(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Increased flexibility of the SARS-CoV-2 RNA-binding site causes resistance to remdesivir [1]

['Shiho Torii', 'Laboratory Of Virus Control', 'Research Institute For Microbial Diseases', 'Osaka University', 'Suita', 'Kwang Su Kim', 'Interdisciplinary Biology Laboratory', 'Iblab', 'Division Of Biological Science', 'Graduate School Of Science']

Date: 2023-05

Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. We confirmed that all the mutant viruses didn’t gain the virus production efficiency without remdesivir treatment. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

Considering the emerging Omicron strain, quick characterization of SARS-CoV-2 mutations is important. However, owing to the difficulties in genetically modifying SARS-CoV-2, limited groups have produced multiple mutant viruses. Our cutting-edge reverse genetics technique enabled construction of eight reporter-carrying SARS-CoV-2 with the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. We confirmed that all the mutant viruses didn’t gain the virus production efficiency without remdesivir treatment. We developed a mathematical model taking into account sequential changes and identified antiviral effects against mutant viruses with differing propagation capacities and lethal effects on cells. In addition to identifying the positions of mutations, we analyzed the structural changes in SARS-CoV-2 NSP12 by computer simulation to understand the mechanism of resistance. This multidisciplinary approach promotes the evaluation of future resistance mutations.

Funding: This study was supported in part by a Grant-in-Aid for JSPS Scientific Research (KAKENHI) (21H02736 to TF, 19K24679 to TF, 18KT0018 to SI, 18H01139 to SI, 16H04845 to SI, 20H04281 to TS); Scientific Research in Innovative Areas (20H05042 to SI, 19H04839 to SI, 18H05103 to SI, 20H04841 to TS); AMED CREST (19gm1310002 to SI, JP22gm1610008 to TF); AMED Japan Program for Infectious Diseases Research and Infrastructure (20wm0225002 to TF, JP20he0822006 to TF, JP20fk0108264 to TF, JP20he0822008 to TF, JP20wm0225003 to TF, JP20fk0108267 to TF, JP19fk0108113 to TF, JP20wm0125010 to TF, 20wm0325007h0001 to SI, 20wm0325004s0201 to SI, 20wm0325012s0301 to SI, 20wm0325015s0301 to SI); AMED Research Program on Emerging and Re-emerging Infectious Diseases (20fk0108401 to TF, 20fk010847 to TF, 21fk0108617 to TF, 20fk0108451 to TF, 19fk0108050h0003 to SI, 19fk0108156h0001 to SI, 20fk0108140s0801 to SI and 20fk0108413s0301 to SI); AMED Program for Basic and Clinical Research on Hepatitis (19fk0210036h0502 to SI); AMED Program on the Innovative Development and the Application of New Drugs for Hepatitis B (19fk0310114h0103 to SI); JST MIRAI to SI; Moonshot R&D (JPMJMS2021 to SI, JPMJMS2025 to SI); Mitsui Life Social Welfare Foundation to SI; Shin-Nihon of Advanced Medical Research to SI; Suzuken Memorial Foundation to SI; Life Science Foundation of Japan to SI; SECOM Science and Technology Foundation to SI; The Japan Prize Foundation to SI; Daiwa Securities Health Foundation to SI. AMED 20fk0108401 and 20fk010847 were the sources of funding for the construction of all mutant SARS-CoV-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we attempted to identify multiple RDV-resistant mutations and examine the mechanisms of RDV resistance by a multidisciplinary approach that integrates state-of-the-art reverse genetics, mathematical modeling, and molecular dynamics analyses. We first predicted the presumed RDV-resistant mutations by in vitro passages of SARS-CoV-2 in the presence of RDV. Next, the recombinant viruses carrying the predicted mutations were generated by the CPER method and the efficiency of infectious virus production and antiviral effects of RDV on the mutants were examined by mathematical modeling. Finally, the conformational changes of NSP12 induced by mutations were analyzed by molecular dynamics simulations to understand the mechanisms of RDV resistance.

To evaluate the effect of each gene mutation on viral propagation, genetically modified viruses should be engineered using the reverse genetics system. We recently established a quick reverse genetics system for SARS-CoV-2 using the circular polymerase extension reaction (CPER) method [ 21 ]. Nine viral genome fragments, which cover the full-length viral genome, and a linker fragment that encodes the promoter sequence were amplified by PCR and connected to obtain the circular viral DNAs by an additional PCR. By direct transfection of the circular DNAs, infectious SARS-CoV-2 was rescued. Introduction of reporters or mutations can be quickly completed by overlapping PCR or plasmid mutagenesis using the desired gene fragments of less than 5,000 base pairs (bp). While other reverse genetics systems for SARS-CoV-2 require specific techniques such as in vitro transcription or in vitro ligation, which are obstacles to mutagenesis [ 22 , 23 ], our method does not need these and has already been applied to the characterization of several viral mutations observed in the different SARS-CoV-2 variants [ 24 , 25 ], allowing us to simultaneously generate multiple mutants [ 26 ].

Remdesivir (RDV) (GS-5734) is the US Food and Drug Administration (FDA)-approved drug for treatment of coronavirus disease 2019 (COVID-19) patients [ 11 , 12 ]. The compound is an intravenously administered adenosine analogue prodrug that binds to the viral RNA-dependent RNA polymerase and inhibits viral replication. It has demonstrated antiviral activities against a broad range of RNA viruses including Ebolavirus, SARS-CoV, MERS-CoV and SARS-CoV-2 [ 13 – 17 ]. RDV has been widely used in the treatment of SARS-CoV-2 patients, however only two amino acid mutations (D484Y and E802D in non-structural protein [NSP]12) were identified from SARS-CoV-2 patients that were administered RDV [ 18 , 19 ]. One mutation (E802D) was also found in in vitro serial passages of the virus under treatment of RDV [ 20 ]. Although studies regarding E802D revealed that the mutation decreased viral susceptibility to RDV [ 19 , 20 ], the mechanisms of how resistance arises have not yet been analyzed in detail. It is critical to elucidate the mechanisms of RDV resistance and to identify further RDV-resistant mutants that may arise in the future to circumvent resistance mutations before they become established in circulating strains.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in 2019 and quickly spread around the world [ 1 ]. Novel SARS-CoV-2 variants have since continued to emerge and the number of virus-infected cases repeats increases and decreases [ 2 ]. The clinical spectrum of SARS-CoV-2 infection ranges from mild to critical. While most infections present mild or minor symptoms (e.g. fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell), severe acute respiratory disease requires admission to intensive care [ 3 – 5 ]. The illness can be observed even after successful vaccination [ 6 ]. Antiviral drugs that can be administered to patients after moderate or severe clinical symptoms have been observed have played important roles in clinical environments. Therefore, it is vital to understand the effectiveness of currently approved antivirals from multiple angles to develop future drugs. In particular, the potential to drive drug resistance should be evaluated because drug-resistant mutations have been observed in several viruses such as influenza A virus, human immunodeficiency virus and hepatitis B virus in the clinical environment [ 7 – 10 ].

Results

In vitro serial passages of SARS-CoV-2 in the presence of remdesivir To identify the genes presumably involved in RDV resistance, we first passaged the SARS-CoV-2 strain SARS-CoV-2/Hu/DP/Kng/19-020 (GenBank: LC528232.2) in HEK293-C34 cells under treatment with RDV (Fig 1A). In order to assess many viral mutations responsible for RDV resistance, we treated the cells with RDV at the highest concentration that would allow the virus to be passed on to the next generation. First of all, the cells were treated with 0.1 μM RDV, however cytopathic effect (CPE) was not observed in 14 days. The cells were then treated with 0.01 μM RDV 0.01 μM and CPE was observed at day 4. The culture supernatants were collected, stored as a Passage 1 (P1) sample (Fig 1A). The concentration of RDV was gradually increased from 0.01 μM to 4.0 μM over 10 passages. Throughout the passages, the virus-infected cells were cultured until CPE was observed (3–8 days). Whereas no CPE was seen for 14 days after 0.1 μM RDV treatment in original viruses (P0), CPE was observed throughout the wells in the presence of 4.0 μM RDV at P10, indicating that the virus decreased susceptibility to RDV during the passages. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Identification of RDV-resistant mutations of SARS-CoV-2. (A) Schematic image of the in vitro serial passage of SARS-CoV-2 in the presence of RDV. Supernatants of virus-infected cells were passaged with gradually increased concentrations of RDV. The virus sequence was examined after 10 passages. (B) The SARS-CoV-2 genome with the locations of mutations, which demonstrated more than 80% of frequency in Miseq sequence and more than 90% of frequency in single virus sequence after 10-time passages of SARS-CoV-2 with RDV. https://doi.org/10.1371/journal.ppat.1011231.g001 After 10 passages with RDV, the culture supernatants were collected (P10 with RDV) and subjected to MiSeq sequencing to determine the full-length viral sequence (DDBJ Accession number: LC742929). We prepared the viruses passaged 10 times without RDV as a control (P10 without RDV). Comparison of the P10 with RDV virus sequence with the original SARS-CoV-2 genome found 14 unique mutation sites and 6 mutation sites in P10 without RDV virus sequence (Fig 1B and S1 Table). Five mutations found in P10 without RDV had a less than 50% mutant frequency, and mutations were not found in NSP7, 8, 12 nor 13, which are involved in the formation of the replication complex. Importantly, amino acid substitutions E796G and C799F in NSP12 were observed only in the P10 with RDV virus and there have been no reports of these two mutations to date, according to Nextstrain [27]. We then conducted the Sanger sequencing of the viruses after the limiting dilution cloning to investigate whether mutations are introduced in the same virus genomes or not. In total of 10 clones were obtained by the limiting dilution cloning after 10 times passage of SARS-CoV-2 with RDV (P10 with RDV). The deletion of nine nucleotides in NSP1 (82GHVM85V) was observed in 9 single viruses, and amino acid substitutions in NSP4 (V294L), NSP6 (L260F) and NSP12 (E796G and C799F) were observed in all 10 viruses, indicating that these mutations had been introduced in a single virus together (S2 Table). In addition, these mutations demonstrated more than 80% of frequency by Miseq sequence. According to Nextstrain, the same mutations had been detected in NSP1, NSP4 and NSP6, but only a few cases of each mutation had been reported. Besides, the deletion of 15 nucleotides in S, start-loss of ORF3A and S194P in N were observed in every single virus and detected by MiSeq sequence as well and these mutations have not been reported in Nextstrain.

Generation of RDV-resistant SARS-CoV-2 We then generated high-affinity NanoBiT (HiBiT)-carrying recombinant SARS-CoV-2 with each mutation to identify the RDV-resistant mutations based on strain JPN/TY/WK-521 (GISAID accession number: EPI_ISL_408667). NanoLuc enzymatic activity can be detected by interaction of HiBiT and large NanoBiT (LgBiT), which constitute a split reporter. The reporter SARS-CoV-2 can be generated by inserting only 11 amino acids into the viral genome, and HiBiT-carrying viruses exhibit similar growth kinetics to wildtype (WT) virus [21]. All recombinant SARS-CoV-2 with HiBiT and mutations were prepared using the CPER method that was previously established by our group. Amino acid substitutions were introduced by overlapping PCR and the full-length sequences of the mutant viruses were confirmed prior to assay by Sanger sequencing. Because RDV acts as a nucleoside analog and targets the RNA-dependent RNA polymerase (RdRp) of coronaviruses, including SARS-CoV-2, in the current study we focused on the mutations in NSP12. We generated recombinant viruses with the E796G or C799F mutations that were observed in our P10 serial virus passage (Table 1). We also prepared recombinant viruses with mutations which demonstrated more than 80% of frequency in MiSeq sequence and was common in more than 9 out of 10 single virus clones after P10 (R10/E796G/C799F) or with mutations except for E796G (R10/C799F). In addition to the mutations observed in this study, we also characterized mutations that have been reported as, or anticipated to be, resistant to RDV, as listed in Table 1. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Table 1. Generation of the recombinant SARS-CoV-2 carrying HiBiT reporter and mutations. https://doi.org/10.1371/journal.ppat.1011231.t001 The amino acid mutation E802D in NSP12 was found during the serial passage of SARS-CoV-2 in vitro in the presence of RDV and another report showed that the same mutation was found in patients receiving RDV [19,20]. The D484Y mutation was also identified in a COVID-19 patient receiving RDV treatment [18]. Previously, amino acid substitutions F476L and V553L were identified as RDV-resistant mutations in the Betacoronavirus murine hepatitis virus (MHV)(15). The two affected amino acid residues (476F and 553L in MHV) are conserved across coronaviruses and correspond to 480F and 557V in the SARS-CoV-2 genome. We attempted to generate recombinant SARS-CoV-2 with either or both mutations but the virus with the single V557L mutation could not be rescued. We then examined the sensitivity of recombinant viruses to RDV (S1 Fig). Viruses were cultured in the presence of RDV at 0–1.0 μM final concentration for 48 hours and luciferase activity was measured and normalized against control without RDV treatment (0 μM final concentration). The 50% effective concentration (EC 50 ) was calculated using the drc package (v3.0–1; R Project for Statistical Computing). All tested mutant viruses showed greater EC 50 than WT virus, although the difference between WT and F480L mutant was small, indicating that the mutations observed in NSP12 led to decreased viral sensitivity to RDV.

Time course analyses of infection with presumed RDV-resistant mutants To investigate the growth efficiency of mutant viruses and the antiviral effects of RDV, we first performed time course analyses of infectious virus production with or without RDV for 96 hours and demonstrated the data up to 72 hours post infection (hpi) because most of the cells have been died and the viral titer have been decreased (Fig 2A and 2B). At all the indicated time points, the infectious titer of each mutant virus was similar or lower than that of the WT virus in the absence of RDV treatment, indicating that the mutant viruses produced the infectious viruses with the same or lower efficiency as WT virus (Fig 2A). Conversely, significant differences were observed in the infectious titers of mutant viruses in the presence of RDV at 0.05 μM final concentration. In the left panel of Fig 2B, the infectious titers of mutant viruses (E796G, C799F, R10/E796G/C799F, and R10/C799F) gradually increased for 48 hours and were significantly higher than that of WT virus at 72 hpi. In the right panel of Fig 2B, the infectious titer of the E802D mutant virus increased rapidly and was significantly higher than that of WT virus at 48 and 72 hpi. The titers of the F484Y and F480L/V557L mutants were also significantly higher than that of WT virus. Meanwhile there were no differences between the titers of F480L mutant and WT viruses at the indicated time points. These results suggest that the sensitivity of all the mutant viruses, except for the F480L virus, to RDV was diminished, which was consistent with the results of the RDV susceptibility test demonstrating minimal change in the EC 50 of the F480L virus (S1 Fig). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Infection kinetics of recombinant SARS-CoV-2 with NSP12 mutations. (A and B) Infectious virus production in the absence (A) and presence (B) of RDV. Supernatants of virus-infected cells were collected from 12–96 hpi and virus titers were determined by titration. Statistical significances were determined by Kruskal–Wallis test with the two stage linear step-up procedure of Benjamini, Kreiger and Yekutieli. Significant differences compared with WT virus are indicated by an asterisk (*p<0.05). (C and D) Infection rate in the absence (C) and presence (D) of RDV. Virus-infected cells were fixed and stained with antibodies for SARS-CoV-2 and cell nuclei. Infection rates were calculated by dividing the numbers of virus-positive cells by the numbers of nuclei. Statistical significances were determined by one-way ANOVA with Dunnett’s test. Significant differences compared with WT virus are indicated by asterisks (***p<0.001). https://doi.org/10.1371/journal.ppat.1011231.g002 Next, we investigated the the ratio of the virus-infected cells (Fig 2C and 2D). HEK293-C34 cells were infected with mutant viruses with and without RDV treatment. Virus-infected cells were harvested and fixed from 12–96 hpi and subjected to immunofluorescent assay using anti-SARS-CoV-2 NP antibody and DAPI. The virus infection rates were then calculated and demonstrated up to 72 hpi. All the mutant viruses demonstrated equivalent or significantly lower virus infection rates compared with WT virus in the absence of RDV. These data suggested that the number of cells infected with mutant viruses increased more slowly compared with WT virus in the absence of RDV treatment, consistent with the data on production of infectious virus particles. Meanwhile, the infection rates of the presumed RDV-resistant mutant viruses, except for D484Y, were higher or significantly higher (R10/E796G/C799F at 48 and 72 hpi, and R10/C799F at 72 hpi) than those of WT virus at 48 and 72 hpi in the presence of RDV, indicating that these mutant viruses can spread more efficiently than WT virus in the presence of RDV.

RDV-resistance mechanisms of the presumed RDV-resistant mutants, analyzed by computer simulation Molecular modeling and molecular dynamics simulations were performed to clarify the structural and property changes caused by amino acid mutations in the NSP12 protein. The representative complete structure of the prepared protein-RNA complex is shown in Fig 4A. In this figure, NSP12, binding RNA, and RDV are shown in cartoon, ball and stick, and van der Waals notation, respectively. RDV is located at the end of the binding RNA and is inside the protein. Then, the root mean square deviations (RMSDs) were compared using molecular dynamics simulation trajectories of each complex of WT or mutant NSP12 protein and RNA-incorporated RDV, as shown in Fig 4B. Most complexes reached thermodynamic stability and plateau in RMSD after 200,000 steps. However, only the V557L mutant structure failed to reach the stabilized structure. When RNA structures for this mutant were superimposed and RMSDs were calculated for RNA and protein separately, the increase in RMSDs for RNA reached a plateau, while the RMSDs for protein continued to increase as before. This behavior means the bonds between protein and RNA tended to move apart. In other words, the RNA-protein complex tended to be unstable, which may correspond with the inability of this mutant virus to multiply, indicating the probable reason for failed rescue of the V557L mutant recombinant SARS-CoV-2 (Table 1). Therefore, we proceeded with the analysis of the other mutants only. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. NSP12-RNA binding structure and comparison of thermodynamic stability. (A) Overall view of NSP12 protein and location of the RDV. (B) RMSD comparison of RNA-binding proteins. (C) Comparison of the molecular vibrations of WT and each mutant. The pink and blue spheres represent regions of large oscillations in the mutant and WT, respectively. https://doi.org/10.1371/journal.ppat.1011231.g004 Using the trajectories obtained from molecular dynamics calculations, we calculated and compared the variation of RMSD for each substructure. The RMSDs of each mutant complex and the WT complex were searched for areas where they differed significantly, and the results are shown in Fig 4C. Locations where the RMSD variation of the mutant is greater than that of the WT are indicated by pink spheres, and conversely, locations where the RMSD variation of the WT is greater than that of the mutant are indicated by blue spheres. In the tested mutations, except for F480L, the molecular vibration of the mutants tended to increase around the RNA-binding site as shown in S4 Fig, indicating that introduction of the mutations increased the flexibility of the RNA binding site. However, in the center of the RNA-binding site (near the RDV-binding site) of the F480L mutant, the molecular vibration of the mutant tended to be small, which is consistent with the small change in antiviral effect (Fig 3C) and RDV sensitivity (S1 Fig). Taken together, NSP12 mutations found in previous studies and in our in vitro virus passages decreased the antiviral effect of RDV, though not to the same degree, and influenced increased flexibility of the RNA-binding site of the NSP12 structure.

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011231

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.