FIG. 2.
EVM005 interacts with HA-cullin-1 during infection. (A) HEK293T cells were infected with VV Copenhagen (VVCop), VV-Flag-EVM005, or VV-Flag-EVM004 and mock transfected or transfected with pSC66-HA-cullin-1. At 12 h postinfection, cells were lysed in NP-40 lysis buffer, subjected to immunoprecipitation (IP) with anti-HA to precipitate cullin-1 (Cul1), and Western blotted (WB) with anti-Flag for the detection of EVM005. (B) EVM005 fails to interact with HA-cullin-1ΔN53. HEK293T cells were infected with VV Copenhagen, VV-Flag-EVM004, or VV-Flag-EVM005 and transfected with HA-cullin-1, HA-cullin-1Δ610-615, or HA-cullin-1ΔN53. Cellular lysates were immunoprecipitated with anti-HA to precipitate cullin-1 and Western blotted with anti-HA or anti-Flag for the detection of cullin-1 or EVM005, respectively. (C) Endogenous Skp1 fails to interact with HA-cullin-1ΔN53 during infection. HEK293T cells were mock transfected or transfected with pSC66-HA-cullin-1, pSC66-HA-cullin-1Δ610-615, or pSC66-HA-cullin-1ΔN53. Cellular lysates were immunoprecipitated with anti-HA to precipitate cullin-1 and Western blotted with anti-HA or anti-Skp1 to detect cullin-1 or endogenous Skp1, respectively. (D) Localization of ectopically expressed cullin-1 and EVM005. HeLa cells (2 × 105) were transfected with HA-cullin-1 or Flag-EVM005 for 16 h, cullin-1 was visualized by anti-HA (a to c), and EVM005 was visualized with anti-Flag (d to f). (E) Flag-EVM005 colocalizes with HA-cullin-1 during infection. HeLa cells (2 × 105) were infected and transfected with pSC66-HA-cullin-1. Cells were costained with anti-cullin-1, anti-Flag, and DAPI to detect viral factories and the nucleus. (a to d) HeLa cells infected with VV-Flag-EVM005. (e to h) HeLa cells infected with VV Copenhagen and cotransfected with pSC66-HA-cullin-1. (i to l) HeLa cells infected with VV-Flag-EVM005 and cotransfected with pSC66-HA-cullin-1. A total of 77% of infected/transfected cells displayed colocalization between Flag-EVM005 and HA-cullin-1. α, anti.