FIG 5.

The K51 of GBP1 is required for the inhibition of CSFV replication. (A) HEK293T cells were transfected with pFlag-GBP1, pFlag-GBP1(R48P), pFlag-GBP1(K51A), or pCMV-Flag (Flag-EV) and were harvested at 36 h posttransfection. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA). (B) Influence of GBP1(K51A) on rCSFV-Fluc replication. PK-GBP1, PK-GBP1(R48P), PK-GBP1(K51A), and PK-EGFP cells were first infected with rCSFV-Fluc at a multiplicity of infection of 0.1 for 48 h and then assayed for luciferase activity using the dual-luciferase reporter assay system (Promega). Error bars represent standard deviations. NS, not significant; *, P < 0.05; **, P < 0.01. RLU, relative light units. (C and D) Influence of GBP1(K51A) on CSFV strain Shimen replication. The cell lines were infected with Shimen at an MOI of 0.1 for 48 h. (C) The viral titers in the supernatants collected at 24 and 48 h postinfection were examined by an immunofluorescence assay and are presented as median tissue culture infective doses (TCID₅₀) per milliliter. (D) The number of genomic copies of CSFV in PK-GBP1(K51A) cells was determined using a quantitative real-time reverse transcription-PCR assay. Each sample was run in triplicate. (E) Cell viability assay of cell lines stably overexpressing wild-type or mutant GBP1.