Mapping of the PI4KIIIα - interaction site within NS5A domain 1.
A: Schematic representation of expression constructs with deletions in NS5A D1 used to identify the PI4KIIIα interaction site. HCV coding sequences are indicated by blue or grey boxes, deleted sequences by black lines. Numbers refer to amino acid positions within the HCV JFH-1 polyprotein, numbers in brackets to amino acid positions within NS5A. The EMCV-IRES is indicated by a schematic RNA secondary structure. T7 Pm, T7 promoter; AH, amphipathic helix; D1, 2, 3, subdomains of NS5A; LCS1, 2, low complexity sequences . B: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) with sub-deletions within NS5A domain 1 with or without HA-tagged PI4KIIIα (HA-PI4K), as indicated at the bottom. Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A (lower panel) or HA-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography and quantified by phosphoimaging. Numbers at the bottom indicate the coprecipitation efficiency of HA-PI4KIIIα with individual mutants compared to NS5A wt. Coprecipitation efficiency was normalized to the total amounts of HA-PI4K for each sample (upper panel). Note that data were not normalized to input NS5A levels due to a consistently high molar excess of NS5A compared to PI4KIIIα (data not shown). Mock: cells transfected with empty pTM vector.