Influenza infections continue to be a global health concern that causing both
seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and
Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses,
which are important for their host cell sialic acid (Sia) receptor binding and
cleaving activities. Although numerous methods have been developed to study the
HA and NA interactions with sialic acid, x-ray crystallography remained the only
method to provide detailed information at atomic resolution.
The aim of this study is to develop and evaluate a novel strategy for the
investigation of influenza virus-receptor interactions, which is able to provide
information about an interaction down to atomic resolution. Influenza virus-like
particles (VLPs) containing HA and NA separately were developed and it was
reported here for the first time that sole expression of NA in mammalian cell led
to VLP formation. Characterization of these VLPs demonstrated that they are
non-infectious, but morphologically and biochemically mimic the native viruses.
Therefore the VLPs can be regarded as an ideal research model to study the
HA-Sia interaction without the interference of NA, or vice versa. Saturation
transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to
determine how a binding-ligand interacts with its target protein. Modification of
STD-NMR methodology was performed to adapt the technique to influenza VLP
system. HA-Sia interaction was investigated in great detail and group epitope
mapping of the interacting ligands was performed by analyzing the STD-NMR
spectra. The data obtained are in a good agreement with the well established
crystallography technique, reflecting the reliability of the STD-NMR technology.
Regarding the NA-Sia interaction, my data demonstrated that
substrate-hydrolysis specificity of NA is dependent on the binding of NA to those
ligands. In addition, using competition experiments with NA inhibitor, a
secondary sialic acid binding site was detected. It is the first direct experimental
evidence that confirms avian, seasonal human and human pandemic swine-origin
influenza virus N1 neuraminidases exhibit a distinct secondary binding site.
In conclusion, here I presented a novel interdisciplinary strategy using VLP
and NMR technology to study the interaction of influenza virus with its receptor.
This method is unique in its ability to provide detailed information on the HA and
NA interactions with sialic acid leading to group epitope mapping of the binding
ligands, which will help us not only to understand the virus tropism but also to
define new therapeutic targets. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174532 |
| Date | January 2011 |
| Creators | Lai, Chun-cheong., 黎振昌. |
| Contributors | Peiris, JSM, Nicholls, JM |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849745 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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