Vpr localizes to autophagosomes, and its autophagy-mediated elimination is counteracted by Nef.

作者：Yuexuan Chen (11424022), Susanne Klute, Anju Bansal (56201), Konstantin M. J. Sparrer, Ruth Serra-Moreno · 发表于：Figshare · 年份：2026 · DOI：10.1371/journal.ppat.1014020.g005 · 研究领域：Chemistry、Molecular biology、Cell biology、Biophysics、Biology、Biochemistry

(A) The subcellular distribution of Vpr relative to autophagosomes (LC3) was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. (B) HEK293T cells were co-transfected with 2,000 ng NL4-3 Vpr-myc and increasing amounts of a plasmid encoding NL4-3 Nef-HA (0-2,000 ng). 44 h later, DMSO or increasing concentrations of Torin2 (5-10 nM) were added. 4 h later, cells were harvested and analyzed by western blot. Membranes were probed for Vpr, Nef, LC3, and SQSTM1. ACTB was included as a loading control. Blots are representative of 3 independent experiments. (C) Vpr protein levels were measured by densitometry, normalized to ACTB and their expression relative to the DMSO and no Nef treatment was calculated. Data correspond to the mean and SEM of 3 independent experiments. Dotted lines represent 2-fold threshold. (D) The degree of co-localization between Vpr and LC3 puncta was examined by fluorescence microscopy in HEK293T cells constitutively expressing EGFP-LC3B in the presence of Nef and treated with Torin2 (10 nM) 4 h before staining. Images are representative of 3 independent experiments. White scale bar: 10 μm. Graph: The Pearson’s correlation coefficient ( R ) for the co-localization of Vpr and EGFP-LC3B in the presence and absence of Nef was calculated from 17 randomly selected fields. Data correspond to ...