Journal: Clinical and Molecular Hepatology

Article Title: Glutamate dehydrogenase 1-dependent α-ketoglutarate promotes hepatitis B virus transcription by modulating histone methylations on the covalently closed circular DNA minichromosome

doi: 10.3350/cmh.2024.0694

Figure Lengend Snippet: Deprivation of glutamine or loss of GDH1 enzymatic activity inhibit HBV replication and transcription in human liver-chimeric Alb-uPA/SCID mice. (A–F) Human liver-chimeric uPA/SCID mice were injected with HBV virions for 6 weeks and grouped (n=6). (A) Serum human albumin levels were determined by ELISA. (B) Serum HBsAg was quantified by chemiluminescent microparticle immunoassay. (C) Serum HBV DNA was measured by real-time PCR. (D–F) HBV DNA, RNA, and cccDNA in liver tissues were measured. (G–L) Human liver-chimeric uPA/SCID mice were injected with HBV virions through tail vein for 6 weeks and grouped randomly (n=6). (G–I) Serum human albumin, HBsAg and HBV DNA were measured. (J–L) HBV DNA, cccDNA and HBV RNA in liver tissues were measured. (M) The functional role of GDH1-derived αKG on HBV transcription and replication. * P <0.05; ** P <0.01; ns, not significant. GDH1, glutamate dehydrogenase 1; HBV, hepatitis B virus; PCR, polymerase chain reaction; cccDNA, covalently closed circular DNA; αKG, α-ketoglutarate; Gln, glutamine; DM-αKG, dimethyl-αKG; EGCG, epigallocatechin gallate; ETV, entecavir; HBc, HBV core protein.

Article Snippet: GDH inhibitor epigallocatechin gallate (EGCG, S2250), transaminases inhibitor aminooxyacetate (AOA, S4989), sodium oxamate (S6871), telaglenastat (CB-839, S7655), entecavir (ETV, S1252), and CP-2 (S8601) were obtained from Selleck Chemicals (Houston, TX, USA).

Techniques: Activity Assay, Injection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Functional Assay, Derivative Assay, Virus, Polymerase Chain Reaction

Journal: PLOS Pathogens

Article Title: ORF45-induced Filamin A phosphorylation promotes cell motility and cell-contact dependent viral infection of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1371/journal.ppat.1013737

Figure Lengend Snippet: A-D. HEK293 cells were transfected with Filamin A WT or S2152A mutated constructs and then infected with empty vector or ORF45-expressing lentiviruses at 8 h after transfection. Twenty-four hours later, the cell morphology was observed by inverted microscopy, and representative images are shown (A). Thirty-six hours after infection, the cells were subjected to analysis for relative adhesion (B), gene expression by western blots (C), and cell migration in the wound healing scratch assay (D). E. Bac16-harboring 239T cells in 6-well plates were transfected with 1.6 μg of Filamin A WT or S2152A construct for 24 h, and then, the cells were induced with 20 ng/ml TPA and 0.3 mm NaB for lytic replication. The supernatants were collected at different time points, and then, the yield of progeny viruses was detected with quantitative PCR analysis.

Article Snippet: To generate FLNA S2152A KI HEK293-mCherry cell line, a donor plasmid, pcDNA3.1-FLNA S2152A, containing homology arms and the point mutation sequence, was linearized with MluI digestion plus gel purification and then co-transfected with the lentiCRISPR v2 plasmid at equimolar concentrations into HEK293-mCherry cells, along with 10 μmol/L SCR7 (S7742, Selleck Chemicals, USA).

Techniques: Transfection, Construct, Infection, Plasmid Preparation, Expressing, Inverted Microscopy, Gene Expression, Western Blot, Migration, Wound Healing Assay, Real-time Polymerase Chain Reaction

Journal: PLOS Pathogens

Article Title: ORF45-induced Filamin A phosphorylation promotes cell motility and cell-contact dependent viral infection of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1371/journal.ppat.1013737

Figure Lengend Snippet: A-B. Filamin WT, S2152A KI or KO HEK293-mCherry cells were transfected with empty vector or ORF45 expressing plasmid for 24 h (A), or left uninfected or infected with Bac16 or STOP45 viral stocks (MOI = 10) for 24 h (B). The cells were collected and whole cell extracted were subjected to Western blotting analysis as indicated. C-E. Filamin WT, S2152A KI or KO HEK293-mCherry cells were left uninfected or infected with iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10). (C) Two hours post infection, the cells were collected by trypsin digestion, and the intracellular viral DNA and cellular DNA genomes were extracted. (D) Eight hours post infection, the cells were collected and nuclear fractions were isolated, and then viral DNA and cellular DNA genome were extracted from nuclear fractions. The viral DNA copy numbers were detected by real-time PCR and GAPDH DNA was used as internal control. (E) Twenty four hours post infection, the total RNA were extracted, reverse-transcribed and detected by real-time PCR. The levels of viral mRNA were normalized to β-actin mRNA.. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.

Article Snippet: To generate FLNA S2152A KI HEK293-mCherry cell line, a donor plasmid, pcDNA3.1-FLNA S2152A, containing homology arms and the point mutation sequence, was linearized with MluI digestion plus gel purification and then co-transfected with the lentiCRISPR v2 plasmid at equimolar concentrations into HEK293-mCherry cells, along with 10 μmol/L SCR7 (S7742, Selleck Chemicals, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Control, Reverse Transcription

Journal: PLOS Pathogens

Article Title: ORF45-induced Filamin A phosphorylation promotes cell motility and cell-contact dependent viral infection of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1371/journal.ppat.1013737

Figure Lengend Snippet: A. Filamin WT, S2152A KI or KO HEK293-mCherry cells were infected with purified iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10) for 24 h. The cells were harvested and analyzed by FACS flow cytometer. The representative images and the percentages of KSHV-infected HEK293-mCherry cells were calculated and shown in three independent experiments. B. After lytic induction for 72 h, iSLK.Bac16 or iSLK.STOP45 cells were collected and washed twice, and directly added to the monolayer of Filamin WT, S2152A KI or KO HEK293-mCherry cells and co-cultured for additional 24 h. Total cells were harvested and analyzed by FACS flow cytometer. The representative images of mCherry-positive cells are shown and the percentages of KSHV-infected HEK293-mCherry cells were calculated in three independent experiments and shown. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.

Article Snippet: To generate FLNA S2152A KI HEK293-mCherry cell line, a donor plasmid, pcDNA3.1-FLNA S2152A, containing homology arms and the point mutation sequence, was linearized with MluI digestion plus gel purification and then co-transfected with the lentiCRISPR v2 plasmid at equimolar concentrations into HEK293-mCherry cells, along with 10 μmol/L SCR7 (S7742, Selleck Chemicals, USA).

Techniques: Infection, Purification, Flow Cytometry, Cell Culture

Journal: PLOS Pathogens

Article Title: ORF45-induced Filamin A phosphorylation promotes cell motility and cell-contact dependent viral infection of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1371/journal.ppat.1013737

Figure Lengend Snippet: A-C. Filamin WT, S2152A KI or KO HEK293-mCherry cells were transfected with empty vector or ORF45 expressing plasmid for 24 h (A), or left uninfected or infected with iSLK.Bac16 or iSLK.STOP45 viral stocks (MOI = 10) for 24 h (B). The cells were subjected to a wound healing scratch assay, and representative images are shown. (C) The relative wound healing abilities were calculated using ImageJ software and shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test. D-E. Bac16 or STOP45-harboring Filamin WT, S2152A KI or KO HEK293-mCherry cells were induced by TPA + NaB for 48 h, and then subjected to a wound healing scratch assay and representative images are shown (D). The relative wound healing abilities were calculated and shown (E). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, t test.

Article Snippet: To generate FLNA S2152A KI HEK293-mCherry cell line, a donor plasmid, pcDNA3.1-FLNA S2152A, containing homology arms and the point mutation sequence, was linearized with MluI digestion plus gel purification and then co-transfected with the lentiCRISPR v2 plasmid at equimolar concentrations into HEK293-mCherry cells, along with 10 μmol/L SCR7 (S7742, Selleck Chemicals, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Wound Healing Assay, Software