(
A) Workflow of SILAC-based proteomic validation time course experiment. CEM-T4 T cells pre-labelled with heavy amino acids were infected with NL4-3-dE-EGFP HIV at an MOI of 10, and control cells pre-labelled with medium amino acids were mock-infected without virus. Aliquots of HIV-infected (heavy) and mock (medium) cells were harvested sequentially at the indicated timepoints and subjected to SILAC-based whole cell proteomic analysis. (
B) Functional annotation clusters enriched amongst proteins from clusters #1–4. Enrichment of Gene Ontology Molecular Function and Biological Process terms against a background of all quantitated proteins was determined using DAVID. Functional annotation clusters with enrichment scores > 1.3 (equivalent to a geometric mean of all included enrichment p values<0.05) were considered significant. Representative Gene Ontology terms are indicated. (
C) Temporal profiles of Vpr targets UNG and HLTF. Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity. (
D) Enlargement of part of cluster #2 from hierarchical cluster analysis (
Figure 2A). The heatmap shows log
2 (fold change in protein abundance compared with uninfected cells). Previously characterised Vpr targets UNG and HLTF are highlighted (red).