Figure 4. MARCH8 Ubiquitinates NS2 In Vitro, in Cells and in HCV RNA-Transfected Cells.
(A) Truncated rNS2 was incubated for 6 hr with ubiquitin alone (lane 3) or with ubiquitin, E1 activating enzyme, and UBE2H (E2 ubiquitin-conjugating enzyme) in the presence of either Huh7.5.1 cell extract (lane 1) or recombinant MARCH8 (rMARCH8; lane 5). Cell extract and MARCH8 incubated in the absence of rNS2 served as controls (lanes 2 and 4, respectively). Shown is a representative membrane blotted with anti-NS2 antibody and quantitative NS2 ubiquitination signal data relative to lane 3.
(B) Lysates of 293T cells transfected with an empty plasmid (lane 1) or ectopically expressing GLuc-NS2 plus either an empty plasmid (lane 2), WT (lane 3), or ligase-dead GLuc-MARCH8 mutant (MARCH8-LD; lane 4) were subjected to IP with anti-NS2 antibody. Shown are representative membranes blotted with antibodies against ubiquitin and NS2 and quantitative data relative to lane 2.
(C) Cell lysates described in (B) were incubated first with FLAG Anti-K63 TUBE reagent, followed by IP with anti-NS2 antibody. Membranes blotted with antibodies against NS2, ubiquitin, FLAG, GLuc, and actin, and quantitative data relative to lane 2 are shown.
(D) Lysates of 293T cells co-transfected with UB-HA-WT (lanes 1, 3, and 4) or UB-HA-K63R mutant (lane 2), WT GLuc-NS2 (lanes 1, 2, and 4) and MARCH8 (lanes 1, 2, and 4), or empty (lane 3) plasmids were subjected to IP with anti-NS2 (lanes 1–3) or IgG (lane 4) antibodies. Membranes blotted with antibodies against HA, NS2, GLuc, MARCH8, and actin, and quantitative data relative to lane 1 are shown. WCL samples in (D) were run on the same gel, from which a few lanes were cut out.
(E) Lysates of 293T cells co-transfected with UB-HA-WT (lanes 1–6), WT GLuc-NS2 (lanes 1, 3, and 5), or 4KE GLuc-NS2 mutant (NS2–4KE; lanes 4 and 6), and MARCH8 (lanes 1, 5, and 6) or empty (lanes 2, 3, and 4) plasmids, were subjected to IP with anti-NS2 (lanes 2–6) or IgG (lane 1) antibodies. Membranes blotted with antibodies against HA, NS2, GLuc, MARCH8, and actin, and quantitative data relative to lane 3 are shown.
(F) Left: MARCH8 protein by western blot in cells transfected with the indicated siRNAs (numbers represent MARCH8 to actin protein ratio relative to the NT control). See Figure S3D for cellular viability data. Right: Lysates of Huh7.5.1 co-transfected with HCV RNA and the indicated siRNAs were subjected to IP with anti-NS2 (lanes 2 and 3) or IgG (lane 1) antibodies. Representative membranes blotted with anti-ubiquitin and NS2 antibodies and quantitative data relative to lane 2 are shown.
(G) Left: MARCH8 protein by western blot in a 293T cell line deleted for MARCH8 via CRISPR/Cas9, this MARCH8KO cell line upon ectopic expression of GLuc NS2 and/or MARCH8, and control (WT) cells. Right: Lysates of this MARCH8KO cell line ectopically expressing GLuc-NS2 (lanes 1–4) and empty control (lanes 1–3) or FLAG-MARCH8 (lane 4) were subjected to IP with anti-NS2 (lanes 2–4) or IgG (lane 1) antibodies. Representative membranes blotted with antibodies against ubiquitin, GLuc, MARCH8, and actin as well as quantitative data relative to lane 2 are shown.
The experiments shown in (A) were conducted three times, and those shown in (B)–(G) were conducted twice. Representative membranes are shown. The numbers below the membranes indicate the signal of NS2 ubiquitination (ladder or smear of bands >75 kDa) normalized to the respective NS2 pull down relative to the respective controls. Molecular weight markers are indicated on the left (kDa). Arrows denote molecular weights of rNS2 (A), GLuc-NS2 (B–E and G), and native NS2 (F). WCLs, whole cell lysates.