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

Unité de Génétique, Papillomavirus et Cancer Humain (GPCH), Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France.
Methods (Impact Factor: 3.65). 08/2012; 58(4). DOI: 10.1016/j.ymeth.2012.07.029
Source: PubMed


Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or "interologs". 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 interaction 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 G. 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.

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Available from: Lionel Tafforeau, Oct 04, 2015
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    • "Gaussia luciferase has been used to measure various cellular processes in cell-based assays as well as in vivo[11]. A split-GLUC system has been developed [12,13] and used for imaging of receptor-ligand interactions [14], virus-host protein interactions [15] and oligomer formation of amyloid beta peptide [16]. A highly useful property of Gaussia luciferase is its stability in body fluids such as blood and urine. "
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    ABSTRACT: Background Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail. Results We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC. Conclusions Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.
    BMC Biochemistry 07/2014; 15(1):14. DOI:10.1186/1471-2091-15-14
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    • "Briefly, system biology is an interdisciplinary experimental and computational field of study that focuses on complex interactions within biological systems with aim to identify novel key features of cell signaling networks. In the last years, it has been widely used in a variety of biomedical contexts, including the deciphering of the network of virus/host interactions (the so call ''interactome'') (Neveu et al., 2012; Sorathiya et al., 2010). The rationale of system biology is that multiple regulatory cascades can be converged into hub-proteins/interactions whose inhibition can affect multiple signaling pathways, commensurate with the administration of multiple drugs that would hopefully cause an overall failure of the ''disease system''. "
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    ABSTRACT: Abstract Despite decades of antiviral drug research and development, viruses still remain a top global healthcare problem. Compared to eukaryotic cells, viruses are composed by a limited numbers of proteins that, nevertheless, set up multiple interactions with cellular components, allowing the virus to take control of the infected cell. Each virus/host interaction can be considered as a therapeutical target for new antiviral drugs but, unfortunately, the systematic study of a so huge number of interactions is time-consuming and expensive, calling for models overcoming these drawbacks. Surface plasmon resonance (SPR) is a label-free optical technique to study biomolecular interactions in real time by detecting reflected light from a prism-gold film interface. Launched 20 years ago, SPR has become a nearly irreplaceable technology for the study of biomolecular interactions. Accordingly, SPR is increasingly used in the field of virology, spanning from the study of biological interactions to the identification of putative antiviral drugs. From the literature available, SPR emerges as an ideal link between conventional biological experimentation and system biology studies functional to the identification of highly connected viral or host proteins that act as nodal points in virus life cycle and thus considerable as therapeutical targets for the development of innovative antiviral strategies.
    Critical Reviews in Microbiology 09/2013; 41(2). DOI:10.3109/1040841X.2013.826177
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    Methods 12/2012; 58(4):315-6. DOI:10.1016/j.ymeth.2013.01.001
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Questions & Answers about this publication

  • Gregory Neveu added an answer in Biochemistry:
    Which would be the ideal biochemical technique to establish that two proteins have interacted and what is the principle?
    Could one use thermodynamics to ascertain the same?
    Gregory Neveu
    I agree with Adrian. It depends on what you need, what is your goal and so on... However I would definitely recommend you depending on what kind of material is available to you. The first one is the yeast two hybrid. Even if this technique is know to screen cDNA library, you can use it also to determine the interaction between 2 proteins of interest and it doesn't require lots of material (technological or biological). The second one is the PCA for Protein-fragment complementation assay. Your 2 proteins of interest are fused to two inactive fragment of the Renilla luciferase. You just need to transfect 100ng of each DNA in cells in 96-well plate and a plate reader to read the Luciferase. This technique exists for 20 years (developed by Michnick in Canada in the mid-90s). I designed, developed and published the update of this technique with the Renilla. I attach below the article. The third one could be the Mass spectrometry but it's much more expansive and not really easy. The last one could also be the Biacore. The advantage of this technique is that it can give you the affinity, kD, etc...
    Finally I would say that indeed the co-IP is the basic thing to do for protein-protein interaction even if the need of antibody can be somewhat annoying (good example in the article of Ashish) with a lot of non specific binding.
    Good luck anyway.