Extended Data Fig. 1: Ubiquitination of flavivirus E protein.
From: Envelope protein ubiquitination drives entry and pathogenesis of Zika virus
a, Proteasome inhibition blocks ZIKV replication. JEG-3 cells were pretreated with DMSO or MG132 (2 h) followed by ZIKV infection (MOI 2, 24 h, visualized by immunofluorescence with anti-E 4G2). b, Ubiquitinated peptides from flavivirus-infected cells identified by mass spectrometry (peptides highlighted in yellow, diglycine residues indicating ubiquitination in red and conserved residues in green). Sequences for strains ZIKV FSS13025, GenBank: KU955593.1; DENV-2 Y98P, JF327392.1; West Nile virus (WNV) NY99, DQ211652; and yellow fever virus (YFV), ANC33490.1. JEG-3 cells were used for ZIKV infections, Huh7 cells for DENV infections and A549 cells for WNV infections (repeated in U2OS cells with identical results, two independent experiments). Representative mass spectra for ubiquitinated peptides found for WNV are shown. b and y ions are indicated in blue and red, respectively. c, Whole-cell extracts from DENV- or ZIKV-infected (MOI 2, 20 h) Huh7 cells transfected with HA–Ub, followed by DMSO or MG132 treatment (6 h) were used for HA immunoprecipitation. Immunoblots are shown. NT, non-treated d, Whole-cell extract from cells transfected with vectors expressing E and wild-type ubiquitin or all K-to-R mutants except for K63 or K48 (only), or Ub(K48R) and Ub(K63R), followed by immunoprecipitation. e, JEG-3 cells pretreated with MG132 or DMSO. Cells were incubated with ZIKV at 4 °C for 30 min, followed by a wash with or without glycine to test virus adsorption. Additional samples were then switched to 37 °C to allow virus internalization. Viral RNA detection by quantitative reverse-transcription PCR (qRT–PCR). Representative of two independent experiments, n = 3 technical replicates, mean ± s.e.m., unpaired t-test, two-sided, *P < 0.05; NS, not significant. All experiments are representatives from two independent experiments, with similar results.
