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RNA-Seq Study Uncovers Transcription Patterns in Large Virus

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – French researchers reported online in Genome Research today that they have characterized the transcriptome of Mimivirus, the largest known virus.

By sequencing RNA from Mimivirus-infected amoeba cells, the team identified transcription start sites, mapped the 5' and 3' ends of messenger RNAs, found promoter elements, and characterized gene expression patterns in the giant virus. In the process, they also turned up dozens of transcripts not found during the initial Mimivirus genome sequencing and analysis effort.

"Not only were we able to confirm the 910 previously predicted protein-coding genes, but we also identified 75 novel genes in the Mimivirus," senior authors Jean-Michele Claverie and Chantal Abergel, researchers with the Mediterranean Institute of Microbiology's Structural and Genomic Information Laboratory, said in a statement.

Claverie led the French research team that sequenced the nearly 1.2 million base Mimivirus genome, published in Science in 2004. The double stranded DNA virus, which infects amoebae in the genus Acanthamoeba, is the largest virus known to date and houses genes coding for close to 1,000 proteins.

For the current study, the researchers used the Roche 454 GS FLX platform to do RNA-Seq from Mimivirus-infected A. castellanii cells at different time points after infection.

Using massively parallel sequencing, they generated more than 633,000 reads from complementary DNA synthesized from polyadenylated RNA in the cells. More than half of these reads mapped to the Mimivirus genome and 85 percent of the other reads mapped to the unfinished, unannotated A. castellanii genome.

They then used 5' and 3' tags to identify the ends of mRNA transcripts and begin characterizing 5' and 3' untranslated regions.

"The long reads generated by the GS FLX System were critical for the assembly of full-length transcripts, identification of polycistronic mRNA and for the discovery of short transcripts, since the 5' and 3' tags were contained within a single read," Claverie and Abergel explained in a statement.

When they sifted through this sequence data, the team turned up all of the Mimivirus protein coding genes predicted in the past. They also found 75 previously undetected transcripts. Among the new transcripts: 26 non-coding RNAs.

And, the researchers noted, mapping the reads to the Mimivirus genome not only refined their view of the intron and exon structures of certain genes, but also served to correct some errors in the genome sequence.

The team's subsequent analyses included comparisons of gene expression patterns in the virus and its host during different stages of infection, classification of Mimivirus genes based on their expression patterns during early, intermediate, and late infection, and investigations of promoters involved in regulating these groups of genes.

For instance, they detected a new Mimivirus promoter element that appears to regulate genes expressed about six hours after amoebae cells are infected by the virus. In addition, the researchers demonstrated that a promoter element also found in a virus called Sputnik — which co-infects Acanthamoeba cells already infected by Mimivirus or a related virus called Mamavirus — mediates the expression of some genes expressed late in infection.

The researchers plan to make data from the current study available to other researchers via an interactive web server.

"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," the researchers wrote.

Because marine metagenomic studies suggest Mimivirus may resemble some viruses infecting planktonic microorganisms, researchers involved with the international TARA-Oceans expedition are reportedly trolling for Mimivirus-like viruses in the ocean.

Other follow-up studies are expected to include transcriptome analyses of Mimiviruses infecting other amoeba hosts as well as studies of the virus after exposure to various inhibitors.

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