Journal: ACS Infectious Diseases

Article Title: Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

doi: 10.1021/acsinfecdis.1c00278

Figure Lengend Snippet: Atovaquone partially requires TMPRSS2 to drive its antiviral action against SARS-CoV-2 and reduces the interaction between the spike protein and its surface receptor ACE2. (A) Schematic of atovaquone administration. (B) VeroE6 hTMPRSS2 cells were treated with atovaquone (10 μM) for 2 h before infection (full time), at the time of infection (entry), or 1 h after infection (postentry), before challenging with original SARS-CoV-2 at an MOI of 0.1. Infection was carried out for 48 h in the presence of the drug and for all conditions. Infection was assessed by immunoblotting of the spike protein within cell lysates. (C) Structure of constructs for a split NanoLuc-based bioreporter. RBD or S1 from either SARS-CoV1 or SARS-CoV2 was linked to Large BiT (LgBiT) on its N-terminus to form LgBiT-RBD or LgBiT-S1; similarly, the Small BiT (SmBiT) peptide was linked to human ACE2 to form SmBiT-ACE2. ACE2 and RBD or S1 constructs were transfected separately or cotransfected into HEK293 cells for 48 h and lysed with a passive lysis buffer. Mixed lysates or lysates from cotransfected cells were incubated with coelenterazine, and the luminescence measured using a plate reader. (D) Following plasmid transfection into HEK293 cells, the cells were lysed in a NanoLuc-compatible passive lysis buffer and lysates were dispensed into a 96-well plate, to which atovaquone was added at a final concentration of 4 μM. The impact of atovaquone on SARS receptor binding was assessed in two ways: (1) with atovaquone added to LgBiT-RBD or LgBiT-S1 for 50 min followed by the addition of an equal quantity of SmBiT-ACE2 for another 10 min (“mixed lysates)” or (2) with atovaquone added to the preformed SmBiT-ACE2 + LgBiT-RBD/S1 complex for 1 h (“cotransfected).” Following incubation, a nanoluciferase substrate was added and the luminescence was measured. HEK293 cells were also transfected with a nanoluciferase control plasmid and lysates were incubated with 4 μM atovaquone. N = 4 per condition. The graph shows % bioreporter luminescence with the highest value in each of the respective untreated conditions taken as 100% ( N = 4 per condition, the p values were determined using a t -test). (E, F). The different cell lines mentioned were subjected to immunoblotting (E) and flow cytometry analysis (F) of TMPRSS2 and ACE2 expression. For ACE2, unstained samples of the matched cell line were used as a control. For TMPRSS2, secondary antibody-stained samples of the matched cell line were used as a control. Controls for the A549 hACE2 cell line are represented in the figure. The same controls were used for all cell lines studied. (G) Vero hTMPRSS2, Calu-3, and A549 hACE2 cells were pretreated with atovaquone (100 μM) for 2 h before infection with the original SARS-CoV-2 (MOI of 0.1). Viral RNA levels were determined 48 h postinfection by qPCR. The data represent the means ± SEM of one experiment performed in biological triplicates. (H) HEK293T cells expressing hACE2 cells were mock-transfected or transfected with a plasmid encoding hTMPRSS2. Twenty-four hours post-transfection, cells were seeded in 96-well plates and preincubated with the different drugs E64d (10 μM), camostat (25 μM), and atovaquone (100 μM) + 5 μg/mL polybrene for 1 h before infection with purified SARS-CoV-2 pseudotypes. LacZ + cells were quantified using the Beta-Glo assay system and luminescence measurement. The data are the means ± SEM of two experiments performed in biological triplicates. Similar results were obtained by X-gal staining. (I) Effector cells (zipV2+) expressing SARS-CoV-2 spike and target cells (zipV1+) expressing ACE2 with or without TMPRSS2 were cocultured for 3 h in the presence of the indicated concentration of drugs or DMSO. Cell–cell fusion was assessed by measuring the fluorescence of the Venus protein complementation (zipV1 + zipV2). Data were normalized to the fusion obtained with target cells expressing ACE2 but not TMPRSS2 (ACE2 + TMPRSS2) and are the means ± SEM of two experiments performed in triplicates. The p values were calculated using a t -test where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For lentiviral production, HEK293T cells were transiently cotransfected with lentiviral packaging plasmid psPAX2 (gift from Didier 99 Trono, Addgene #12260), lentiviral vector encoding LacZ (LV-Lac, gift from Inder Verma (Addgene plasmid # 12108)), and pcDNA3.1 encoding the D614G SARS-CoV-2 spike at a 1:1:1 ratio using a jetPRIME transfection reagent (Polyplus-transfection) according to the manufacturer’s protocol.

Techniques: Infection, Western Blot, Construct, Transfection, Lysis, Incubation, Plasmid Preparation, Concentration Assay, Binding Assay, Control, Flow Cytometry, Expressing, Staining, Purification, Glo Assay, Fluorescence

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Expressing, Negative Control, Control, Transduction, Amplification, Quantitative RT-PCR, Two Tailed Test

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Labeling

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Biomarker Discovery, Genome Wide