FIG 8.

Ectopic expression of DDX60L using lentiviral vectors. (A) Titers of lentiviral vectors encoding DDX60L or PI4KIIIα compared to empty vectors. pWPI constructs encoding the indicated inserts and packaging plasmids were transfected into 293T cells to generate lentiviral vectors. Forty-eight hours after transfection, supernatants were harvested. All data represent mean values with SDs (n = 4). Infectious titers were determined by transduction of HeLa cells and selection for the appropriate antibiotic resistance (puro, puromycin; blr, blasticidin). CFU per milliliter were calculated by counting cell colonies 4 days posttransduction. (B) Western blotting for HIV-1 Ca protein in lentiviral vector-producing 293T cell lysates. Transferrin receptor was used as a loading control. Shown is a representative blot of three independent experiments. (C) Titers of lentiviral vectors encoding doxycycline-inducible DDX60L are not strongly reduced. Lentivirus was generated and titers were determined as described for panel A, with pLVX-based inducible expression vectors encoding DDX60L, HA- or eGFP-tagged DDX60L, or eGFP as a control. Data represent mean values with SDs (n = 2). (D) Expression of DDX60L by the inducible pLVX system. Huh-7-Lunet cells transduced with lentiviral vectors encoding the inducible DDX60L-HA or with an empty vector were transfected with HCV Fluc reporter replicons. Twenty-four hours after transfection, cells were treated with doxycycline (Dox) to induce expression of DDX60L. Seventy-two hours after transfection, total RNA was isolated and DDX60L mRNA levels were determined by qRT-PCR. DDX60L mRNA levels were normalized to that of the empty vector control (−Dox). Shown are mean values with SDs (n = 2). (E) Inducible overexpression of DDX60L reduces HCV replication. Cells were treated as described for panel D. Seventy-two hours after transfection, HCV replication was determined by luciferase assay. Shown are mean values normalized to that of the empty vector control (−Dox) with SDs (n = 4). (F) Absence of impact of inducible DDX60L overexpression on ISG response. IFIT1 mRNA levels were determined in total RNA samples from panel D. Shown are mean values with SDs (n = 2).