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Targeting Norovirus Receptor Interactions at Atomic Resolution

Final Report Summary - NOROCARB (Targeting Norovirus Receptor Interactions at Atomic Resolution)

Summary description of the project objectives
Acute gastroenteritis is the second largest burden of all infectious diseases. It is estimated that it accounts for 89.5 million disability-adjusted life-years (DALYs) and 1.45 million deaths worldwide every year. Norovirus (NoV) infections are the leading cause for acute gastroenteritis across all age groups, and there is an urgent need for drugs or vaccines to combat NoV infections. So far, neither drugs nor vaccines are available. Epidemic outbreaks are difficult to control due to the viruses' highly contagious nature and their resistance to disinfectants. Therefore, NoVs have been classified as B-agents in the NIH/CDC biodefense program. Human NoVs recognize histo blood group antigens (HBGAs) as cellular attachment factors. We have proposed to study the interaction of NoV coat proteins with HBGAs at atomic resolution in order to establish a firm basis for the design of potent entry-inhibitors. In fact, this project has led to a major breakthrough in the field. We have uncovered that human NoV attachment to HBGAs is a cooperative multistep process, which is in contrast to all current structural models. Moreover, the number of binding sites for HBGAs is twice a high as anticipated (Fig. 1, see attachment). Our findings represent a novel paradigm for NoV-host recognition and will have a major impact on any research into NoV infection, especially on the development of entry-inhibitors.

Work performed since beginning of the project
1 The expression of VP1 P-dimers of two human NoV strains, GII.10 Vietnam026 and the currently predominant GII.4 Saga has been extremely successful in terms of yields and stability of the dimeric protein. Therefore, we have abandoned the initial target, a related GII.4 strain Ast6139. With our optimized yields, any stable isotope labeling scheme is now affordable. We have also succeeded in preparing 13C-methyl labeled P-dimers using the ILV labeling scheme. These samples will be subjected to NMR experiments into the protein dynamics.
2 Along with the project we have established cell free protein synthesis in our lab, which now can be routinely done. In fact, classical 3D triple resonance NMR spectra of uniformly 2H,15N,13C labeled P-dimers obtained at the EU large scale NMR facility in Utrecht (Instruct) are of such a high quality that costly combinatorial labeling became superfluous and cell free protein synthesis is not necessary at this point. The backbone assignment is underway.
3 Our studies show that surface plasmon resonance experiments are unsuitable for the investigation of NoV P-dimers or corresponding VLPs. The experimental setup (immobilization of the protein) leads to a loss of cooperativity of binding. Only competitive binding studies can be performed. ITC experiments have been postponed because of the cost of the ligands. Instead we have established STD NMR titration protocols that deliver binding data under physiological conditions. We have also started a collaboration with the laboratory of Dr. Charlotte Uetrecht (FEL/HPI Hamburg) to determine binding affinities via native mass spectrometry. This is a novel approach that requires only very small amounts of sample and that has also been instrumental to determine the number of binding sites per P-dimer.
4 A combination of 1H,15N-TROSY-HSQC chemical shift titrations and STD NMR titrations was instrumental for the discovery of the cooperative multi-step binding mechanism. For these experiments we have used uniformly 2H,15N-labeled P-dimers.
5 A novel approach has been followed for the assignment of the 13C methyl groups of the ILV labeled P-dimers. In collaboration with the laboratory of Javier Castells (Complutense University, Madrid) we have synthesized compounds as shown in Fig. 2 (see attachment). These compounds are loaded with lanthanide ions and allow the almost complete assignment of ILV residues from simple titration experiments. The results are subject to two manuscripts in preparation.
6 The synthesis of entry-inhibitors has to be completely redesigned since the old binding models that the proposal was based upon are all incomplete. We have teamed up with the group of Laura Hartmann (University of Düsseldorf, Germany) in order to synthesize novel multivalent ligands. This work will be subject of new grant proposals.
7 In a side project we have investigated the use of metabolic precursors such as ManNAz or GalNAz for in vivo metabolic labeling of viral coat proteins. The project aims at a novel approach to identify unknow receptors. For this project the synthetic expertise of Alvaro Mallagaray has been instrumental.

Main results
• A novel paradigm for NoV-host cell interactions has been uncovered and most likely extends into the complete family of Caliciviridae. Our findings will inspire any future studies into NoV-host interactions and has strong implications for the design of entry-inhibitors.
• A highly optimized high-yield expression protocol has been established for stable isotope labeling of NoV P-dimers.
• A novel approach to assign 13C methyl groups of ILV labeled samples has been established.

Expected final results and their potential impact and use
• A complete NMR assignment of NoV P-dimers is in reach and corresponding experiments are underway. A collaboration has been established with the laboratory of Rolf Boelens (University of Utrecht, Netherlands) giving us access to the high field NMR center in Utrecht. This will open up many possibilities to study HBGA binding and protein dynamics as a functions of HBGA binding. Finally, these experiments may uncover the complete mechanism by which NoV enters host-cells.
• The newly discovered binding mechanism has paved the way for any successful strategy to synthesize potent entry inhibitors. Finally, this will be beneficial to anyone suffering from NoV infection and in turn may reduce the socio-economic burden that such infections currently represent.