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1.
Commun Biol ; 5(1): 516, 2022 05 30.
Article in English | MEDLINE | ID: covidwho-1947507

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Bronchi , Humans , Organoids
2.
Commun Biol ; 5(1): 483, 2022 05 19.
Article in English | MEDLINE | ID: covidwho-1852521

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Viral Proteins/metabolism , Animals , Antiviral Agents , Biological Transport , Cricetinae , Viral Proteins/genetics , Virus Replication , alpha Karyopherins/genetics , alpha Karyopherins/metabolism
3.
Sci Transl Med ; 14(650): eabn7737, 2022 Jun 22.
Article in English | MEDLINE | ID: covidwho-1807308

ABSTRACT

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.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Animals , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , Humans , Immunization, Passive , Mice , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2
5.
Nat Commun ; 12(1): 3802, 2021 06 21.
Article in English | MEDLINE | ID: covidwho-1387351

ABSTRACT

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.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/pharmacology , COVID-19/drug therapy , Mutation , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cricetinae , Crystallography, X-Ray , Disease Models, Animal , Humans , Male , Molecular Dynamics Simulation , Protein Binding , Protein Engineering/methods , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
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