Figure 1.
Characterization of E2 proteins expressed in HT-GPCA conditions.
(A) Schematic representation of the HT-GPCA method. This assay is based on the reconstitution of a luciferase activity upon co-expression of interacting partners in fusion with two inactive fragments of the Gaussia princeps luciferase (designated GL1 and GL2). The reconstituted Luciferase activity is estimated from a Normalized Luminescence Ratio (NLR) (B) 293T cells were transfected with the pTK6E2BS-Luc reporter and the GL2-E2 expressing plasmids. Fold activation is given relative to TK6E2BS-Luc in absence of E2. (C) E2-Firefly luciferase fusion proteins were expressed in 293T cells and the firefly luciferase activity was determined 24 h post-transfection. The results are expressed as a percentage of the activity obtained with the firefly luciferase only.
Figure 2.
Interaction of E2 with gold standards by HT-GPCA.
(A) Heat maps representing the interactions between the 12 E2 proteins (by columns) and the gold standards (rows). The colour represents Normalized Luminescence Ratio (NLR) obtained by HT-GPCA, from no interaction (black) to strong interactions (light blue). The red rectangles indicate interactions identified in the literature (LCE2-PPI) (B) Heat maps representing the interactions between the 12 E2 proteins (by columns) and the negative random set (rows). (C) Interaction between BRD4 CTD and mutated E2 proteins (16E2I73A and 18E2I77A) tested by HT-GPCA. The results are displayed relative to BRD4 CTD interaction with the wild-type E2 proteins.
Figure 3.
Interaction map between the 12 E2 proteins and the 121 cellular proteins by HT-GPCA and hierarchical clustering.
(A) Heat maps representing the complete dataset of interactions between the 12 E2 proteins (by columns) and the 121 cellular proteins (by rows). The intensity of interaction is represented by the colour, from black (no interaction) to light blue (strong interactions) based on Normalized Luminescence Ratio (NLR). The E2-PPI profiles were clustered according to their similarities by hierarchical clustering (tree above the heat map). (B) Interaction dendrogram generated from the hierarchical clustering of E2-interaction profiles and phylogenetic tree based on E2 sequences alignment.
Table 1.
HR-specific interactions of the E2 proteins from HPV16 and HPV18.
Figure 4.
Topological analysis of the E2 interaction network.
(A) Cumulative distribution of node degree of a reconstructed human interactome (black curve) and the E2 interactome (red curve). The fraction of proteins under the estimated average degree of the human interactome (8) is represented. The characteristics of each interactome are given in the inset. (B) Distribution of degree probability of the human (black) and the E2 interactome (red). P(degree) is the probability to connect K other proteins in the network. For the human interactome, the straight line represents the linear regression fit of the data (with a correlation coefficient R2 = 0.91). For the E2 interactome, we could not fit the data to a linear regression (R2 = 0.34).
Figure 5.
E2-targeted functional families.
Cellular proteins (nodes) classified into enriched families based on the Gene Ontology annotations are colored according to the associated GO functions. Proteins shared by different families are bi-coloured. The network representation was generated by Cytoscape.
Table 2.
Functional enrichment analysis of E2 targets.
Figure 6.
Validation of HR-specific interactions.
(A) HeLa cells were transfected by pTK6E2BS-Luc reporter and HPV16 or HPV18 E2 expression plasmids. Where indicated, GTF2B was added. Fold activation is given relative to TK6E2BS-Luc in the absence of E2. (B) HeLa cells were transfected with a pool of four siRNA targeting GTF2B or control siRNA (Scramble). 48 h post silencing, pTK6E2BS reporter plasmid was transfected along with E2 expression plasmids. Results are given as a fold activation relative to TK6E2BS basal activity in the presence of the same siRNA. Experiments were performed in triplicate with each bar representing the mean ± SD. The stars (***) indicate a statistical significant difference between fold activation by 16E2 with a scramble siRNA or a GTF2B-directed siRNA directed (p-value<0,001) (C) HaCaT cells were co-transfected by GFP-E2 proteins from HPV16 or HPV18 and mCherry-VPS39. 24 h later, cells were fixed in 4% paraformaldehyde, stained with DAPI and subjected to fluorescence microscopy.
Figure 7.
Fluorescence analysis of interactions between E2 proteins and intracellular transport proteins.
(A–D) HaCaT cells were cotransfected with expression plasmids for the indicated GFP-E2 proteins and mCherry-VPS52 (A), mCherry-VPS39 (B), mCherry-CLTA (C), and mCherry-KIF20A(D). After fixation, the cells were subjected to fluorescence microscopy after counterstaining of the nucleus with DAPI.