A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated a continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 Δ3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging Δ3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 Δ3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using the rSARS-CoV-2 Δ3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2.

Importance: Small-molecule antiviral drugs have been used for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. However, drug-resistant SARS-CoV-2 mutants to currently United States Food and Drug Administration-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remains limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins (Δ3a7b-Nluc) to identify nirmatrelvir-resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using Δ3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 Δ3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 Δ3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

Keywords: SARS-CoV-2; attenuated virus; drug-resistance mutations; nanoluciferase; nirmatrelvir.

Fig 1

Generation and in vitro characterization of Δ3a7b-Nluc. (A) Schematic representation of the viral genomes of rSARS-CoV-2 WT (top), Δ3a7b (middle), and Δ3a7b-Nluc (bottom). (B) The plaque phenotype of rSARS-CoV-2 WT (top), Δ3a7b (middle), and Δ3a7b-Nluc (bottom) in Vero-AT was determined by crystal violet staining (left), Nluc expression (middle), and N protein staining (right). (C) Vero-AT cells (six-well plate format, triplicates) were infected (MOI 0.01) with rSARS-CoV-2 WT (yellow), Δ3a7b (brown), or Δ3a7b-Nluc (blue). At the indicated hours post-infection, cell culture supernatants were collected, and the presence of virus was determined by plaque assay. LoD: limit of detection. (D) Cell culture supernatants from (C) were used to evaluate the presence of Nluc. Data are presented as mean ± SD. The statistical significance was analyzed with one-way analysis of variance, followed by Tukey’s test (***P < 0.001).

Fig 2

Antiviral activity of nirmatrelvir and remdesivir against Δ3a7b-Nluc. The antiviral activity of nirmatrelvir (A) and remdesivir (B) against Δ3a7b and Δ3a7b-Nluc in Vero-AT cells (96-well plate format, quadruplicates) was evaluated using a PRNT assay. EC₅₀ values of the different antivirals were calculated using GraphPad Prism. Data are presented as means ± SD. Dotted line indicates 50% of viral inhibition.

Fig 3

Selection of nirmatrelvir drug-resistant mutants (DRM-N). (A) 3a7b-Nluc was cultured in the presence of increasing concentrations of nirmatrelvir for 10 serial passages (P0–P10). RNA from Δ3a7b-Nluc P10 DRM-N was isolated and used for NGS and Sanger sequencing to identify DRM-N. Then, Δ3a7b-Nluc containing the identified individual and combined mutations was rescued using BAC-based reverse genetics. (B) Vero-AT cells (six-well plate format) were infected (MOI 0.001) with P0 Δ3a7b-Nluc (top) and P10 Δ3a7b-Nluc DRM-N (bottom). After viral adsorption, infectious media were replaced by post-infection media containing the indicated concentrations of nirmatrelvir (0, 2.5, 10, and 40 µM). At 48 h post-infection, cells were fixed and permeabilized, and the presence of viral N protein was determined using the SARS-CoV 1C7C7 cross-reactive monoclonal antibody and an anti-mouse FITC-conjugated secondary antibody. The cell nucleus was stained with DAPI. Scale bars = 200 µm.

Fig 4

P10 Δ3a7b-Nluc DRM-N resistance to nirmatrelvir and remdesivir. The antiviral activity of nirmatrelvir (A–C) and remdesivir (D–F) against parental (blue) and P10 DRM-N (red) Δ3a7b-Nluc in Vero-AT cells (96-well plate format, quadruplicates) was evaluated using PRNT (A and D), OD₅₆₀ (B and E), and Nluc (C and F) assays. EC₅₀ values of the different antivirals were calculated by using GraphPad Prism. Data are presented as means ± SD. The dotted line indicates 50% of viral inhibition.

Fig 5

Identification of P10 Δ3a7b-Nluc DRM-N mutations. (A) NGS was performed on RNA isolated from Δ3a7b-Nluc that was cultured in the presence of PBS or increasing concentrations of nirmatrelvir for 10 serial passages, P10 PBS, and P10 DRM-N. Identified amino acid changes and their protein locations are indicated. DRM-N mutations L50F and R188G located in the NSP5 gene are indicated with a red rectangle. (B and C) RNA used for NGS was used for the RT-PCR amplification of NSP5 as well as for Sanger sequencing to confirm the presence of L50F (B) and R188G (C) mutations in P10 Δ3a7b-Nluc DRM-N, which are indicated with red rectangles.

Fig 6

Characterization of Δ3a7b-Nluc L50F, R188G, and L50F + R188G mutants. (A) Vero-AT cells (six-well plate format) were infected with Δ3a7b-Nluc parental, L50F, R188G, and L50F + R188 viruses. At 72 h post-infection, plates were incubated with crystal violet (top) or stained with Nluc substrate (bottom). (B) Vero-AT cells (six-well plate format, triplicates) were infected (MOI 0.01) with Δ3a7b-Nluc parental, L50F, R188G, and L50F + R188 viruses. At the indicated hours post-infection, cell culture supernatants were collected, and the presence of virus was determined by plaque assay. LoD: limit of detection. The antiviral activity of nirmatrelvir (C) and remdesivir (D) against parental Δ3a7b-Nluc and DRM-N L50F, R188G, and L50F + R188 viruses was determined in Vero-AT cells (96-well plate format, quadruplicates) by Nluc activity. The EC₅₀ values of the different antivirals were calculated using GraphPad Prism. Data are presented as means ± SD. The dotted line indicates 50% of viral inhibition. Data are presented as mean ± SD. The statistical significance was analyzed with one-way analysis of variance, followed by Tukey’s test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 7

Schematic ribbon diagram of Mpro-nirmatrelvir binding. Amino acids L50 (red) and R188 (blue) in Mpro (green) are shown. Nirmatrelvir is indicated in orange. The Mpro-nirmatrelvir complex was obtained from PDBID: 7VH8.