Expression of Ebolavirus glycoprotein on the target cells enhances viral entry

Abstract

Background: Entry of Ebolavirus to the target cells is mediated by the viral glycoprotein GP. The native GP exists as a homotrimer on the virions and contains two subunits, a surface subunit (GP1) that is involved in receptor binding and a transmembrane subunit (GP2) that mediates the virus-host membrane fusion. Previously we showed that over-expression of GP on the target cells blocks GP-mediated viral entry, which is mostly likely due to receptor interference by GP1.

Results: In this study, using a tetracycline inducible system, we report that low levels of GP expression on the target cells, instead of interfering, specifically enhance GP mediated viral entry. Detailed mapping analysis strongly suggests that the fusion subunit GP2 is primarily responsible for this novel phenomenon, here referred to as trans enhancement.

Conclusion: Our data suggests that GP2 mediated trans enhancement of virus fusion occurs via a mechanism analogous to eukaryotic membrane fusion processes involving specific trans oligomerization and cooperative interaction of fusion mediators. These findings have important implications in our current understanding of virus entry and superinfection interference.

Figure 1

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.

Figure 2

GP enhancement is correlated with the entry susceptibility of the target cells. (A) EGP expression in HeLa cells enhances EGP/HIV transduction. HeLa Tet-On cells with EGP, ΔEGP or control vector were induced with dox and challenged with pseudotyped viruses. The luciferase activities were measured 48 h post-infection and are shown as percentage of the uninduced cells (100%). Data represents an average of at least three independent experiments. Bars, standard deviations. (B) EGP expression in Jurkat Tet-On cells does not enhance EGP/HIV transduction. Jurkat Tet-On cells expressing EGP or ΔEGP or control vector were challenged with luciferase reporter virus and luciferase activity in the cell lysates are shown in relative light units (RLU). Data represents an average of at least three independent experiments. Bars, standard deviations. (C) Enhancement of EGP/MLV pseudovirus transduction in HeLa Tet-On cells. HEK Tet-On cells with EGP, ΔEGP or control vector induced with dox were challenged with ΔEGP/MLV pseudovirion carrying a GFP reporter. The percentage of GFP expressing cells are shown as inserts in each panel.

Figure 3

Analysis of Ebola GP mutants in trans enhancement process. (A) HEK Tet-On cells expressing Wt EGP were challenged with EGP mutant viruses, GP1 mutants, relative infectivity of Wt and GP1 mutant viruses. Fusion peptide mutants, relative infectivity of Wt and fusion peptide mutant viruses. Coiled-coil mutants, relative infectivity of Wt and GP2 mutant viruses. (B) Matrix analysis of trans enhancement by EGP. Wt or mutant EGP expressing HEK Tet-On was either uninduced or induced with 1 μg/ml of Dox. After 24 h, the cells challenged with pseudovirus particles carrying Wt or mutant EGP. The luciferase activities in infected cells are represented as relative percentage of luciferase activity in uninduced cells. Data represents an average of three independent experiments. For clarity of the chart we have omitted the error bars in panel B.

Figure 4

β-lactamase based Ebola VLP fusion assay. (A) Schematic representation of VP40 and BlaM-VP40 chimera. (B) BlaM based VLP fusion assay. HEK cells were infected by spinoculating at 1,500 rpm for 2 h (4°C) followed by incubation at 37°C for 3 h. The cells were loaded with CCF4-AM dye and analyzed by fluorescence microscopy. The percentage of infected cells were quantified by flow cytometry and shown as inserts. (C) BlaM VLP fusion assay in EGP expressing cells. EGP expressing cells challenged with Wt or mutant EGP carrying BlaM-VLP. The cells were loaded with CCF4-AM dye and analyzed by fluorescence microscopy. The percentage of infected cells were quantified by flow cytometry and shown as inserts.

Figure 5

A proposed model for EGP-mediated enhancement. Step 1. Receptor binding. EGP binds to its cell surface receptor(s). The bound virus is endocytosed into the cell, where it undergoes cleavage by cysteine proteases (cathepsin B and L) under low pH environment. Step 2. Fusion peptide insertion. The fusion peptide from the virion EGP is inserted into the host membrane. Also, EGP of the target cell inserts to the viral membrane through the fusion peptide. Step 3. Initiation of membrane fusion. Conformational changes occur on EGPs and lead to direct contact and interaction between the viral membrane-anchored EGP and the cell membrane-anchored EGP, forming an oligomeric complex. Membrane fusion ensues. Step 4. Membrane fusion. Fusion pore forms and host-viral contents mix.