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. 2018 Mar 14;92(7):e02182-17. doi: 10.1128/JVI.02182-17

FIG 4.

FIG 4

The transmembrane domain upstream of the NS2 cysteine protease domain is essential for DVG replication. (A) Schematic of three transmembrane segments of NS2. The numbers start with the first amino acid of NS2. Six DVGs that have a deletion site in NS2 are marked. (B) Schematics of the JFH1-based DVG JD533 and its NS2 protease-defective mutant (JD533-C184A) and of the JFH1-based DVG JD835 and its mutant with a deletion of the entire TMD (JD835-ΔTMD). (C) NS5A (green) immunostaining and nuclei (blue) in Huh7.5.1 cells transfected with in vitro-transcribed DVG RNA at day 2 posttransfection. (D) HCV RNA levels in Huh7.5.1 cells transfected with in vitro-transcribed DVG RNAs. The HCV RNA levels were determined by RT-qPCR and are expressed as values relative to the cellular actin mRNA levels. The error bars represent standard deviations from three independent experiments. *, P < 0.05. (E) Schematic of plasmids expressing core-NS3 of JD835 (835), JD835 with wild-type NS2 (835-TMD), or JD835 with TMD-deleted NS2 (835-ΔTMD). (F) Western blot analysis of NS2-NS3 cleavage in HEK293 cells. (G) Schematic of plasmids expressing core-NS3 of JD835 (835), JD835 with wild-type NS2 (835-TMD), JD835 with TMS1-deleted NS2 (835-ΔTMS1), or JD835 with TMD-deleted NS2 (835-ΔTMD). (H) Western blot analysis of NS2-NS3 cleavage in HEK293 cells. (I) Schematic of JFH1-based DVG JD835 and its mutant with TMS1-deleted NS2 (JD835-ΔTMS1). (J) HCV RNA levels in Huh7.5.1 cells transfected with in vitro-transcribed DVG RNAs. The HCV RNA levels were determined by RT-qPCR and are expressed as values relative to the cellular actin mRNA levels. The error bars represent standard deviations from three independent experiments. *, P < 0.05.