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. 2014 Jan;88(1):82–98. doi: 10.1128/JVI.01957-13

FIG 7.

FIG 7

Establishment of a BVDV DI-388mCherry replicon. (A) Schematic representation of the BVDV DI-388mCherry replicon. The mCherry coding sequence was introduced after amino acid 288 of NS5A (numbering refers to BVDV CP7 NS5A). The resulting organization of NS5A-mCherry is shown below. (B) Visualization of fluorescent NS5A-mCherry. Huh7-T7 cells were infected with vaccinia virus MVA-T7pol and transfected with pT7-DI-388, pT7-DI-388Δ288-297, or pT7-DI-388mCherry. Live cell images were acquired 24 h posttransfection. (C) NS5A-mCherry does not affect polyprotein processing. Transfected cells used for live cell imaging were harvested 24 h posttransfection and analyzed for NS3, NS4A, and NS5A/NS5A-mCherry expression by Western blotting. (D) The BVDV DI-388mCherry RNA is replication competent. MDBK cells electroporated with BVDV DI-388mCherry replicon RNA were fixed at 48 h pe. Cells were stained with NS3-specific antibody and a secondary Alexa Fluor 488-labeled antibody (center). NS5A-mCherry was directly visualized by fluorescence microscopy (right). Nuclei were counterstained with DAPI (left).