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. 2016 Apr 14;90(9):4412–4426. doi: 10.1128/JVI.02718-15

FIG 9.

FIG 9

The CSFV NS5A protein antagonizes the antiviral activity of GBP1 by inhibiting its GTPase activity. (A and B) Expression levels of GBP1 in transfected cells. HEK293T cells were transfected with the indicated plasmids and collected at 48 hpt. The expression level of GBP1 was detected by Western blotting (WB) (A) or quantitative real-time reverse transcription-PCR (B). Error bars represent standard deviations. (C) Effects of NS5A on the GTPase activity of GBP1. HEK293T cells were cotransfected with the indicated plasmids and harvested at 36 hpt. GTPase activity was measured by an enzyme-linked inorganic phosphate assay (ELIPA), and the expression of the indicated proteins was verified by Western blotting using antibodies against the Myc tag, the Flag tag, or GAPDH. (D and E) Influence of NS5A overexpression on CSFV in PK-GBP1 cells. PK-GBP1 or PK-EGFP cells were transfected with pMyc-NS5A, pMyc-NS5A(1-268), pMyc-NS5A(269-497), or pCMV-Myc (pMyc-EV). At 24 hpt, the transfected cells were infected with rCSFV-Fluc or CSFV strain Shimen at a multiplicity of infection of 0.1. The cells were collected at 48 h postinfection for analysis of luciferase activity using a luciferase reporter assay system (Promega) (D) and for virus titration using an immunofluorescence assay (E). RLU, relative light units. Each sample was run in triplicate. *, P < 0.05; **, P < 0.01.