DNAJC14 inhibits a post entry step.

(A) and (B) V1-GFP- (GFP) or V1-hDNAJC14-FL (FL) transduced SW13 cells were electroporated 2 d later with in vitro transcribed YF-17D RNA to bypass the entry step. Cells and media were harvested at the indicated times after electroporation. (A) Western blot analysis was performed on equal volumes of the cell extracts using the antibodies indicated to the right; actin serves as a loading control and antibodies to myc detect the tagged DNAJC14 protein. Migration of size standards (in kDa) is indicated to the left. (B) Virus present in the medium from V1-GFP- (GFP, black circles) and V1-hDNAJC14-FL (FL, red triangles) transduced cells was enumerated by plaque assay. A single separate well was utilized for each time point. Pfu, plaque forming units. (C) Schematic of the YFV replicon construct, which expresses Renilla luciferase (RLuc) in place of the structural proteins. The locations of the YFV nonstructural proteins are indicated in the polyprotein, which is targeted to the ER by a signal sequence (red bar). UTR, untranslated region; 2A (black bar), the foot and mouth disease virus 2A autoproteolytic peptide. (D) V1-GFP- (GFP, circles) and V1-hDNAJC14-FL- (FL, triangles) transduced SW13 cells were electroporated with the wild type YF replicon RNA (filled symbols, solid lines) or with replication incompetent RNA containing a mutation in the RNA-dependent RNA polymerase (ΔDD, open symbols, dashed lines). At various times after electroporation, the cells were harvested and luciferase activity was determined. Data represents the mean luciferase value of triplicate samples; error bars indicate the standard deviation and are sometimes obscured by the symbol. RLU, relative light units. Similar results were obtained in an independent experiment utilizing Huh7.5 cells.