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. 2020 Jun 9;226(3):407–419. doi: 10.1093/infdis/jiaa325

Figure 3.

Figure 3.

HULC is transcriptionally upregulated by NS5A. A, Huh-7.5 cells were transfected with plasmids encoding NS3, NS4A, NS4B, NS5A, and NS5B from genotype 1a H77S.3 or empty vector (EMV). At 72 hours after transfection, total RNA was extracted, HULC and β-actin levels were quantified by reverse-transcription quantitative polymerase chain reaction, and HULC levels were normalized to those of β-actin. Furthermore, the relative HULC levels from plasmid-transfected cells were normalized to the relative HULC levels from EMV-transfected cells, which were set to 1. The differences of means between EMV-transfected cells and each NS protein-encoding plasmid were analyzed by one-way analysis of variance (ANOVA). B, The promoter regions of HULC, which are located approximately 400 and 900 nucleotides upstream from the transcription start site of HULC, were introduced into the pGL3-Basic vector encoding a firefly luciferase gene whose expression was regulated by the introduced HULC promoter regions, to create HULC-400 and HULC-900, respectively. Those plasmids were transfected into Huh-7.5 cells with a reporter plasmid containing a Renilla luminescent reporter gene. At 24 hours later, the cells were infected with cell culture–derived hepatitis C virus from HJ3-5 at multiplicities of infection (MOIs) 0.2 and 1 or mock. At 48 hours postinfection, firefly and Renilla luciferase activity was measured, and firefly luciferase activity was normalized to that of Renilla. Furthermore, activity was normalized to the activity of pGL3-Basic-transfected or mock-infected cells, which was set to 1. The differences of means at each condition were analyzed by 2-way ANOVA. C, Huh-7.5 cells were transfected with pGL3-Basic, HULC-400, or HULC-900 and then transfected with plasmids encoding NS3, NS4A, NS4B, NS5A, or NS5B from genotype 1a H77S.3 or EMV. At 48 hours after transfection, firefly and Renilla luciferase activity was measured, and firefly luciferase activity was normalized to that of Renilla. Furthermore, activity was normalized to that from pGL3-Basic-transfected cells for each nonstructural protein-coding plasmid, which was set to 1. The differences of means between pGL3-Basic and HULC-400 or HULC-900 at each NS protein overexpression were analyzed by 2-way ANOVA. ***P < .001.