Analysis of SARS-CoV-2 Mutations after Nirmatrelvir Treatment in a Lung Cancer Xenograft Mouse Model.

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (Mpro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in Mpro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the Mpro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC50 values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

A mouse xenograft long-term replication yields a SARS-CoV-2 Delta mutant with increased lethality.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2.

Introduction: The spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection. Methods: We established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice. Results: After infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus. Discussion: These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model.

Influenza is a major cause of highly contagious respiratory illness resulting in high mortality and morbidity worldwide. Annual vaccination is an effective way to prevent infection and complication from constantly mutating influenza strains. Vaccination utilizes preemptive inoculation with live virus, live attenuated virus, inactivated virus, or virus segments for optimal immune activation. The route of administration also affects the efficacy of the vaccination. Here, we evaluated the effects of inoculation with ultraviolet (UV)-inactivated or live influenza A virus strains and compared their effectiveness and cross protection when intraperitoneal and intramuscular routes of administration were used in mice. Intramuscular or intraperitoneal inoculation with UV-inactivated Influenza A/WSN/1933 provided some protection against intranasal challenge with a lethal dose of live Influenza A/WSN/1933 but only when a high dose of the virus was used in the inoculation. By contrast, inoculation with a low dose of live virus via either route provided complete protection against the same intranasal challenge. Intraperitoneal inoculation with live or UV-inactivated Influenza A/Philippines/2/1982 and intramuscular inoculation with UV-inactivated Influenza A/Philippines/2/1982 failed to produce cross-reactive antibodies against Influenza A/WSN/1933. Intramuscular inoculation with live Influenza A/Philippines/2/1982 induced small amounts of cross-reactive antibodies but could not suppress the cytokine storm produced upon intranasal challenge with Influenza A/WSN/1993. None of the tested inoculation conditions provided observable cross protection against intranasal challenge with a different influenza strain. Taken together, vaccination efficacy was affected by the state and dose of the vaccine virus and the route of administration. These results provide practical data for the development of effective vaccines against influenza virus.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells.

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

Abiraterone Acetate Attenuates SARS-CoV-2 Replication by Interfering with the Structural Nucleocapsid Protein.

The drug repurposing strategy has been applied to the development of emergency COVID-19 therapeutic medicines. Current drug repurposing approaches have been directed against RNA polymerases and viral proteases. Recently, we found that the inhibition of the interaction between the SARS-CoV-2 structural nucleocapsid (N) and spike (S) proteins decreased viral replication. In this study, drug repurposing candidates were screened by in silico molecular docking simulation with the SARS-CoV-2 structural N protein. In the ChEMBL database, 1994 FDA-approved drugs were selected for the in silico virtual screening against the N terminal domain (NTD) of the SARS-CoV-2 N protein. The tyrosine 109 residue in the NTD of the N protein was used as the center of the ligand binding grid for the docking simulation. In plaque forming assays performed with SARS-CoV-2 infected Vero E6 cells, atovaquone, abiraterone acetate, and digoxin exhibited a tendency to reduce the size of the viral plagues without affecting the plaque numbers. Abiraterone acetate significantly decreased the accumulation of viral particles in the cell culture supernatants in a concentration-dependent manner. In addition, abiraterone acetate significantly decreased the production of N protein and S protein in the SARS-CoV-2-infected Vero E6 cells. In conclusion, abiraterone acetate has therapeutic potential to inhibit the viral replication of SARS-CoV-2.

Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43.

Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with "common colds", infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies.

The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.

Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein.

SARS-CoV-2 infections continue to spread quickly by human-to-human transmission around the world. Therefore, developing methods to rapidly detect SARS-CoV-2 with high sensitivity are still urgently needed. We produced a monoclonal antibody that specifically detects the N protein of SARS-CoV-2 and recognizes N protein in cell lysates of SARS-CoV-2-infected Vero cells but not in cell lysates of MERS-CoV- or HCoV-OC43-infected Vero cells. This antibody recognized N protein in SARS-CoV-2 clades S, GR, and GH and recognized N protein in all the SARS-CoV-2 clades to ∼300 pfu. Previously, we reported that the coronavirus N protein interacts with the C-terminal domain of the spike protein (Spike CD). In this study, we developed an ELISA-based "bait and prey" system to confirm the interaction between SARS-CoV-2 Spike CD and N protein using recombinant fusion proteins. Furthermore, this system can be modified to quantitatively detect SARS-CoV-2 in culture media of infected cells by monitoring the interaction between the recombinant Spike CD fusion protein and the viral N protein, which is captured by the N protein-specific antibody. Therefore, we conclude that our N protein-specific monoclonal antibody and our ELISA-based bait and prey system could be used to diagnose SARS-CoV-2 infections.

Apoptosis Enhances the Replication of Human Coronavirus OC43.

Human coronavirus OC43 (HCoV-OC43) is one of the coronaviruses causing a mild common cold, but few studies have been made on this strain. Here, we identified the molecular mechanisms involved in HCoV-OC43-induced apoptosis and its implications for viral reproduction in Vero cells and MRC-5 cells. HCoV-OC43 infection induced apoptosis that was accompanied by cleavage of caspase-3 and PARP, degradation of cyclin D1, and cell cycle arrest at S and G2M phases. Dephosphorylation of STAT1 and STAT3, induced by HCoV-OC43 infection, was also associated with HCoV-OC43-mediated apoptosis. The pan-caspase inhibitor effectively prevented HCoV-OC43-induced apoptosis and reduced viral replication, suggesting that apoptosis contributes to viral replication. Collectively our results indicate that HCoV-OC43 induces caspase-dependent apoptosis to promote viral replication in Vero cells and MRC-5 cells.

MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein.

Background: The molecular interactions between viral proteins form the basis of virus production and can be used to develop strategies against virus infection. The interactions of the envelope proteins and the viral RNA-binding nucleocapsid (N) protein are essential for the assembly of coronaviruses including the Middle East respiratory syndrome coronavirus (MERS-CoV). Methods: Using co-immunoprecipitation, immunostaining, and proteomics analysis, we identified a protein interacting with the spike (S) protein in the cells infected with MERS-CoV or SARS-CoV-2. To confirm the interaction, synthetic peptides corresponding to the C-terminal domain of the S protein (Spike CD) were produced and their effect on the interaction was investigated in vitro. In vivo effect of the Spike CD peptides after cell penetration was further investigated using viral plaque formation assay. Phylogeographic analyses were conducted to deduce homology of Spike CDs and N proteins. Results: We identified a direct interaction between the S protein and the N protein of MERS-CoV that takes place during virus assembly in infected cells. Spike CD peptides of MERS-CoV inhibited the interaction between the S and N proteins in vitro. Furthermore, cell penetration by the synthetic Spike CD peptides inhibited viral plaque formation in MERS-CoV-infected cells. Phylogeographic analyses of Spike CDs and N proteins showed high homology among betacoronavirus lineage C strains. To determine if Spike CD peptides can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we used the same strategy and found that the SARS-CoV-2 Spike CD peptide inhibited virus replication in SARS-CoV-2-infected cells. Conclusions: We suggest that the interaction between the S protein and the N protein can be targeted to design new therapeutics against emerging coronaviruses, including SARS-CoV-2.