Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors

Abstract

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (M^pro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the M^pro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where M^pro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC₅₀ assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by M^pro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that M^pro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the M^pro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Keywords: RNA-dependent RNA polymerase; antiviral drugs; antiviral resistance; coronavirus; main protease; pandemic.

FIG 1

Drug-resistant strains were obtained under the pressure of passaging with GC376. (A) Chemical structure of GC376. (B) GC376 passaging method for resistance. After 79-1146 was inserted into the 60-mm cell culture dish, it was passaged with increasing concentrations of GC376. (C and D) Inhibitory effect of GC376 for WT and P50 (a–e: GC376 added at concentrations of 0, 1, 5, 10, and 20 μM; f: EC₅₀ of GC376 for WT and P50).

FIG 2

A mutation in S925P in RdRp mediated partial resistance in the presence of GC376. (A) Ten nonsynonymous mutations in P50 nonstructural proteins. (B) EC₅₀ of WSU 79-1146 strains against GC376 for 10 nonsynonymous mutations of nonstructural proteins. Single-site viral rescue of 10 nonsynonymous mutations in nonstructural proteins of P50 based on an infectious cloning platform tested their EC₅₀ for GC376, which showed an 8-fold difference in EC₅₀ for P50 compared to the WT strain, for nsp12-S925P, there was a 3-fold difference, which was extremely significant. Error bars represent standard error of the mean (SEM). Statistical significance compared to the WT strain at each concentration was determined by one-way ANOVA and indicated with an asterisk (*); ***, P < 0.001. (C, D) Inhibitory effect and EC₅₀ of GC376 for nsp12-WT and nsp12-S925P (a–e: GC376 was added at concentrations of 0, 1, 2, 3, 4 μM; f: EC₅₀ of GC376). (E, F) Inhibitory effect and EC₅₀ of nirmatrelvir for nsp12-WT and nsp12-S925P (a–e: nirmatrelvir was added at concentrations of 0, 2, 4, 8, 10 μM; f: EC₅₀ of nirmatrelvir).

FIG 3

FIPV nsp12-P925 has better proliferation ability. (A) Growth kinetics of WT and nsp12-S925P strains, significantly different at 8 h and 40 h; error bars represent SEM. Statistical significance compared to the WT strain at each concentration was determined by t test and indicated with an asterisk; *, P < 0.05. (B) The expression level of nsp12 in nsp12-S925P was significantly higher than that in the WT strain. (C) IFA showed that the expression level of nsp12 in nsp12-S925P was significantly higher than that in the WT strain, and the dsRNAs were expressed at 10 h postinfection (hpi).

FIG 4

Nsp12-925 Pro has a higher nsp5 cleavage efficiency than Ser. (A) Comparison of the activity of FIPV M^pro in hydrolyzing different substrates. MOCK has no substrate added, FIPV-4/5 is the hydrolysis substrate nsp4 and nsp5, FIPV-12/13 S is the hydrolysis substrate nsp12 and nsp13, P5 site is Ser, FIPV-12/13 P is nsp12 and nsp13, and P5 is the hydrolysis substrate of Pro. (B) The binding mode of the M^pro with STVLQAAG complex. (C) The binding mode of the M^pro with PTVLQAAG complex. (D) Molecular docking results of polypeptides and proteins. Red boxes indicate differential amino acid structures.

FIG 5

Increasing the expression of nsp12 attenuated the inhibitory effect of GC376. (A) Construction of nsp12-overexpressing cell lines. Red indicates the expression of nsp12. (B–D) The proliferation ability of FIPV on different cell lines was detected by IFA, TCID50, and WB assays. Error bars represent SEM. Statistical significance compared to the WT strain at each concentration was determined by one-way ANOVA with Dunnett’s multiple comparison post hoc test and indicated with an asterisk; *, P < 0.05. (E) Inhibitory effect of GC376 on 79-1146 in different cell lines.

FIG 6

rQS-79 nsp12-S925P has the same resistance to GC376. (A) Comparison of nsp12 C-terminal amino acid homology between 79-1146 and rQS-79. All 925 positions are S. (B) Growth kinetics of rQS-79 nsp12-WT and rQS-79 nsp12-S925P, which were significantly different at 8 h, 16 h, 24 h, 32 h, and 40 h. Error bars represent SEM. Statistical significance compared to WT at each concentration was determined by t test and indicated with an asterisk; **, P < 0.01. (C) Inhibitory effect of GC376 on rQS-79 nsp12-WT and rQS-79 nsp12-S925P. (D, E) EC₅₀ of GC376 for rQS-79 nsp12-WT and rQS-79 nsp12-S925P.

FIG 7

GC376 is unable to treat rQS-79 nsp12-S925P-induced FIP. (A) Over time, a cat’s body temperature responds to viral infection. The green arrow is the start time of GC376 treatment, which lasted for 2 weeks; the same is true below. (B) The cat’s clinical status score (add 1 point for any of the following conditions and 5 points for death) was determined as follows: 1. cats with fever (>39°C); 2. cats with anorexia; 3. cats with lethargy; 4. cats with secretions from eyes and nose; and 5. abnormal excretion. (C) Mean relative weight at euthanasia or 21 days after inoculation. On day 0 before challenge, the body weight was 100%. (D) Survival of different cats in each group. (E) During GC376 treatment, the viral load in each organ in the dead cats in the nsp12-S925P group was detected by virus-specific qRT–PCR. (F) Gross necropsy and histopathological analysis of cats that died in the nsp12-S925P group during GC376 treatment. Gross necropsy revealed gross lesions consistent with FIP, such as parenchymal and serous pyogranulomas of various sizes in affected organs. In these FIP cats, fibrinogenic to granulomatous serositis affected the spleen, liver, and kidney. In the analysis of hematoxylin and eosin (HE) staining of different tissue sections, granuloma-related lesions were surrounded by dense inflammatory infiltrates. Immunohistochemical antirabbit antiserum against FIPV N protein (brown) detected viral antigens in the macrophages in cat lesions in the nsp12-S925P group.