A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage

Edward Emmott,Trevor R Sweeney,Ian Goodfellow

doi:10.1074/jbc.m115.688234

Edward Emmott, Trevor R Sweeney + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m115.688234

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Abstract

The viral protease represents a key drug target for the development of antiviral therapeutics. Because many protease inhibitors mimic protease substrates, differences in substrate recognition between proteases may affect their sensitivity to a given inhibitor. Here we use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein. Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. These altered the cleavage efficiency of specific cleavage sites both within and between genogroups. The differences observed between these proteases may affect sensitivity to protease inhibitors and the suitability of MNV as a model system for testing such molecules against the human norovirus protease. Finally, we demonstrate that replacement of MNV polyprotein cleavage sites with the GI or GII equivalents, with the exception of the NS6-7 junction, leads to the production of infectious virus when the MNV NS6 protease, but not the GI or GII proteases, are present.

Highlights

Noroviruses generate the viral replicase through polyprotein cleavage by a viral protease
Because the viral protease performs an enzymatic activity not seen in uninfected cells, it represents a key target for antiviral therapy
We demonstrate that a sensor based on loss of FRET signal upon cleavage by the norovirus protease represents a rapid and robust means to characterize protease function and examine protease specificity

Summary

Background

Noroviruses generate the viral replicase through polyprotein cleavage by a viral protease. We use a cell-based FRET sensor to investigate the activity of different norovirus proteases upon cleavage of various norovirus cleavage sites inserted into a linker region separating cyan fluorescent protein and yellow fluorescent protein Using this system, we demonstrate that differences in substrate processing exist between proteases from human noroviruses (genogroups I (GI) and II) and the commonly used murine norovirus (MNV, genogroup V) model. Recent work has led to the observation that HuNoV can replicate, albeit to relatively moderate levels, in immortalized human B cells (5) and in immunocompromised mice (6) These latter experimental tools pave the way toward experimental systems to enable a detailed understanding of the HuNoV life cycle. The NS6 protease is a 3C-like cysteine protease with residues His-30, Glu-54 (Asp-54 in MNV), and Cys-139 comprising the active

The abbreviations used are

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