STD-NMR as a novel method to study influenza virus-receptor interactions

Abstract

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.