EGP expression in target cells enhances EGP/HIV transduction. (A) Cell surface expression of EGP. EGP Tet-On cells were seeded in 12-well plates (6 × 104 cells/well) and EGP expression was induced with indicated concentrations of dox. After 24 h post-induction, cell surface EGP levels were analyzed by flow cytometry using an EGP monoclonal antibody. (B) Western blot analysis of EGP expression in Tet-On cells. EGP Tet-On cells were seeded in 12-well plates and induced with indicated concentrations of dox. Forty-eight hours post-induction, cell lysates were subjected to SDS-PAGE followed by immunoblotting using a EGP monoclonal antibody. (C) HIV pseudotyping constructs. HIV packaging construct encodes gag/pol genes required for virion assembly. Envelope expression construct encodes genes for EGP or MGP or VSV-G under the control of a CMV promoter. HIV reporter construct encodes the viral genomic RNA, carrying a luciferase or a GFP reporter gene. (D) Enhancement of EGP/HIV transduction by EGP expression in target cells. EGP or control Tet-On cells were seeded in 24-well plates (3 × 104 cells/well) and induced with varying concentrations of dox. After 24 h post-induction, cells were challenged with EGP/HIV, MGP/HIV or VSV-G/HIV pseudovirions carrying a luciferase reporter gene. The luciferase activities in the cell lysates were measured 48 h post-infection and are presented as percentage of the uninduced cells (100%). Data represents an average of at least three independent experiments. Bars, standard deviations. (E) The mucin-like region in EGP is not required for enhancement. Tet-On stable cells with EGP mutant lacking the mucin-like region (ΔEGP) were induced with dox and challenged with pseudotyped virions carrying luciferase reporter. The luciferase activities are shown as relative percentage of the uninduced cells (100%). Data represents an average of at least three independent experiments. Bars, standard deviations. (F) EGP Tet-On cells infected with EGP/HIV pseudovirions carrying a GFP reporter. The percentage of GFP expressing cells, shown in each panel as inserts, were quantified by flow cytometry.