Extended Data Fig. 4: Entry efficiencies of filovirus pseudoviruses into three human target cell types, measured using a double-normalization strategy to control for GP expression levels.
From: Structures of Marburgvirus glycoprotein and its complex with NPC1 receptor
Retroviruses pseudotyped with EBOV, RAVV, or Musoke GPs were used to infect Huh7 (hepatoma) (a), HUVEC (primary vascular endothelial) (b), and THP-1–derived macrophages (c), all major cellular targets of filoviruses. For each GP, both wild-type and C-terminally C9-tagged versions were packaged, yielding six pseudoviruses in total. Entry efficiencies were measured in four biological replicates per pseudovirus and cell type. Normalization was performed in two steps: first, wild-type pseudoviruses were standardized to their C9-tagged counterparts using GP-specific neutralizing sera; second, C9-tagged RAVV and Musoke pseudoviruses were normalized to C9-tagged EBOV pseudoviruses using anti-C9 antibodies. This double-normalization ensured equivalent GP expression levels across all pseudoviruses, allowing direct comparison of entry efficiencies mediated by the three wild-type GPs. Data are presented as mean ± SEM (n = 4 biologically independent samples). Statistical differences between wild-type EBOV GP and wild-type MBV GPs (RAVV and Musoke), and between C9-tagged EBOV GP and C9-tagged MBV GPs (RAVV and Musoke), were determined using two-tailed unpaired Student’s t-tests. No adjustment was made for multiple comparisons. The P values are as follows: wild-type GP—Huh7, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P < 0.0001; HUVEC, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P < 0.0001; THP-1-derived macrophages, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P = 0.0001; C9-tagged GP—Huh7, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P < 0.0001; HUVEC, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P < 0.0001; THP-1-derived macrophages, EBOV vs RAVV P < 0.0001, EBOV vs Musoke P = 0.0002. Asterisks above the bars indicate statistical significance (****P < 0.0001, ***P < 0.001). Uncropped Western blot images with molecular weight markers are shown in Supplementary Fig. 1. All experiments were repeated three times with independently prepared pseudovirus stocks, yielding consistent results.
