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Peroxisomes support human herpesvirus 8 latency by stabilizing the viral oncogenic protein vFLIP via the MAVS-TRAF complex

Fig 8

Functional vFLIP is targeted to peroxisomes in a PEX19-dependent manner.

(A) Alignment of putative mPTSs of yeast Pex8 and vFLIP (residues 34–45). Identical and similar residues are highlighted in red and blue, respectively. (B) In vitro GST pull-down assay using purified GST, GST-vFLIP, and GST-vFLIP mPTSX proteins and the extracts from 293T cells expressing HA-PEX19. (C) Immunoblots of extracts from MAVS KO 293T cells transfected with V5-vFLIP WT and mPTSX along with the indicated amounts of MAVS-Pex vectors. Relative V5-vFLIP band intensities normalized by β-actin are shown below the blot. (D) Co-IP assay using extracts from 293T cells transfected with V5-vFLIP WT and mPTSX and treated with 10 μM MG132 for 8 h. (E) Immunoblots of extracts from WT and PEX19 KO 293A cell lines transfected with vectors expressing V5-vFLIP and GST with or without MAVS-Pex vectors. Relative V5-vFLIP band intensities normalized by GST are shown below the blot. “Endo.MAVS” indicates endogenous MAVS. Relative MAVS-Pex and endogenous MAVS band intensities are shown above and below the MAVS immunoblot panel, respectively. (F) Immunostaining of 293A cells transfected with V5-vFLIP and V5-vFLIP-Pex. DMSO or MG132 was added at 8 h to the cell culture before fixation. The insets show enlarged portions of the images, revealing co-localization or juxtaposition of V5-vFLIP with peroxisomal marker PMP70. (G) NF-κB reporter assay in 293T cells transfected with empty vector, V5-vFLIP, and V5-vFLIP-Pex vectors together with an NF-κB-luciferase reporter plasmid. The corresponding cell extracts were then analyzed by immunoblotting with anti-V5 and β-actin antibodies. Data are presented as mean ± SD of triplicate samples. (H) Co-IP assay using extracts from 293T cells transfected with Myc-IKKγ expression plasmid and empty vector, V5-vFLIP, or V5-vFLIP-Pex vectors.

Fig 8

doi: https://doi.org/10.1371/journal.ppat.1007058.g008