Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance

Abstract

Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.

FIG 1

Treatment with 3CLpro inhibitor GRL-001 selects for resistant strains of MHV-A59. (A) Chemical structure of GRL-001 and dose-dependent inhibition of MHV-induced cell death. DBT cells were infected with infectious clone WT MHV (icMHV) at an MOI of 0.1 and treated with GRL-001 at serial concentrations. Cell viability was determined by cell titer Glo assay (Promega). The EC₅₀s were determined using nonlinear regression program with Prism 5 software. DMSO, dimethyl sulfoxide. (B) Scheme for selection of inhibitor-resistant viruses and evidence of selection for viruses that induce cytopathic effect in the presence of GRL-001. (C) Frequency of genotypes detected in plaque-purified MHV isolates. RNA was isolated from 31 randomly isolated plaques and the 3CLpro region was amplified and sequenced.

FIG 2

MHVs with two substitutions in 3CLpro are resistant to GRL-001. (A) Antiviral-activity assay reveals range of sensitivities to GRL-001. DBT cells were infected with virus at an MOI of 1, followed by addition of GRL-001 at the indicated concentrations. Cells were evaluated for viability using the cell titer Glo assay at 24 h postinfection. (B) EC₅₀s of WT and 3CLpro mutant viruses were calculated based on the results of the antiviral-activity assay. (C) Production of WT and mutant viruses produced in the presence of 3CLpro inhibitor GRL-001. DBT cells were incubated with viral inoculum for 1 h, and then the medium was replaced with fresh medium containing DMSO or GRL-001 inhibitor at 20 μM or 50 μM and cells were incubated for 17 h. The infectious virus titer in the supernatant was determined by plaque assay on DBT cells.

FIG 3

Inhibitor-resistant virus exhibits deficiencies in viral replication. (A) Representative plaques formed by WT and 3CLpro mutant MHVs at 48 h postinfection at 37°C. The relative areas of at least 12 single plaques of each virus were measured by Photoshop CS software to determine average area. n.s., not significant; *, P < 0.01; **, P < 0.001. (B and C) Growth kinetics of WT and 3CLpro mutant viruses in DBT cells infected at an MOI of 0.1. The error bars represent SD from the results of three plaque assays.*, P < 0.01.

FIG 4

Inhibitor-resistant viruses have delayed replicase processing. (A) Schematic diagram of 3CLpro-mediated replicase processing. (B to E) Comparison of proteolytic processing of WT and resistant viruses (infectious clone MHV-T26I/D65G [B and C] and plaque-purified isolate of inhibitor-resistant MHV [MHV-IR] that harbors T26I and A298D mutations [D and E]) by pulse-chase analysis. DBT cells were infected with WT or resistant viruses, and newly synthesized proteins were pulse-labeled with [³⁵S]Met for 20 or 30 min at 4.5 h postinfection. Labeling medium was removed and replaced with medium containing excess unlabeled methionine and cysteine, and cells were harvested at 30-min intervals during the chase period. Cell lysates were subjected to immunoprecipitation with antisera for nsp5 (B and D) and nsp8 (C and E), and the products were analyzed by 10% SDS-PAGE and visualized by autoradiography.

FIG 5

MHV-T26I/D65G inhibitor-resistant virus is highly attenuated. (A) C57BL/6 (WtB6) mice succumb to wild-type MHV (WT) but survive infection with mutant MHV (T26I/D65G). Four-week-old wild-type C57BL/6 mice were intracranially infected with 600 PFU of the WT (n = 5) or MHV-T26I/D65G mutant (n = 5). (B) Pathogenesis of MHV-T26I/D65G is delayed compared to that of WT MHV in highly susceptible type I IFN receptor knockout (IFNAR^−/−) mice. Fourteen-week-old IFNAR^−/− mice were intraperitoneally inoculated with 50 PFU of the WT (n = 6) or MHV-T26I/D65G (n = 6). Body weight loss was monitored daily. The statistical differences in survival were analyzed by Prism 5 software using the log rank test. (C) Morbidity and mortality in C57BL/6 mice following intracranial administration of WT and MHV-T26I/D65G. p.i., postinfection.

FIG 6

T26I/D65G MHV 3CLpro has reduced enzymatic efficiency and is not efficiently blocked by GRL-001. The enzymatic efficiency (k_app) (A) and inhibition by GRL-001 (IC₅₀) (B) of both WT and T26I/D65G 3CLpro were determined at ambient temperature (25°C) and 37°C. (A) The initial reaction rates (V_i) were determined by calculating the initial slope of the progress curve, which was then converted to the amount of product (μM) produced per minute using the experimentally determined value of the fluorescence extinction coefficient for UIVT3. Plotting Vi/[E] versus [UIVT3] gave a linear correlation, the slope of which was taken to be the k_app. (B) The inhibition of the 3CLpro by GRL-001 was monitored by following the change in RFUs over time, using the initial slope of the progress curve to determine the initial rate. The percent inhibition of the 3CLpro enzymes was then plotted as a function of inhibitor concentration. (C) k_app and IC₅₀s were determined using the nonlinear regression program SigmaPlot.

FIG 7

Esterase activities of WT and T26I/D65G MHV 3CLpro toward GRL-001 inhibitor. (A) Mechanism of GRL-001 hydrolysis catalyzed by MHV 3CLpro where the catalytic cysteine (Cys₁₄₅) is indicated, the inhibitor (GRL-001) is shown in bold type, and intermediates and hydrolysis products are labeled with Roman numerals and identified in the bottom left corner. (B) GRL-001 hydrolysis time point assay at 25°C, where the restoration of enzymatic activity correlates to the enzymatic rate of hydrolysis of GRL-001. WT and T26I/D65G MHV 3CLpro enzymes were incubated in a 1:2 enzyme/GRL-001 ratio at the appropriate temperature, and enzymatic activity was monitored by measuring the fluorescence intensity of the reaction after addition of the UIVT3 substrate at each time point and determined as a percentage of the appropriate uninhibited enzyme at each time point. Note that in the absence of coincubation with both the UIVT3 substrate and GRL-001, T26I/D65G MHV 3CLpro is substantially slower at GRL-001 hydrolysis than with the WT MHV 3CLpro enzyme. (C) GRL-001 hydrolysis time point assay at 37°C. Note the enhanced rates of both the WT and T26I/D65G MHV 3CLpro toward GRL-001 hydrolysis.

FIG 8

Context of sites that confer resistance to 3CLpro inhibitor GRL-001. (A) Alignment of 3CLpro amino acid sequence from selected CoVs with MUSCLE algorithm. Residues of 3CLpro associated with resistance in MHV (T26, D65, and A298) and corresponding sites among selected CoVs are highlighted. The Protein Data Bank identification numbers (PDB ID) of available structures of 3CLpros are listed. (B) Structural alignment of SARS-CoV 3CLpro (cyan, 2V6N) and HKU1 3CLpro (green, 3D23) to model the position identified in MHV 3CLpro that confers resistance to GRL-001. Catalytic residues Cys₁₄₅ and His₄₁ are labeled and colored (SARS-CoV 3CLpro, orange; HKU1 3CLpro, olive), and resistance-associated residues are labeled and colored (SARS-CoV 3CLpro, red, and HKU1 3CLpro, magenta).