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Mimivirus, a giant DNA virus infecting Acanthamoeba, is revealing an increasing list of unique features such as a 1.2-Mb genome with numerous genes not found in other viruses, a uniquely conserved promoter signal, and a particle of unmatched complexity using two distinct portals for genome delivery and packaging. Herein, we contribute a further Mimivirus distinctive feature discovered by sequencing a panel of viral cDNAs produced for probing the structure of Mimivirus transcripts. All Mimivirus mRNAs are polyadenylated at a site coinciding exactly with unrelated, but strongly palindromic, genomic sequences. The analysis of 454 Life Sciences (Roche) FLX cDNA tags (150,651) confirmed this finding for all Mimivirus genes independent of their transcription timings and expression levels. The absence of a suitable palindromic signal between adjacent genes results in transcripts encompassing multiple ORFs in the same or even in opposite orientations. Surprisingly, Mimivirus tRNAs are expressed as polyadenylated messengers, including an ORF/tRNA composite mRNA. To our knowledge, both the nature and the stringency of the "hairpin rule" defining the location of polyadenylation sites are unique, raising once more the question of Mimivirus's evolutionary origin. The precise molecular mechanisms implementing the hairpin rule into the 3'-end processing of Mimivirus pre-mRNAs remain to be elucidated.

The amoeba-infecting Mimivirus is the largest known double-stranded DNA virus, with a 400 nm particle size, comparable to that of mycoplasma. The complete sequence of its 1.2 Mbp genome has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes that were not expected to be found in a virus, such as genes encoding translation components, including 4-amino-acyl tRNA synthetases and homologues to various translation initiation, elongation and termination factors. A comprehensive structural and functional study of these Mimivirus gene products was initiated, as they may hold important clues about the origin of DNA viruses. Here, the first preliminary crystallographic and functional results obtained on one of these targets, Mimivirus TyrRS, are reported. Preliminary phasing was obtained using an original combination of homology modelling and normal mode analysis. Experimental evidence that Mimivirus tyrosyl tRNA synthetase recombinant gene product does indeed activate tyrosine is also presented.

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. [ABSTRACT FROM AUTHOR]