Comparative analysis of virus–host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase

Abstract

Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or “iginterologs”. Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus-host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interation assays: yeast two-hybrid (Y2H), and our recently described “High-Throughput Gaussia princeps Protein Complementation Assay” (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split Gaussia princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus-host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism-host interactions.

Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

“…After 24 h, cells were transfected using jetPEI (Polyplus) with 100 ng of each recombinant vector. At 24 h post-transfection, after a washing step in PBS, cells were harvested with 30 l of Renilla lysis buffer (Promega, E2820) for 30 min, and luciferase enzymatic activity was measured in triplicates using a Berthold Centro XS LB960 luminometer by injecting 100 l of Renilla luciferase assay reagent (Promega, E2820) into cell lysates and counting luminescence for 10 s. Results were expressed as a fold-change normalized over the sum of controls (normalized luminescence ratio) (37,45).…”

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

“…This large-scale comparative interactomic approach has been recently successfully applied to three early proteins of the Human Papillomaviruses (HPV): E2, E6 and E7 originating from different genotypes representative of the HPV natural diversity in tropisms and pathologies 13,14 . For each of the early proteins, hierarchical clustering of the interaction profiles mostly recapitulates HPV phylogeny, proving the robustness of the approach and the pertinence of interaction datasets (Figure 3 adapted from references 13 and 14).…”

Convergent evolution and mimicry of protein linear motifs in host–pathogen interactions

“…Linear motif repertoire has been linked to changes in viral phenotypic traits such as virulence [65][66][67], persistence [68,69,70 ] tropism [62 ], oncogenicity [26,71,72], and response to therapy [73]. The association may be inferred from purely statistical correlation between motif repertoire and phenotypic traits [62 ,67,68,73], such as the correspondence between the motif repertoire in the HIV proteome and response of patients to antiretroviral therapy [73].…”

“…The association may be inferred from purely statistical correlation between motif repertoire and phenotypic traits [62 ,67,68,73], such as the correspondence between the motif repertoire in the HIV proteome and response of patients to antiretroviral therapy [73]. The association may be further strengthened by experimental demonstration of biochemical properties modulated by the motif [26,69,71,72]. For example, specific substitutions in E7 proteins of high-risk oncogenic types, compared to low-risk types, lead to 30-fold increases in the thermodynamic and kinetic stability of the complexes formed between E7 and the retinoblastoma cell cycle regulator [26].…”