title: mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.
abstract: Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.
authors.affiliation: Structural & Genomic Information Laboratory, Centre National de la Recherche Scientifique, UPR2589, Mediterranean Institute of Microbiology IFR88, Aix-Marseille University, Parc Scientifique de Luminy, FR-13288 Marseille, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Legendre
authors.forename: Stéphane
authors.initials: S
authors.lastname: Audic
authors.forename: Olivier
authors.initials: O
authors.lastname: Poirot
authors.forename: Pascal
authors.initials: P
authors.lastname: Hingamp
authors.forename: Virginie
authors.initials: V
authors.lastname: Seltzer
authors.forename: Deborah
authors.initials: D
authors.lastname: Byrne
authors.forename: Audrey
authors.initials: A
authors.lastname: Lartigue
authors.forename: Magali
authors.initials: M
authors.lastname: Lescot
authors.forename: Alain
authors.initials: A
authors.lastname: Bernadac
authors.forename: Julie
authors.initials: J
authors.lastname: Poulain
authors.forename: Chantal
authors.initials: C
authors.lastname: Abergel
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Claverie
mesh_terms: D000048:Acanthamoeba / Q000821:virology*; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D016679:Genome, Viral; D008854:Microscopy, Electron; D056686:Mimiviridae / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins