Journal: Microorganisms

Article Title: Guanylate-Binding Protein 2 Exerts GTPase-Dependent Anti-Ectromelia Virus Effect.

doi: 10.3390/microorganisms11092258

Figure Lengend Snippet: Figure 2. Overexpression of GBP2 inhibits ECTV replication. (a) The relative cell viability was measured using the CCK8 kit. (b,c) BSR-T7 cells were transfected with 1.5 µg/mL pcDNA3.1-Flag- GBP2 (Flag-GBP2) or the empty vector (EV) for 16 h. Afterwards, the cells were infected with ECTV (MOI = 1). At specified time points, the cell lysate and cell culture supernatant were harvested. The mRNA levels of EVM003 (b) and viral load (c) were measured using qRT-PCR and plaque assay, respectively. The viral mRNA relative expression, normalized to GAPDH, is presented as bar plots representing the mean ± S.D of technical repetition. (d–f) BSR-T7 cells were transfected with increasing doses of Flag-tagged GBP2 (0.5, 1.0, and 1.5 µg/mL) or the empty vector (1.5 µg/mL). The 0.5 and 1.0 µg/mL groups were supplemented with the empty vector to reach a final concentration of 1.5 µg/mL. After 16 h, cells were infected with ECTV (MOI = 1) for an additional 24 h. Samples were collected separately to determine the viral RNA transcripts (d), virus titer (e), and the expression of H3L protein (f). All three biological replicates showed similar results. * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: GBP2 shRNA Lentiviral Particles (sc-41708-V, Santa Cruz, CA, USA) were utilized, which containing 3 target-specific constructs that encode 19–25 nt (plus hairpin) shRNA designed to knock down GBP2 expression in mouse cells.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Cell Culture, Quantitative RT-PCR, Plaque Assay, Expressing, Concentration Assay, Virus

Journal: Microorganisms

Article Title: Guanylate-Binding Protein 2 Exerts GTPase-Dependent Anti-Ectromelia Virus Effect.

doi: 10.3390/microorganisms11092258

Figure Lengend Snippet: Figure 3. Knockdown of GBP2 enhances ECTV replication. (a) qRT-PCR analysis of GBP2 knockdown in sh-GBP2 and negative control (sh-Con) screening RAW264.7 cells. GAPDH was used as a loading control. (b) CCK8 tests of relative cell viability in sh-GBP2 and sh-Con screening RAW264.7 cells. (c–e) sh-GBP2 or sh-Con RAW264.7 cells were infected with ECTV at a MOI of 1 for 4, 12 and 24 h. The levels of viral mRNA, virus titers, and viral H3L protein expression were determined by RT-qPCR (n = 3) (c), plaque assay (n = 4) (d), and immunoblotting (e), respectively. Immunoblotting was performed to analyze cell lysates using antibodies against viral H3L protein and Tubulin. GBP2 antibody was used to detect the expression of endogenous GBP2 in RAW264.7 cells. * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: GBP2 shRNA Lentiviral Particles (sc-41708-V, Santa Cruz, CA, USA) were utilized, which containing 3 target-specific constructs that encode 19–25 nt (plus hairpin) shRNA designed to knock down GBP2 expression in mouse cells.

Techniques: Knockdown, Quantitative RT-PCR, Negative Control, Control, Infection, Virus, Expressing, Plaque Assay, Western Blot

Journal: Microorganisms

Article Title: Guanylate-Binding Protein 2 Exerts GTPase-Dependent Anti-Ectromelia Virus Effect.

doi: 10.3390/microorganisms11092258

Figure Lengend Snippet: Figure 4. The GTPase domain of GBP2 is essential for its antiviral effect. (a) Schematic presentation of full-length and truncated domain of GBP2. (b–d) BSR-T7 cells were transfected with Flag-tagged full-length and the three truncated GBP2 plasmids (1.5 µg/mL) for 16 h, respectively, the empty vector (EV) plasmid (1.5 µg/mL) was used as control, then infected with ECTV at a MOI of 1 for 24 h. The cells were collected separately for H3L protein expression (b), viral DNA copies (c), and virus titers analysis (d). The gray value in (b) were normalized to the EV control (set as 1). (e) The relative cell viability was measured using the CCK8 kit. *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: GBP2 shRNA Lentiviral Particles (sc-41708-V, Santa Cruz, CA, USA) were utilized, which containing 3 target-specific constructs that encode 19–25 nt (plus hairpin) shRNA designed to knock down GBP2 expression in mouse cells.

Techniques: Transfection, Plasmid Preparation, Control, Infection, Expressing, Virus

Journal: Microorganisms

Article Title: Guanylate-Binding Protein 2 Exerts GTPase-Dependent Anti-Ectromelia Virus Effect.

doi: 10.3390/microorganisms11092258

Figure Lengend Snippet: Figure 5. The K51 residue of GBP2 is the key site for anti-ECTV activity. (a) Schematic presentation of the full-length and the two mutants of GBP2. (b–d) BSR-T7 cells were transfected with either full-length or mutant plasmids of GBP2, while an empty vector served as the control (EV). The cells were then infected with ECTV (MOI = 1) for 24 h. Separate collections of cells were used for immunoblotting (b), qPCR (n = 3) (c), and plaque assay (n = 4) (d) analysis. The gray value in (b) were normalized to the EV control (set as 1). (e) The relative cell viability was measured using the CCK8 kit. * p < 0.1; ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: GBP2 shRNA Lentiviral Particles (sc-41708-V, Santa Cruz, CA, USA) were utilized, which containing 3 target-specific constructs that encode 19–25 nt (plus hairpin) shRNA designed to knock down GBP2 expression in mouse cells.

Techniques: Residue, Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Control, Infection, Western Blot, Plaque Assay