Figure 3. GPS2 is essential for HCV RNA replication.

(A) Huh7.5.1 cells were transduced with the indicated short hairpin RNA (shRNA)-expression retroviral constructs or left untreated. GPS2 mRNA levels were determined by real-time qRT-PCR in these cells. The amount of GPS2 mRNA to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were calculated and graphed. (B) All cell lines were seeded respectively in 6-well plates with the same number and cell numbers were calculated at the indicated time point and graphed. (C) Huh7.5.1 cells expressing shRNAs were infected with JFH1 at a multiplicity of infection (MOI) of 0.5. At 72 h postinfection, the levels of intracellular HCV RNA and GPS2 mRNA were analyzed by qPCR. (D) Total cell lysates harvested from these cells were immunoblotted with the indicated antibodies. (E) Extracellular RNAs isolated from the culture supernatant were quantified by qPCR. Experiments were carried out in duplicate. Error bars indicate standard deviations. (F) Cell culture supernatants were used to infect naive Huh7.5.1 cells plated in 96-well plate. At 72 h postinfection, cells were stained for HCV core with anti-core antibody and stained with the DAPI before confocal microscopy.