Journal: Cell Reports Medicine

Article Title: Picolinic acid is a broad-spectrum inhibitor of enveloped virus entry that restricts SARS-CoV-2 and influenza A virus in vivo

doi: 10.1016/j.xcrm.2023.101127

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Article Snippet: HEK293T cells in a 6wp were transfected per well with 1 μg pWPI-IRES-Puro-Ak-ACE2-TMPRSS2 (Addgene #154987), 0.6 μg Gag-Pol and 0.4 μg VSVG plasmid using Lipofectamine 2000 as per manufacturer’s instructions.

Techniques: Virus, Produced, Expressing, Luciferase, Recombinant, Modification, Saline, Staining, Transfection, One Step RT-PCR, Western Blot, Reporter Assay, Plasmid Preparation, Software, Transmission Assay, Microscopy, Flow Cytometry, Imaging, Real-time Polymerase Chain Reaction, Blocking Assay, Multiplex Assay, Spectrophotometry, Membrane, Pore Size

Journal: mBio

Article Title: Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry

doi: 10.1128/mbio.02327-24

Figure Lengend Snippet: CRISPRa gain-of-function screen identifies TMPRSS2 and TMPRSS13 as sufficient for HPIV3 infection. HEK293/dCas9-VP64 + MPH cells were transfected with CRISPRa plasmids or tracrRNA:crRNA complexes for proteases significantly enriched in the lentiviral screen. Transfected cells were then infected with HPIV3 F E108 with JS strain background and infectious HPIV3 released by cells were titered. * P ≤ 0.05, ** P ≤ 0.01 by two-way analysis of variance and Kruskal-Wallis post hoc test. Values are means and SEM from three biological replicates.

Article Snippet: Receptor-bearing cells expressing the omega peptide of β-Gal were mixed with cells co-expressing envelope glycoproteins [HN T193A ( ) and F (E108 or K108)], control or TMPRSS2 CRISPRa plasmid (Santa Cruz Biotechnology), and the α peptide of β-Gal.

Techniques: Infection, Transfection

Journal: mBio

Article Title: Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry

doi: 10.1128/mbio.02327-24

Figure Lengend Snippet: TMPRSS2 expression is sufficient for HPIV3 HN-F-mediated cell-cell fusion. Fusion activity measured with β-galactosidase complementation assay. HEK293T cells were transfected with the α subunit of β-galactosidase and HPIV3 F E108/HN (blue), HPIV3 F E108/HN/TMPRSS2 (red), HPIV3 F K108/HN (green), or HPIV3 F K108/HN/TMPRSS2 (purple). ( A ) Cells were incubated for 16 hours with HEK293T cells expressing the Ω subunit of β-galactosidase. ( B ) Cells were then incubated for 16 hours with HEK293T-TMPRSS2 (TMPRSS2 in trans ) cells expressing the Ω subunit of β-galactosidase. Values are means and SEM from three biological replicates. Schematic created with BioRender.com.

Article Snippet: Receptor-bearing cells expressing the omega peptide of β-Gal were mixed with cells co-expressing envelope glycoproteins [HN T193A ( ) and F (E108 or K108)], control or TMPRSS2 CRISPRa plasmid (Santa Cruz Biotechnology), and the α peptide of β-Gal.

Techniques: Expressing, Activity Assay, Transfection, Incubation

Journal: mBio

Article Title: Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry

doi: 10.1128/mbio.02327-24

Figure Lengend Snippet: TMPRSS2 and TMPRSS13 are not necessary for infectious viral particle production by Calu-3 cells. WT, TMPRSS2 KO, TMPRSS13 KO, and TMPRSS2/13 KO Calu-3 cells were inoculated with HPIV3 F E108 or HPIV3 F K108 with JS strain background. HPIV3 titers in Calu-3 culture media. Values are means and SEM from three to six biological replicates.

Article Snippet: Receptor-bearing cells expressing the omega peptide of β-Gal were mixed with cells co-expressing envelope glycoproteins [HN T193A ( ) and F (E108 or K108)], control or TMPRSS2 CRISPRa plasmid (Santa Cruz Biotechnology), and the α peptide of β-Gal.

Techniques: