Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

A computationally designed ACE2 decoy has broad efficacy against SARS-CoV-2 omicron variants and related viruses in vitro and in vivo.

SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a 'decoy' to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy, FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments. FLIF displayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) against FLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys like FLIF may be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants.

SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response.

Reports of post-acute COVID-19 syndrome, in which the inflammatory response persists even after SARS-CoV-2 has disappeared, are increasing1, but the underlying mechanisms of post-acute COVID-19 syndrome remain unknown. Here, we show that SARS-CoV-2-infected cells trigger senescence-like cell-cycle arrest2,3 in neighboring uninfected cells in a paracrine manner via virus-induced cytokine production. In cultured human cells or bronchial organoids, these SASR-CoV-2 infection-induced senescent cells express high levels of a series of inflammatory factors known as senescence-associated secretory phenotypes (SASPs)4 in a sustained manner, even after SARS-CoV-2 is no longer detectable. We also show that the expression of the senescence marker CDKN2A (refs. 5,6) and various SASP factor4 genes is increased in the pulmonary cells of patients with severe post-acute COVID-19 syndrome. Furthermore, we find that mice exposed to a mouse-adapted strain of SARS-CoV-2 exhibit prolonged signs of cellular senescence and SASP in the lung at 14 days after infection when the virus was undetectable, which could be substantially reduced by the administration of senolytic drugs7. The sustained infection-induced paracrine senescence described here may be involved in the long-term inflammation caused by SARS-CoV-2 infection.

SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses.

Many patients with severe COVID-19 suffer from pneumonia and the elucidation of the mechanisms underlying the development of this severe condition is important. The in vivo function of the ORF8 protein secreted by SARS-CoV-2 is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2 and found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrated that the serum ORF8 protein levels are well-correlated with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a SARS-CoV-2 viral cytokine involved in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

Stimulation of interferon-ß responses by aberrant SARS-CoV-2 small viral RNAs acting as retinoic acid-inducible gene-I agonists.

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5' ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5' end svRNAs are RIG-I agonists and induce the IFN-ß response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5'-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5' end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs.

Phenothiazines inhibit SARS-CoV-2 cell entry via a blockade of spike protein binding to neuropilin-1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells using angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP-1) as the primary receptor and entry co-factor, respectively. Cell entry is the first and major step in initiation of the viral life cycle, representing an ideal target for antiviral interventions. In this study, we used a recombinant replication-deficient vesicular stomatitis virus-based pseudovirus bearing the spike protein of SARS-CoV-2 (SARS2-S) to screen a US Food and Drug Administration-approved drug library and identify inhibitors of SARS-CoV-2 cell entry. The screen identified 24 compounds as primary hits, and the largest therapeutic target group formed by these primary hits was composed of seven dopamine receptor D2 (DRD2) antagonists. Cell-based and biochemical assays revealed that the DRD2 antagonists inhibited both fusion activity and the binding of SARS2-S to NRP-1, but not its binding to ACE2. On the basis of structural similarity to the seven identified DRD2 antagonists, which included six phenothiazines, we examined the anti-SARS-CoV-2 activity of an additional 15 phenothiazines and found that all the tested phenothiazines shared an ability to inhibit SARS2-S-mediated cell entry. One of the phenothiazines, alimemazine, which had the lowest 50% effective concentration of the tested phenothiazines, exhibited a clear inhibitory effect on SARS2-S-NRP-1 binding and SARS-CoV-2 multiplication in cultured cells but not in a mouse infection model. Our findings provide a basis for the development of novel anti-SARS-CoV-2 therapeutics that interfere with SARS2-S binding to NRP-1.

Phenothiazines inhibit SARS-CoV-2 cell entry via a blockade of spike protein binding to neuropilin-1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells using angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP-1) as the primary receptor and entry co-factor, respectively. Cell entry is the first and major step in initiation of the viral life cycle, representing an ideal target for antiviral interventions. In this study, we used a recombinant replication-deficient vesicular stomatitis virus-based pseudovirus bearing the spike protein of SARS-CoV-2 (SARS2-S) to screen a US Food and Drug Administration-approved drug library and identify inhibitors of SARS-CoV-2 cell entry. The screen identified 24 compounds as primary hits, and the largest therapeutic target group formed by these primary hits was composed of seven dopamine receptor D2 (DRD2) antagonists. Cell-based and biochemical assays revealed that the DRD2 antagonists inhibited both fusion activity and the binding of SARS2-S to NRP-1, but not its binding to ACE2. On the basis of structural similarity to the seven identified DRD2 antagonists, which included six phenothiazines, we examined the anti-SARS-CoV-2 activity of an additional 15 phenothiazines and found that all the tested phenothiazines shared an ability to inhibit SARS2-S-mediated cell entry. One of the phenothiazines, alimemazine, which had the lowest 50% effective concentration of the tested phenothiazines, exhibited a clear inhibitory effect on SARS2-S–NRP-1 binding and SARS-CoV-2 multiplication in cultured cells but not in a mouse infection model. Our findings provide a basis for the development of novel anti-SARS-CoV-2 therapeutics that interfere with SARS2-S binding to NRP-1.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Engineering ACE2 decoy receptors to combat viral escapability.

Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

SARS-CoV-2 ORF6 disrupts nucleocytoplasmic trafficking to advance viral replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin ß1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-ß but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-ß. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.