Receptor binding studies of H5 haemagglutinins
Lead Research Organisation:
Medical Research Council
Department Name: UNLISTED
Abstract
Receptor binding is the first step in influenza virus infection. We will analyse this process for recent H5N1 viruses to provide information on virus variation, virus surveillance and the potential for trans-species infection.
Technical Summary
Influenza haemagglutinin (HA) mediates the first step in virus infection sialic acid receptor
binding [1], is a principal factor in cell and species specificity [2], and, as the target of
infectivity neutralizing antibodies [3] is the major object of influenza surveillance [4]. We
have determined the structure of A/Duck/Singapore/3/97 Singapore (H5N1) HA [5]; [6] to
compare an avian HA of the H5 subtype with avian and human HAs of H1 and H3 subtypes,
and investigate in particular its receptor-binding properties. Because of the widespread
distribution of H5 viruses and their persistence in avian populations, we now intend to
examine the structures of HAs from three recent H5N1 viruses from humans. Our proposal is
in three inter-related parts.
1. We will determine by X-ray crystallography the structures of the HAs from two viruses
from Turkey that were recently isolated from clinical samples in the WHO Influenza Centre
at NIMR, and from one of the 2004 Vietnamese viruses that is in a different H5 antigenic
group. The structures will be determined as complexes with pentasaccharide analogues of
human and avian receptors.
2. The Turkish viruses differ from each other at two residues in the receptor binding site
SS227N and N158D. The substitution at 227 has been observed before in viruses from Hong
Kong and Vietnam and shown to influence haemagglutination of differently derivatized
erythrocytes, and receptor binding in an ELISA binding assay [7]; [8]; [9]. We also have
preliminary evidence that the Turkish mutant virus is resistant to oseltamivir and this is
consistent with the haemagglutination and ELISA experiments. To establish the basis of
these properties we will determine the specificities and affinities of the HAs using BIAcore
procedures that we have established and refined for HA-sialic acid interactions.
3. We intend to mutate the H5 HA binding site using a vaccinia-HA recombinant virus
system that we have used extensively [10], and to determine the effects of mutations on
receptor binding properties. Subsequently we will determine the structures of appropriate
mutant HAs.
By these experiments we will have determined the binding properties of H5 HAs, understand
their molecular basis, and established a receptor binding database for interpretation of the
results of future surveillance of H5 subtype viruses in relation to their potential for crossspecies
transfer and for an aspect of anti-neuraminidase drug resistance mediated by
mutant HAs.
binding [1], is a principal factor in cell and species specificity [2], and, as the target of
infectivity neutralizing antibodies [3] is the major object of influenza surveillance [4]. We
have determined the structure of A/Duck/Singapore/3/97 Singapore (H5N1) HA [5]; [6] to
compare an avian HA of the H5 subtype with avian and human HAs of H1 and H3 subtypes,
and investigate in particular its receptor-binding properties. Because of the widespread
distribution of H5 viruses and their persistence in avian populations, we now intend to
examine the structures of HAs from three recent H5N1 viruses from humans. Our proposal is
in three inter-related parts.
1. We will determine by X-ray crystallography the structures of the HAs from two viruses
from Turkey that were recently isolated from clinical samples in the WHO Influenza Centre
at NIMR, and from one of the 2004 Vietnamese viruses that is in a different H5 antigenic
group. The structures will be determined as complexes with pentasaccharide analogues of
human and avian receptors.
2. The Turkish viruses differ from each other at two residues in the receptor binding site
SS227N and N158D. The substitution at 227 has been observed before in viruses from Hong
Kong and Vietnam and shown to influence haemagglutination of differently derivatized
erythrocytes, and receptor binding in an ELISA binding assay [7]; [8]; [9]. We also have
preliminary evidence that the Turkish mutant virus is resistant to oseltamivir and this is
consistent with the haemagglutination and ELISA experiments. To establish the basis of
these properties we will determine the specificities and affinities of the HAs using BIAcore
procedures that we have established and refined for HA-sialic acid interactions.
3. We intend to mutate the H5 HA binding site using a vaccinia-HA recombinant virus
system that we have used extensively [10], and to determine the effects of mutations on
receptor binding properties. Subsequently we will determine the structures of appropriate
mutant HAs.
By these experiments we will have determined the binding properties of H5 HAs, understand
their molecular basis, and established a receptor binding database for interpretation of the
results of future surveillance of H5 subtype viruses in relation to their potential for crossspecies
transfer and for an aspect of anti-neuraminidase drug resistance mediated by
mutant HAs.
Organisations
People |
ORCID iD |
| Steve Gamblin (Principal Investigator) | |
| John Skehel (Co-Investigator) |