Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) PPP2R1A overexpression promotes RTA dephosphorylation. iSLK.RGB-Vector and iSLK.RGB -PPP2R1A cells were induced with doxycycline at different time points as indicated. Cells were then lysed, and cell lysates were immunoprecipitated with an anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (B) PP2A activity was measured among iSLK.RGB-Vector and iSLK.RGB-PPP2R1A cells. Bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). ****P < 0.0001. (C) Phosphatase PP2A agonist Forskolin promotes RTA dephosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (D) Phosphatase PP2A inhibitor LB-100 enhances RTA phosphorylation. iSLK.RGB cells were induced with doxycycline for 48 hours in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (E) Phosphatase PP2A agonist Forskolin cannot promote RTA dephosphorylation when PPP2R1A expression was suppressed with siRNAs. iSLK.RGB cells were transfected with siRNA as indicated. At 24 hours after transfection, cells were induced by doxycycline for 48 hours in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). WCLs were immunoprecipitated with anti-RTA antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity gel (Sigma, A2220), Lipofectamine 2000 (ThermoFisher Scientific, 11668019), MG132 (MedChemExpress, HY-13259), cycloheximide (CHX) (MedChemExpress, HY-12320), protease inhibitor cocktail (Sigma, P8340), phosphatase PP2A inhibitor (LB-100, MedChemExpress, HY-18597) and phosphatase PP2A agonist (Forskolin, MedChemExpress, HY-15371).

Techniques: Over Expression, De-Phosphorylation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Activity Assay, Phospho-proteomics, Expressing, Transfection

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) Phosphatase PP2A agonist Forskolin suppresses the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription of viral genes. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). RNA was extracted from cells to investigate the transcriptional level of several KSHV genes: ORF71, ORF45, ORF57 and K8.1. (C) Phosphatase PP2A agonist Forskolin suppresses virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A agonist Forskolin (40 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. (D) Phosphatase PP2A inhibitor LB-100 promotes virus production. iSLK.RGB cells were induced with doxycycline at different time points as indicated in the absence and presence of phosphatase PP2A inhibitor LB-100 (5 μM). Extracellular virion DNA were extracted from cell supernatants. Then, the KSHV genomic DNA copy numbers were quantified by qPCR analysis. For A to D, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity gel (Sigma, A2220), Lipofectamine 2000 (ThermoFisher Scientific, 11668019), MG132 (MedChemExpress, HY-13259), cycloheximide (CHX) (MedChemExpress, HY-12320), protease inhibitor cocktail (Sigma, P8340), phosphatase PP2A inhibitor (LB-100, MedChemExpress, HY-18597) and phosphatase PP2A agonist (Forskolin, MedChemExpress, HY-15371).

Techniques: Virus

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: (A) Phosphatase PP2A agonist Forskolin suppresses the transcription activity of RTA. (B) Phosphatase PP2A inhibitor LB-100 promotes the transcription activity of RTA. For A and B, HEK293T cells were transfected with ORF57 (left) or PAN (right) reporter plasmids (1 μg) and expression plasmids containing RTA (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (A) or 5 μM PP2A inhibitor LB-100 (B) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity and cell lysates were immunoprecipitated with an anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (C) PP2A enzymatic activity was measured in two peptides of RTA containing phosphorylated Thr-42 or Thr-678 sites respectively and one random peptide. (D and E) Phosphatase PP2A agonist Forskolin (D) or inhibitor LB-100 (E) has no effect on the phosphorylation status of RTA mutants containing T42A and T678A sites. For D and E, HEK293T cells were transfected with wildtype RTA or RTA mutants as indicated. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (D) or 5 μM PP2A inhibitor LB-100 (E) for 48 hours. Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting analysis using anti-pan Phospho-Serine/Threonine antibodies. Phosphorylated RTA was quantified by densitometry and normalized to the RTA level. (F) The transcriptional activity of three RTA mutants was impaired. HEK293T cells were transfected with K8 (left) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 48 hours after transfection, cells were then lysed to detect dual luciferase reporter activity. (G and H) Phosphatase PP2A agonist Forskolin (G) or inhibitor LB-100 (H) has no effect on the transcriptional activity of RTA mutants containing T42A and T678A sites. For G and H, HEK293T cells were transfected with K8 (left panel) or ORF59 (right) reporter plasmids (1 μg) and expression plasmids containing wildtype RTA or RTA mutants as indicated (1 μg) or empty vector (1 μg) as a control. At 6 hours after transfection, cells were treated with 40 μM PP2A agonist Forskolin (G) or 5 μM PP2A inhibitor LB-100 (H) for 48 hours. Cells were then lysed to detect dual luciferase reporter activity. For A to C and F to H, bars represent means ±SEM of triplicates from three independent experiments. The P values were calculated using Student’s t-test (two sides). **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates not significant.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity gel (Sigma, A2220), Lipofectamine 2000 (ThermoFisher Scientific, 11668019), MG132 (MedChemExpress, HY-13259), cycloheximide (CHX) (MedChemExpress, HY-12320), protease inhibitor cocktail (Sigma, P8340), phosphatase PP2A inhibitor (LB-100, MedChemExpress, HY-18597) and phosphatase PP2A agonist (Forskolin, MedChemExpress, HY-15371).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: PLOS Pathogens

Article Title: Phosphatase PP2A promotes RTA dephosphorylation to impair KSHV lytic replication

doi: 10.1371/journal.ppat.1013731

Figure Lengend Snippet: The scaffold protein PPP2R1A interacted with RTA, inducing RTA dephosphorylation mediated by phosphatase PP2A, which seriously destroyed the transcription activity of RTA and thereby greatly inhibiting KSHV lytic replication. In turn, RTA promoted PPP2R1A degradation through ubiquitin-proteasome pathway, counteracting the antiviral activity of phosphatase PP2A and ensuring a complete lytic replication of KSHV.

Article Snippet: The other used reagents and their sources were as follows: recombinant protein A agarose (Invitrogen, 15948–014), recombinant protein G agarose (Invitrogen, 15920–010), anti-Flag M2 affinity gel (Sigma, A2220), Lipofectamine 2000 (ThermoFisher Scientific, 11668019), MG132 (MedChemExpress, HY-13259), cycloheximide (CHX) (MedChemExpress, HY-12320), protease inhibitor cocktail (Sigma, P8340), phosphatase PP2A inhibitor (LB-100, MedChemExpress, HY-18597) and phosphatase PP2A agonist (Forskolin, MedChemExpress, HY-15371).

Techniques: De-Phosphorylation Assay, Activity Assay, Ubiquitin Proteomics