Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: Elongator complex loss reduces SARS-CoV-2 and ZIKV infection. ( A ) Schematic representation of U34 tRNA modification pathway in FD (ELP1-deficient) cells. The Western blot shows ACE2 receptor and actin levels in cells transduced by ACE2-expressing lentivector (VLP ACE2 ). ( B ) Quantification of U34 modification levels (ncm 5 U, mcm 5 U, and mcm 5 s 2 U) in wild-type ( wt ) versus FD cells determined by mass spectrometry on tRNA-enriched RNA fractionations. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR in wt and FD cells at different multiplicities of infection (MOI) 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR in wt and FD cells at different MOIs 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Infection, Modification, Western Blot, Expressing, Mass Spectrometry, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: ALKBH8 and CTU1 knockdown individually impairs SARS-CoV-2 and ZIKV infection. ( A ) Schematic showing shRNA-mediated knockdown approach targeting U34 ALKBH8 and CTU1 components of U34 modification pathway. The Western blot analysis of ACE2 expression in A549 cells with and without lentiviral vector expressing ACE2 (VLP ACE2 ). ( B ) Western blot validation of ALKBH8 and CTU1 knockdown efficiency with corresponding actin loading controls. Numbers indicate relative protein levels. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR following knockdown of the indicated genes 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR following knockdown of the indicated genes 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Knockdown, Infection, shRNA, Modification, Western Blot, Expressing, Plasmid Preparation, Biomarker Discovery, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Susceptibility of various cell lines to CPE following infection by HCoVs

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of OC43-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) OC43-infected HCT-8 cells (red). CD13, CD147, CD326, ACE2, and GD3 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected HCT-8 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of two biological replicates for all antigens except GD3, where three biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of 229E-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) 229E-infected MRC-5 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected MRC-5 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD147 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of NL63-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) NL63-infected LLC-MK2 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected LLC-MK2 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD13 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Soluble ACE2 protein and anti-ACE2 monoclonal blocking antibody inhibit NL63 infection in LLC-MK2 cells. ( A ) Treatment with soluble ACE2 or control protein ovalbumin at 5 and 10 mg/mL was pre-incubated with NL63 virus at MOI 0.1 (virus titer: 8.89 × 10 5 TCID 50 /mL) for 1 hour, and the protein and virus mixture complex was added to the LLC-MK2 cells. ( B ) Similarly, cells were incubated for 1 hour with human anti-ACE2 monoclonal blocking antibody (10, 40, and 120 µg/mL) or unrelated control antibody (anti-DC-SIGN; 40 µg/mL) followed by addition of NL63 virus (MOI 0.1). Five days post-infection, both culture supernatants and cell lysates were harvested for quantification of nucleocapsid (N) and spike (S) vRNA copies by ddPCR. Percent relative viral inhibition following treatment with (C) sACE2 or (D) anti-ACE2 monoclonal antibody compared to their respective untreated control. Data represent three biological replicates for sACE2 or four (anti-ACE2) with three technical replicates per experiment. Statistical analysis was performed using an unpaired t -test with Welch’s correction ( P < 0.05) relative to untreated control. n.s. denoted as no statistical difference; * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: The following antibodies and isotypes were used to quantify the surface marker expression and receptor modulation in the uninfected (control) and infected cells: PE anti-human CD13, anti-human CD147 (isotype-control antibody mouse IgG1 κ, BioLegend); and anti-human CD326 (isotype-control antibody mouse IgG2a κ; BioLegend); human anti-ACE2 antibody (isotype-control antibody Goat IgG; R&D Systems) and anti-GD3, 9-O-acetyl (clone UM4D4; isotype-control antibody mouse IgM κ; Millipore Sigma).

Techniques: Blocking Assay, Infection, Control, Incubation, Virus, Inhibition