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. 2016 Nov 14;90(23):10670–10681. doi: 10.1128/JVI.01634-16

FIG 2.

FIG 2

Expression of predicted hepaciviral NS3/4A proteases and of mammalian MAVS transmembrane domains (TMDs). (A) Diagram illustrating the N-terminally HA-tagged hepaciviral NS3/4A constructs. (B) Detection of HA-NS3 expression in Huh7-Lunet hCD81 cells transduced by lentiviral pseudoparticles (PP) harboring each distinct nonhuman hepaciviral NS3/4A construct. The expression of HA-tagged HCV NS3/4A (1st column) serves as a comparison control, and human calnexin (2nd row) serves as an ER marker. The images shown are representative of two individual experiments. (C) Diagram illustrating the RFP-NLS reporter constructs. The dotted lines indicate the MAVS C-terminal domain from each indicated MAVS species fused to the reporter C terminus. The red arrow and yellow box indicate the predicted cleavage site and hydrophobic TMD of MAVS, respectively, for each indicated species. (D) Expression profiles of reporter constructs harboring the C-terminal domain and TMD from the indicated species (2nd row) in comparison to the expression of endogenous Hu MAVS stained with an N-terminus-specific antibody (1st row) in Huh7-Lunet hCD81 cells. (E) Expression profiles of the indicated reporter constructs (2nd row) in the presence of HCV or nonhuman hepaciviral NS3/4A protease (1st row). Cleavage of the MAVS TMD by the viral NS3/4A proteases causes relocalization of the RFP-NLS reporter to the nuclei. Dotted white lines outline the HA-positive (NS3/4A-expressing) cells. The images shown are representative of two individual experiments.