Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

Abstract

Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl 2 and N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents. IMPORTANCEHuman noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc, we demonstrated a cell typespecific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.

Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

“…NV stool RNA isolation and transfection. Purification of NV particles from NV stool filtrate using rabbit anti-NV VP1 antibody-coated magnetic beads (Bio-Rad) and isolation of NV RNA from purified virus particles by a modified QIAamp viral RNA minikit (Qiagen) protocol have been described previously (41). The yield of NV stool RNA from 20-ml stool filtrate was typically 60 l of RNA at a concentration of 60 to 70 ng of RNA/l (mostly carrier RNA) containing 3 ϫ 10 7 to 4 ϫ 10 7 NV genome copies/l.…”

Antiviral targets of human noroviruses

“…In addition to potentially higher stability to metabolic enzymes these inhibitors exhibit increased cellular permeability. Further studies should be anticipated that are directed at optimizing the design strategies and improving the pharmacokinetic properties and metabolic stability of HuNoV protease inhibitors using structural analysis and cell-based assays [74–76]. Considering that the active-site residues are highly conserved between the HuNoV proteases in various genogroups and picornavirus proteases, there is a distinct possibility of designing broad-spectrum protease inhibitors as antivirals [77].…”

Structural Biology of Noroviruses

“…They further suggest that peptido-mimetics with suitable warheads, a Glu-like chemical entity at P1 for optimal interactions with S1, and an appropriate combination of hydrophobic residues at P2 and P4 that maximizes the interactions with S2 and S4, have a direct impact on the potency of the inhibitors. Further studies should be anticipated that are directed at optimizing the design strategies and improving the pharmacokinetic properties of such inhibitors as antivirals using structural analysis and cell-based assays (Qu et al, 2014;Chang et al, 2006).…”