Journal: Virus research

Article Title: Glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3 transactivate HSV-1 ICP0 promoter sequences but a GC-rich binding antibiotic, Mithramycin A, impairs reactivation from latency.

doi: 10.1016/j.virusres.2024.199487

Figure Lengend Snippet: Fig. 2. Transactivation of ICP0 CRM constructs by GR, DEX, and Sp1 or Sp3. Panel A: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM fragment constructs (0.5 μg DNA), a plasmid that expresses the mouse GR-α protein (1.0 μg DNA), DEX and Sp1 (1.0 μg DNA) as described in the Materials and Methods. Panel B: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM fragment constructs (0.5 μg DNA), a plasmid expressing the mouse GR-α protein (1.0 μg DNA), DEX and Sp3 (1.0 μg DNA) as described in the Materials and Methods. Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h post-transfection, MEM was changed, and denoted cultures were treated with water-soluble DEX (10 μM). Cells were harvested 48 h post- transfection, and luciferase activity measured. Basal transcriptional activity of cells transfected with the respective CRM construct plus empty expression vector normalized to 1, and fold activation values for other samples calculated. The results are the average of three separate experiments, and error bars denote standard errors. Asterisks denote significant differences (*, p ≤0.05; **, p ≤0.01) between the indicated treatments as determined by Student’s t-test.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Construct, Transfection, Plasmid Preparation, Luciferase, Expressing, Activity Assay, Activation Assay

Journal: Virus research

Article Title: Glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3 transactivate HSV-1 ICP0 promoter sequences but a GC-rich binding antibiotic, Mithramycin A, impairs reactivation from latency.

doi: 10.1016/j.virusres.2024.199487

Figure Lengend Snippet: Fig. 3. Transactivation of ICP0 CRM-A mutants by GR, DEX, and Sp1 or Sp3. Panel A: Schematic of CRM-A wt fragment and mutants that were previously described (Wijesekera et al., 2022). Panel B: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM-A fragment constructs (0.5 μg DNA), a plasmid that expresses the mouse GR-α protein (1.0 μg DNA), DEX and Sp1 (1.0 μg DNA) as described in the Materials and Methods. Panel C: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM-A fragment constructs (0.5 μg DNA), a plasmid expressing the mouse GR-α protein (1.0 μg DNA), DEX and Sp3 (1.0 μg DNA) as described in the Materials and Methods. Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h post-transfection, MEM was changed, and the denoted cultures were treated with water-soluble DEX (10 μM). Cells were harvested 48 h post-transfection, and luciferase activity measured. Basal transcriptional activity of cells transfected with the respective CRM construct plus empty expression vector normalized to 1, and fold activation values for other samples calculated. The results are the average of three separate experiments, and error bars denote standard errors. Asterisks denote significant differences (*, p ≤0.05; **, p ≤0.01) between the indicated treatments as determined by Student’s t-test.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Expressing, Activity Assay, Activation Assay

Journal: Virus research

Article Title: Glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3 transactivate HSV-1 ICP0 promoter sequences but a GC-rich binding antibiotic, Mithramycin A, impairs reactivation from latency.

doi: 10.1016/j.virusres.2024.199487

Figure Lengend Snippet: Fig. 4. Transactivation of ICP0 CRM-B mutants by GR, DEX, and Sp1 or Sp3. Panel A: Schematic of CRM-B wt fragment and mutants that were previously described. Panel B: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM-B fragment constructs (0.5 μg DNA), a plasmid that expresses the mouse GR-α protein (1.0 μg DNA), DEX and Sp1 (1.0 μg DNA) as described in the Materials and Methods. Panel C: Neuro-2A cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), denoted CRM-A fragment constructs (0.5 μg DNA), a plasmid expressing the mouse GR-α protein (1.0 μg DNA), DEX and Sp3 (1.0 μg DNA) as described in the Materials and Methods. Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h post-transfection, MEM was changed, and the denoted cultures treated with water-soluble DEX (10 μM). Cells were harvested 48 h post-transfection, and luciferase activity measured. Basal transcriptional activity of cells transfected with the respective CRM construct plus empty expression vector normalized to 1, and fold activation values for other samples calculated. The results are the average of three separate experiments, and error bars denote standard errors. Asterisks denote significant differences (*, p ≤0.05; **, p ≤0.01) between the indicated treatments as determined by Student’s t-test.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Expressing, Activity Assay, Activation Assay

Journal: Virus research

Article Title: Glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3 transactivate HSV-1 ICP0 promoter sequences but a GC-rich binding antibiotic, Mithramycin A, impairs reactivation from latency.

doi: 10.1016/j.virusres.2024.199487

Figure Lengend Snippet: Fig. 5. ChIP studies to examine GR and Sp1 or Sp3 that occupy the ICP0 promoter. Panel A. Schematic of ICP0 promoter that includes sequences amplified by the PCR primer used in this study. Panels B and E: Neuro-2A cells were grown in MEM containing 2 % charcoal-stripped FBS following transfection with plasmid containing just the FL ICP0 promoter construct (1.5 μg DNA). Panel C: Neuro-2A cells were grown in MEM containing 2 % charcoal-stripped FBS following transfection with plasmids containing the FL ICP0 promoter construct (1.5 μg DNA) and a Sp1 expression plasmid (3.0 μg DNA). Panel D: Neuro-2A cells were grown in MEM containing 2 % charcoal-stripped FBS followed by transfection with plasmids containing the FL ICP0 promoter construct (1.5 μg DNA), Sp1 expression plasmid (3.0 μg DNA), GR-α expression plasmid (3.0 μg DNA), and DEX. Panel F: Neuro-2A cells were grown in MEM containing 2 % charcoal-stripped FBS following transfection with plasmids containing the FL ICP0 promoter construct (1.5 μg DNA) and a Sp3 expression plasmid (3.0 μg DNA). Panel G: Neuro-2A cells were grown in MEM containing 2 % charcoal-stripped FBS followed by transfection with plasmids containing the FL ICP0 promoter construct (1.5 μg DNA), Sp3 expression plasmid (3.0 μg DNA), GR-α expression plasmid (3.0 μg DNA), and DEX. Designated samples were treated with DEX (10 μM) for 4 h prior to harvesting cells (48 h after transfection). Cells were cross-linked with formaldehyde and harvested as described in the materials and methods. ChIP was performed as described in the materials and methods using the isotype control antibody, GR antibody, Sp1 antibody, or Sp3 antibody. Target DNA was amplified using PCR primers that amplify the ICP0 ¡95 region (forward, 5′-GCCATTGGGGGAATCGTC-3′; reverse, 5′-TGTGGTGATGCGGAGAGG-3′) that were previously described (Ostler et al., 2019). Individual bands were quantified using Image Lab software and presented as a percentage of the input sample, representing 13.3 % of the cell lysate used for each sample. Data presented are the means from two separate transfection studies that were performed on different days. Asterisks denote a significant difference between the GR-, Sp1-, or Sp3-specific antibody and respective isotype control, and as indicated between samples (*, p ≤0.05; **, p ≤0.01; ***, p ≤0.001; ns, not significant). Student’s t-test was performed to determine significant differences.

Article Snippet: The Sp1 (plasmid #27,264) and Sp3 (plasmid #24,541) expression vectors were purchased from Addgene.

Techniques: Amplification, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software