The NS4A-2K-4B precursor polyprotein is the main interaction partner of NS1.

(A) Huh7 cells stably expressing carboxy-terminally HA tagged or non-tagged (nt) NS1 were infected with the DVR2ApΔNS1 virus (MOI = 1) or mock infected and cell lysates prepared 72 h after infection were subjected to immunoprecipitation (IP) using beads coated with HA-specific antibodies as shown in Fig 3A. Samples were analyzed by western blot using NS4B-, NS4A- or NS1-specific antisera. Mature forms of NS4B and NS4A migrate with an apparent molecular weight of ~25 kDa and ~11 kDa, respectively (indicated by red arrows on the left side of each panel). The NS4A-2K-4B precursor bands migrate at ~30 kDa and ~35 kDa and are highlighted with yellow dots. (B) Huh7 cells were infected with the DVR2A wildtype (WT) or the DVR2A variant encoding an internally HA-tagged NS1 (NS1_HA*) and cell lysates prepared 72 h post infection were subjected to anti-HA immunoprecipitation followed by western blot as described in (A). (C) Huh7-Lunet_T7 cells were transfected with an NS1 to 5 polyprotein expression construct encoding either wildtype or internally HA-tagged NS1 under control of the T7 RNA polymerase promoter. Cells were lysed 20 h post transfection and lysates were subjected to anti HA immunoprecipitation as described in (A).