Singapore/3/97) (black), wild-type Kan-1 (green), and the L129V/A134V mutant (red) as the results of molecular dynamics simulations indicated an altered binding conformation in the L129V/A134V mutant (c). The modeling showed that SAα2,6Gal in a cis conformation in the wild-type Kan-1 HA pockets had long distances between O6 and Gln222 (3.47 Å) and between 4-OH and Gly221 (7.72 Å) (left). However, in the L129V/A134V mutant, the glycoside was stabilized with the Gly221 and Gal interactions as observed by the shorter distance between 4-OH and Gln221 (2.02 Å) (right) (d).