Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions

Abstract

Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes.

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.

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.

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.