Etude de la relation hôte pathogène du système Acanthamoeba/Mimivirus – MIMIVIRUS

The discovery of Mimivirus and deciphering of its 1.2Mb genome sequence sent a shock wave through the community of virologists and evolutionists. The size, gene content, and phylogenetic characterization of the virus genome challenged many accepted ideas about what virus should look like, and where they might come from. Even though large DNA virus genome sequences accumulated steadily in the databases since 1990, Mimivirus represented a quantum leap by two accounts: quantitatively it was the first endowed with a gene content larger than cellular microorganisms, and qualitatively it was breaking the "conceptual mold" by exhibiting several central components of the protein translation system, such as amino-acyl tRNA synthetases. Until then, protein translation was thought to be an absolute criterion distinguishing all cellular organisms from all viruses. Despite its exceptional features, Mimivirus harbors the characteristic core gene set of previously described DNA virus families such as Poxviruses, Iridoviruses, or Phycodnaviruses (algal virus). The analysis of metagenomic data also revealed that the newly defined family of Mimiviridae represents the most abundant eukaryotic viruses in marine environments. Mimivirus could thus be interpreted as some kind of "living fossil," suggesting that large DNA viruses might have originated from ancestors as complex as primitive cellular organisms, by a process of reductive evolution (i.e. progressive loss of genes observed in intracellular parasitic bacteria). The unexpected characteristics of Mimivirus, together with its controversial anchoring in the Tree of Life, triggered a healthy debate, whereby new and old hypotheses on the origin of viruses and their role in the emergence of the eukaryote were reassessed. Outrageous ideas such as 'viruses invented DNA', 'the nucleus derived from an ancient virus', 'DNA viruses emerged from primitive nuclei', or 'DNA virus are the parasitic remains of a 4th cellular domain of life', are now reevaluated on this new research front. The present proposal aims at fueling this theoretical debate with real data on the life cycle of Mimivirus, and its interaction with its laboratory host Acanthamoeba focusing on the eclipse phase. First, a comprehensive transcriptomic study of the pathogen-host interaction will be performed using two complementary techniques: 454-cDNA pyrosequencing and Agilent-tiling arrays. These experiments will set up the stage for a series of in vivo studies aiming at elucidating the cellular mechanisms at work throughout the whole Mimivirus replication cycle, with an emphasis on two specific questions: how does this cytoplasmic virus emulate typical nuclear functions, and how and which other host cellular functions are hijacked by Mimivirus, with a particular interest for energy production and protein synthesis. Our overall goal, is to understand the complex pathogen-host relationship leading to the setting up of 'virus factories', and to what extent these transient organelles resemble a putative primitive nucleus and eventually recapitulate its evolution. These in vivo studies will be performed in parallel on infected and non infected Acanthamoeba and will include: 1) the expression and differential localization of Mimivirus selected gene products, 2) interference experiments using RNAi to inhibit Mimivirus gene expression and study the consequences on the virus cycle and host behavior, 3) protein-protein interaction studies. These studies will take advantage of the proteomic and imagery platforms available in our Institute (fluorescence and electron microscopy). The proposed project establish a simple experimental model for the study of host/pathogen interactions between an eukaryotic host, Acanthamoeba, and the most complex representative of large dsDNA viruses, Mimivirus. The two comprehensive transcriptomic analyses that we proposed will provide a detailed spatial (5' to 3') and temporal maps of Mimivirus gene expression, including potential non-coding RNAs. This will result in the highest quality of genome annotation ever achieved for a large virus. The bioinformatic clustering of the genes exhibiting similar expression patterns, will allow the discovery of new regulatory signals, setting up the stage for a really 'systemic' understanding of Mimivirus life-cycle. The in situ analysis of the temporal/spatial pattern of Mimivirus genes and proteins expression in the host will provide the detailed experimental data needed to decipher their roles in the replication cycle and suggest the mechanisms by which they interfere with the metabolism of Acanthamoeba. Finally, discovering the molecular strategies used by Mimivirus to control its amoebal host, might open new avenues in the therapy of cellular malfunctions at the origin of many diseases. 60% of Mimivirus genes correspond to unknown functions. It is likely that a number of them participate in hijacking the host cell in totally original ways.