Extended Data Figure 5: Only full-length SEC14L2 supports replication of wild-type HCV.

a, Huh-7.5 cells stably expressing human or murine SEC14L2 (93% amino acid identity) were lysed and protein expression was confirmed by immunoblotting. These cells were then transfected with S52/SG-neo and selected with G418. The resulting cell colonies were stained with crystal violet. b, c, Only isoform 1 of SEC14L2 supports HCV RNA replication. The SEC14L2 gene is comprised of 12 exons and results in 3 alternatively spliced transcript variants encoding 3 different protein isoforms. Isoform 1 was identified in cDNA screening. b, Schematic representation of SEC14L2 isoforms. Coding exons are shown as green blocks and the amino acid length of each protein is shown on right. c, Cell lysates from Huh-7.5 cells stably expressing 3 SEC14L2 isoforms were analysed by 4–12% SDS–PAGE and immunoblotting was performed with SEC14L2 mouse monoclonal antibody. The bands highlighted with asterisks might be a cleavage product of SEC14L2. UC, untransduced cells. These cells were transfected with S52/SG-neo and selected with G418 for 3 weeks. The resulting cell colonies were stained with crystal violet. d, SEC14L3 and SEC14L4 (86% and 80% amino acid similarity to SEC14L2, respectively)¹¹ are not expressed in Huh-7.5 cells. Cell lysates from Huh-7.5 cells stably expressing SEC14L2, SEC14L3, or SEC14L4 were analysed by 4–12% SDS–PAGE followed by immunoblotting with the indicated antibodies (the signal generated by SEC14L3 antibody in SEC14L2-expressing cells most likely reflects the cross-reactivity of SEC14L3 antibody). These cells were then tested for their ability to support replication of S52/SG-neo. e, f, Deletion mutants of SEC14L2 do not support HCV RNA replication. e, Schematic representation of N-terminal EGFP-tagged SEC14L2 deletion mutants. f, Cell lysates from Huh-7.5 cells stably expressing the full-length SEC14L2 and the deletion mutants were analysed by 4–12% SDS–PAGE followed by immunoblotting with the indicated antibodies. These cells were then transfected with S52/SG-neo and selected with G418 for 3 weeks. The resulting cell colonies were stained with crystal violet. g, h, As N-terminal deletion mutants of SEC14L2 were unstable in Huh-7.5 cells (they formed protein aggregates), we generated chimaeric constructs by fusing C-terminal ends of SEC14L2 with the corresponding N-terminal sequences from SEC14L4 and tested their ability to support HCV replication. g, Schematic representation of C-terminal EGFP-tagged chimaeric constructs. h, Huh-7.5 cells stably expressing the indicated chimaeric constructs were lysed and protein expression was confirmed by immunoblotting with anti-GFP antibody. The cells were then transfected with S52/SG-neo and selected with G418 for 3 weeks.