Abstract
ABSTRACTHuman norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro. Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.
Highlights
Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking
The replicon cells were passaged on days 3, 8, and 12, and the levels of replicon RNA were quantified by quantitative reverse transcription-PCR (qRT-PCR)
The EC50 and CC50 values of rupintrivir in the HGT-NV replicon system were 1.3 Ϯ 0.1 M and Ͼ100 M, respectively, which were comparable to those presented in a previous report describing a study with an HG23 cell system based on the same aac.asm.org 9 replicon in the Huh-7 hepatocellular carcinoma cell line [24]
Summary
Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. The lack of a robust cellular system for the analysis of viral replication has hampered antiviral research against HuNoV infection for many years [5] This situation has been changed by the recent development of enterocytes derived from stem cell and B cell culture systems [6, 7]. We sought to examine whether resistant replicons could be identified following prolonged culture in the presence of a suitable antiviral To achieve this aim, we utilized rupintrivir (AG7088), an irreversible inhibitor of the human rhinovirus (HRV) 3C protease. We concluded that mutations around the norovirus protease active site lead to the generation of rupintrivir resistance; some of these mutations appear to compromise viral fitness, at least in the context of the MNV infection model
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