Journal: PLoS Pathogens

Article Title: Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

doi: 10.1371/journal.ppat.1009246

Figure Lengend Snippet: ( A) Schematic illustration of SARS-CoV-2 S including receptor binding domain (RBD) in green and proteolytic cleavage sites (S1/S2, S2’). Amino acid sequences around the S1/S2 recognition sites of SARS-CoV-2 S are indicated while the multibasic site is highlighted in purple. Amino acid mutations are highlighted in light blue while deletions are marked with orange dashes. (B) Overall structure of the SARS-CoV-2 S protein (PDB: 6VYB). RBD core is shown in green. Pro-Arg-Arg-Ala-Arg residues are shown in yellow. (C,D) Representative western blots of HIV Pseudoviruses (C) and Virus Like Particles (VLPs) (D) harbouring the indicated SARS-CoV-2 S protein mutants (detected with anti-S antibody) and produced in 293-wt and 293T-ΔFURIN cells. Expression of HIV capsid protein (p24) (C) and SARS-CoV-2 nucleoprotein (D) is shown as loading control. (E) Representative western blot analysis of spike and nucleoprotein present in SARS-CoV-2 viral particles produced in 293T-hACE2 and 293T-hACE2-ΔFURIN after 42 hours post infection. The cleaved S in (C) (D) and (E) identifies the S2 subunit.

Article Snippet: For the generation of hACE2 over-expressing 293T cells, the human hACE2 ORF was PCR amplified from Addgene plasmid 1786 and C-terminally fused with the porcine teschovirus-1-derived P2A cleavage sequence (ATNFSLLKQAGDVEENPGP) followed by the blasticidin resistance gene.

Techniques: Binding Assay, Western Blot, Virus, Produced, Expressing, Control, Infection

Journal: PLoS Pathogens

Article Title: Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

doi: 10.1371/journal.ppat.1009246

Figure Lengend Snippet: (A) Infection of 293T-hACE2 cells with GFP expressing HIV pseudotyped with SARS-CoV-2 S mutants, measured as proportion of cell area expressing GFP. Viruses were produced in either 293T-wt or 293T-ΔFURIN cells. (B) and (C) Infection data of 293T-hACE2 cells with HIV pseudotyped with SARS-CoV-2 S mutants as in (A), with cells pre-treated for 2 hours with either DMSO or 25μM lysosomal inhibitor E64d as indicated. Statistical analysis was performed using Student t -test * P< 0.05; ** P< 0.01, “ns” not significant. (D) Representative western blot of viral particles produced from SARS-CoV-2 infection of 293T-hACE2 and 293T-hACE2-ΔFURIN cells at 72 hours post infection. Spike and nucleoprotein are detected (left panel). Total protein content of virus preparation by Coomassie staining (right panel). (E) Plaque assay of Vero-hACE2-TMPRSS2 infected with viruses produced in D. (F) and (G) Immunofluorescence images displaying infection of Caco2 BVDV-Npro cells (F) and 293T-hACE2 (G) with equalised amounts of SARS-CoV-2 virus produced in 293T-hACE2 and 293T-hACE2-ΔFURIN cells. Spike (green) and nuclei (blue) are shown. Scale bar, 50 μm. (H) and (I) Quantification of SARS-CoV-2 infected cells shown in (F) and (G). * P< 0.05; ***, P< 0.001 analysed using Student t -test. Data are expressed as mean +/- SEM (n = 2).

Article Snippet: For the generation of hACE2 over-expressing 293T cells, the human hACE2 ORF was PCR amplified from Addgene plasmid 1786 and C-terminally fused with the porcine teschovirus-1-derived P2A cleavage sequence (ATNFSLLKQAGDVEENPGP) followed by the blasticidin resistance gene.

Techniques: Infection, Expressing, Produced, Western Blot, Virus, Staining, Plaque Assay, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

doi: 10.1371/journal.ppat.1006868

Figure Lengend Snippet: A. Schematic representation of the promoter of human CASP1 . IRF8 consensus binding site is highlighted in green. The ATG of CASP1 is highlighted in red. B. The pGL2-CASP1p constructs (with or without IRF8 consensus site) and the IRF8 consensus site mutated construct were co-transfected into 293T cells with either vector control or IRF8 expression vectors. Luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. C. The pGL2-CASP1p1 construct was co-transfected into 293T cells with either vector control, wild-type IRF8 (WT) or IRF8 DNA binding mutant (K108E) expression vectors and luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. D. The pGL2-CASP1p1 construct was co-transfected into 293T cells with either vector control or IRF8 and IRF1 expression vectors and luciferase assays were performed 36 hrs post-transfection. The value of cells transfected with empty vectors was set as 1. The results were presented as mean ± standard deviation of triplicate assays. *** p<0.001.

Article Snippet: 293T cells (a gift from Diane Hayward, Johns Hopkins University) were grown in DMEM media supplemented with 10% FBS.

Techniques: Binding Assay, Construct, Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Standard Deviation, Mutagenesis

Journal: PLoS Pathogens

Article Title: Interferon regulatory factor 8 regulates caspase-1 expression to facilitate Epstein-Barr virus reactivation in response to B cell receptor stimulation and chemical induction

doi: 10.1371/journal.ppat.1006868

Figure Lengend Snippet: A. Caspase-1 depletion suppresses KAP1 degradation. Protein extracts form were analyzed by western blot using antibodies against KAP1, PAX5, DNMT3A and Caspase-8 (CASP8). B. Caspase-1 and -8 cleave KAP1 in vitro . HA-KAP1 and the antibody recognition sites are labeled as indicated. HA-tagged KAP1 was immuoprecipitated from transfected 293T cells using HA magnetic beads. The beads-bound HA-KAP1 was incubated with individual recombinant caspase for 2 hrs at 37°C. WB was performed using either anti-HA or anti-KAP1 antibodies. The relative positions of cleaved fragments were labeled as indicated.

Article Snippet: 293T cells (a gift from Diane Hayward, Johns Hopkins University) were grown in DMEM media supplemented with 10% FBS.

Techniques: Western Blot, In Vitro, Labeling, Transfection, Magnetic Beads, Incubation, Recombinant

Journal: Nucleic Acids Research

Article Title: Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits

doi: 10.1093/nar/gkv048

Figure Lengend Snippet: Expression of scAlu RNA reduces the translational capacity of HEK 293T cells. ( A ) Constructs used for luciferase reporter assay. Sequences of ncRNAs were cloned downstream of the 7SL gene enhancer element (Enh). ( B ) Northern blot analysis of transiently transfected HEK 293T cells. Membranes containing 0.5 μg of total RNA per lane were hybridized with [ 32 P]-labeled oligonucleotides complementary to 4.5S RNA, 7SL RNA, scAluY NF1 RNA and 18S RNA (loading control). ( C ) Translation efficiency of reporter mRNAs in HEK 293T cells expressing different ncRNAs. Luciferase activity was measured 24 h post-transfection, normalized to μg of protein in the cell lysate and expressed as a percentage of the value obtained in control cells (ctrl) transfected with the empty vector pDL7Enh. Error bars are shown as SEM ( n ≥ 5). * P < 0.05, ** P < 0.01 by Student's t -test. See Supplementary Figure S8A for northern blot analysis of reporter mRNAs. ( D ) Bioluminescence recordings of HEK 293T cells expressing the estradiol-induced luciferase reporter gene along with pDLscAlu (scAlu) or pDL7enh (Ctrl) following stimulation with 100 nM estradiol. ( E ) Relative total protein synthesis in HEK 293T cells after arsenite treatment (top panel). Control cells were treated with 500 μM sodium arsenite for 30 min at 48 h post-transfection, allowed to recover for the indicated times and pulse labeled with [ 35 S]-methionine/cysteine for 15 min. 100 μg of total protein was precipitated in 10% TCA, [ 35 S]-incorporation was determined by scintillation counting and expressed as a percentage of that in untreated cells. Error bars are shown as SEM ( n ≥ 5). Bottom panel shows total protein synthesis in untreated cells (-Ars) and cells expressing scAluY NF1 or 4.5S RNA treated for 30 minutes with 500 μM sodium arsenite and allowed to recover for 60, 90 and 120 min (+Ars). Total protein synthesis is expressed as a percentage of that in control cells measured in parallel. Error bars are shown as SEM ( n ≥ 5). * P < 0.05, ** P < 0.01 by Student's t -test. Expression of scAluY NF1 RNA is shown in Supplementary Figure S8D. ( F ) Representative autoradiograph following SDS-PAGE of VSV-infected cell lysates. HEK 293T cells expressing scAluY NF1 , 4.5S RNA or control cells were infected with VSV for 6 h, pulse labeled with [ 35 S]-methionine/cysteine for 15 min and 100 μg of total protein was displayed by 5–20% gradient SDS-PAGE. Bands corresponding to viral proteins are indicated. Coomassie-blue staining (bottom panel) was used to check uniform loading. Quantification of the viral protein synthesis is shown on the right. Viral protein synthesis in cells expressing scAluY NF1 RNA or 4.5S RNA was calculated as the sum of the intensities of the viral protein bands and expressed as a percentage of the control, which was set to 100%. Error bars are shown as SD ( n ≥ 3). ** P < 0.01 by Student's t -test. Expression of scAluY NF1 RNA is shown in Supplementary Figure S8E.

Article Snippet: HEK 293T cells were transfected with TransIT-T2020 reagent (Mirus) according to manufacturer's protocol.

Techniques: Expressing, Construct, Luciferase, Reporter Assay, Clone Assay, Northern Blot, Transfection, Labeling, Control, Activity Assay, Plasmid Preparation, Autoradiography, SDS Page, Infection, Staining