Journal: PLoS Pathogens

Article Title: Functional Interchangeability of Late Domains, Late Domain Cofactors and Ubiquitin in Viral Budding

doi: 10.1371/journal.ppat.1001153

Figure Lengend Snippet: (A) Yeast two-hybrid analysis of the interaction between the indicated class E VPS factors and ubiquitin containing either an intact (Ub WT) or disrupted (Ub I44A) hydrophobic patch. β-galactosidase expression was measured (as optical density at 540nm (OD540)) in Y190 cells transformed with the indicated Gal4-DNA binding domain-ubiquitin and VP16 activation domain (-HBP, -ALIX, and -UBPY) fusion constructs. Absence of a bar indicates background levels of β-galactosidase expression. A single representative of two independent experiments is shown. (B) Validation of siRNAs targeting ubiquitin-binding ESCRT-complexes or other class E VPS factors. Lysates of 293T cells transfected with GFP-Tsg101 or YFP-Hrs, -ALIX, -UBPY, or -Eap45 expression plasmids and siRNAs targeting either luciferase (−) or the specified class E VPS factors (+) were probed with an αGFP monoclonal antibody. (C) L-domain-specific inhibition of Gag budding by class E factor depletion. Quantitative Western blot analysis of VLP production from 293T cells transfected with plasmids expressing Lck-Gag(PSAP), EIAV Gag, Lck-Gag-Ub, Lck-Gag-PY or MLV Gag-HA and siRNAs directed against the indicated class E VPS protein. Corresponding cell lysates were also probed with antibodies to PFV, EIAV, HA, Tsg101 and/or ALIX, as appropriate. (D) Quantitation of VLP release following knockdown of the indicated class E VPS proteins. Values are plotted the mean+SD of two or three independent experiments and represent the levels of particles released relative to those released from cells transfected with control luciferase siRNAs, which was assigned a value of 1.

Article Snippet: Previous work has shown that Mason-Pfizer monkey virus particle release, which is dependent on a PPxY motif, is quite strongly inhibited by depletion of Tsg101 and that budding of a EIAV Gag-ubiquitin fusion protein is modestly inhibited by Tsg101 or ALIX depletion .

Techniques: Ubiquitin Proteomics, Expressing, Transformation Assay, Binding Assay, Activation Assay, Construct, Biomarker Discovery, Transfection, Luciferase, Inhibition, Western Blot, Quantitation Assay, Knockdown, Control