Figure 5: Gag interacts with eEF2.

(a) Flowchart of the proteomics analyses used to define Gag-binding proteins (top). Venn diagram exhibiting the number of overlapping hits between Gag-binding partners with SGDFs (bottom)⁶. (b) HeLa cells were transfected with 5 μg of Gag-EGFP or GFP alone and 48 h later were exposed to PatA, lysed and cell extracts were subjected to IP with an anti-GFP antibody. The presence of endogenous eEF2 in the immunoprecipitates was assessed by western blotting. Ten per cent of total cell lysates used in IP are shown as input. (c,d) HEK293T cells were co-transfected with mCherry or mCh-Gag and Flag-eEF2 and collected for IP with (c) anti-RFP (to detect mCherry) or (d) anti-Flag followed with immunoblot analysis. (e) Cell lysates were incubated in the presence (+) or absence (−) of RNase A for 30 min before IP analysis. Data shown are representative of three experiments. (f) HeLa cells were transfected with HIV-1 WT (pNL4-3) or CA mutants’ (Q7A/Q9A, G89A) provirus and collected for IP with anti-p24 (to detect Gag). (g) Relative quantitation of eEF2 co-IP with Gag. Ratio of eEF2/Gag for n=3 experiments. Data are normalized to 1 for Gag WT and represented as mean±s.d. (h) Cells from f were subjected to PLA. Nuclei stained with DAPI (blue); PLA performed for Gag and eEF2 (red). Scale bar, 15 μm. (i) Relative quantitation of Gag–eEF2 association. Spots per cell were counted. Data represented as mean±s.e.m. of three independent experiments. Statistical analysis was performed with a one-way ANOVA combined with the Bonferroni’s post test. (j) HIV-1-expressing HeLa cells were treated with a WT or Q7A/Q9A peptide for 1 h and subjected to PatA. PLA was performed. Top panel: nuclei stained with DAPI (blue); PLA performed for Gag and eEF2 (red). Scale bar, 15 μm. (k) Relative quantitation of Gag–eEF2 association. Spots per cell were counted. Data are represented as mean±s.e.m. of three independent experiments. Statistical analysis was performed with a one-way ANOVA combined with the Bonferroni’s post test. (l) HEK293T cells were co-transfected with Gag-deletion mutants (MA/CA; MA; CA) fused to Rluc and Flag-eEF2. Cell lysates were IP with anti-RLuc and immunoblotted with anti-Flag (*nonspecific band).