Cell type dependent stability and virulence of a recombinant SARS-CoV-2, and engineering of a propagation deficient RNA replicon to analyze virus RNA synthesis.

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu-1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV-2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening.

SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Nature of viruses and pandemics: Coronaviruses.

Coronaviruses (CoVs) have the largest genome among RNA viruses and store large amounts of information without genome integration as they replicate in the cell cytoplasm. The replication of the virus is a continuous process, whereas the transcription of the subgenomic mRNAs is a discontinuous one, involving a template switch, which resembles a high frequency recombination mechanism that may favor virus genome variability. The origin of the three deadly human CoVs SARS-CoV, MERS-CoV and SARS-CoV-2 are zoonotic events. SARS-CoV-2 has incorporated in its spike protein a furine proteolytic site that facilitates the activation of the virus in any tissue, making this CoV strain highly polytropic and pathogenic. Using MERS-CoV as a model, a propagation-deficient RNA replicon was generated by removing E protein gene (essential for viral morphogenesis and involved in virulence), and accessory genes 3, 4a, 4b and 5 (responsible for antagonism of the innate immune response) to attenuate the virus: MERS-CoV-Δ[3,4a,4b,5,E]. This RNA replicon is strongly attenuated and elicits sterilizing protection after a single immunization in transgenic mice with the receptor for MERS-CoV, making it a promising vaccine candidate for this virus and an interesting platform for vector-based vaccine development. A strategy could be developed for the design of RNA replicon vaccines for other human pathogenic coronaviruses.

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents.

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.

Alu retrotransposons modulate Nanog expression through dynamic changes in regional chromatin conformation via aryl hydrocarbon receptor.

Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator protein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation.