Project description:We used a limited proteolysis-coupled mass spectrometry approach to pinpoint binding sites of cortisol on SARS-CoV-2 S1. Binding target identification is based on the principle that small-ligand binding alters (increases or decreases) the protease accessibility of the target protein37,38. The binding of the ligand (cortisol) to the target protein (SARS-CoV-2 S1) induces perturbations/conformational changes at the binding sites which can lead to either facilitating or preventing proteolysis by non-specific proteases (thermolysin and trypsin)37,38. We subjected SARS-CoV-2 S1 incubated with either cortisol or vehicle to proteolysis by thermolysin followed by trypsin digestion and mass spectrometry-based identification of the resultant peptides. We found that cortisol either facilitated or prevented S1 proteolytic cleavage at discrete sites. Six unique peptides were identified that were only present for SARS-CoV-2 S1 incubated with either cortisol or vehicle.
Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. RNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. RNA sequencing libraries were prepared using KAPA RNA HyperPrep Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >120 for RNA samples, respectively. RNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129). All parts of RNAseq data analysis were conducted in R, version 4.3.2. and RNAseq read counts were normalized using R package DESeq2. Fold change (FC) and log2FC were calculated from normalized reads, nontransformed RPE-PPM1D-T1 cells were considered a reference. Significance of differential expression for each gene was evaluated by Fisher's t-test with simulated p-values and Holm's p-value correction for multiple comparisons.
Project description:To search for host factors regulating SARS-COV-2 infection, we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid human ESCs. The regulators were identified by the quantification of enrichment of their mutant clones within a pooled loss-of-function library upon SARS-COV-2 infection.
Project description:Otx2 has been shown to be non cell autonomously required for photoreceptor cell survival in the adult mouse RPE. This study aims to identify Otx2 DNA binding profile in both RPE and neural retina to i) identify direct targets of Otx2 in the RPE ii) compare Otx2 binding profile in neural retina and RPE to unveil hidden functions in the neural retina. WT and GFP antibodies were used to perform two independent ChIP-seq experiments using Illumina GAIIx.
Project description:SARS-CoV-2 has spread globally and caused the COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 are in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected mice. Whereas Fc effector functions are fully dispensable when mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact but not LALA-PG loss of Fc effector function variant mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and enhanced tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Our study demonstrates that therapeutic neutralizing mAbs require Fc effector functions to reduce SARS-CoV-2 infection and modulate protective immune responses.
Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. DNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. DNA sequencing libraries were prepared using KAPA EvoPlus Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >35 DNA samples, respectively. DNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). Copy number variations (CNV) were analyzed using CNVkit version 0.7.4. Areas with median coverage >20 were included in the analysis. RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129).
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that can cause severe respiratory disease in humans. Identification of the host factors that are necessary for viral infection and virus-induced cell death is critical to our understanding of the viral life cycle and can potentially aid the development of new treatment options. Here, we report CRISPR screen results of both SARS-CoV and MERS-CoV infections in derivatives of the human hepatoma cell line Huh7. Our screens identified the known entry receptors ACE2 for SARS-CoV and DPP4 for MERS-CoV. Additionally, the SARS-CoV screen uncovered several components of the NF-κB signaling pathway (CARD10, BCL10, MALT1, MAP3K7, IKBKG), while the MERS-CoV screen revealed the polypyrimidine tract-binding protein PTBP1, the ER scramblase TMEM41B, furin protease and several transcriptional and chromatin regulators as candidate factors for viral replication and/or virus-induced cell death. Together, we present several known and unknown coronavirus host factors that are of interest for further investigation.
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which, since 2019 in China, has rapidly become a worldwide pandemic. The aggressiveness and global spread were enhanced by the many SARS-CoV-2 variants that have been isolated up to now. These mutations affect mostly the viral glycoprotein Spike (S), the capsid protein mainly involved in the early stages of viral entry processes, through the recognition of specific receptors on the host cell surface. In particular, the subunit S1 of the Spike glycoprotein contains the Receptor Binding Domain (RBD) and it is responsible for the interaction with the angiotensin-converting enzyme 2 (ACE2). Although ACE2 is the primary Spike host receptor currently studied, it has been demonstrated that SARS-CoV-2 is also able to infect cells expressing low levels of ACE2, indicating that the virus may have alternative receptors on the host cells. The identification of the alternative receptors can better elucidate the pathogenicity and the tropism of SARS-CoV-2. Therefore, we investigated the Spike S1 interactomes, starting from host membrane proteins of non-pulmonary cell lines, such as human kidney (HK-2), normal colon (NCM460D), and colorectal adenocarcinoma (Caco-2). We employed an affinity purification-mass spectrometry (AP-MS) to pull down, from the membrane protein extracts of all cell lines, the protein partners of the recombinant form of the Spike S1 domain. The purified interactors were identified by a shotgun proteomics approach. The lists of S1 potential interacting proteins were then clusterized according to cellular localization, biological processes, and pathways, highlighting new possible S1 intracellular functions, crucial not only for the entrance mechanisms but also for viral replication and propagation processes.