Brevia

A Giant Virus in Amoebae

See allHide authors and affiliations

Science  28 Mar 2003:
Vol. 299, Issue 5615, pp. 2033
DOI: 10.1126/science.1081867

During a study following a pneumonia outbreak in 1992, a microorganism growing in amoebae and resembling a small Gram-positive coccus (Fig. 1A) was isolated from the water of a cooling tower in Bradford, England. Despite attempts with various extraction protocols and low-stringency polymerase chain reaction, no amplification product was obtained with universal 16S rDNA bacterial primers (1).

Figure 1

(A) Mimivirus (arrows) in cytocentrifuged A. polyphaga as Gram-positive particles. (B) Electronic microscopy of Mimivirus and U. urealyticum. (C) Phylogenetic tree from alignment of ribonucleotide reductase small subunit sequences (9). Similar trees are obtained with ribonucleotide reductase large subunits and topoisomerase 2.

Study of this microorganism within Acanthamoeba polyphaga (2) revealed a characteristic viral morphology with mature particles of 400 nm in diameter and surrounded by an icosahedral capsid. This structure is consistent with the finding that Mimivirus is not filterable through 0.2-μm pore size filters. No envelope was observed, but 80-nm fibrils attached to the capsid were visible (fig. S1). A typical virus developmental cycle, including an eclipse phase, was observed (fig. S2). As it resembles a bacterium on Gram staining, it was named Mimivirus (for Mimicking microbe) (Fig. 1A). DNA digestion by Sal I and Sac II treatment of purified particles (2), followed by pulsed-field gel electrophoresis, demonstrated that Mimivirus has a double-stranded DNA circular genome of about 800 kilobase pairs (kbp). Its genome is thus larger than the sequenced genomes of several bacteria, including Mycoplasma genitalium (580 kbp), Ureaplasma urealyticum (752 kbp),Buchnera sp. (641 kbp), and Wigglesworthia brevipalpis (698 kbp) (3). Consistent with this large genome, Mimivirus particles have a size comparable to that of small bacteria such as U. urealyticum (Fig. 1B). The viruses with the largest genomes previously described are a Phycodnavirus infectingPyramimonas algae (560 kbp) and phage D of Bacillus megaterium (670 kbp) (4, 5).

Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV). This group of viruses includes four other families, including the enveloped Poxviridae, which infect vertebrates (Chordopoxvirinae) and insects (Entomopoxvirinae). The three others are also icosahedral.Iridoviridae and Phycodnaviridae are aquatic viruses, and Asfarviridae infect vertebrates (6). Whole genome shotgun sequencing is under way. Two libraries (5-kb and 9-kb inserts obtained by mechanic shearing, cloned in pcdna 2.1 with Bst XI adaptators) were constructed. Plasmid inserts were sequenced from both ends with flanking vector sequences and dye terminator primers. The preliminary assembly [using the Phred/Phrap software (7)] of 6X coverage shotgun data confirmed that the Mimivirus genome is about 800 kbp (734 kbp of preliminary sequence data with phrap score >20 is available in the WGS section of GenBank, accession # AABV01000000). More than 900 open reading frames (ORFs) longer than 100 amino acids were identified, representing ∼82.4% of the available genome, a coding fraction comparable to other NCLDVs. Comparisons to DNA and protein sequence databases (GenBank, Swissprot, and Tr-EMBL) did not reveal any sign of amoebal or other contamination.

Following Iyer et al. (8), we compared Mimivirus ORFs with viral proteins only, allowing greater sensitivity in relating it to one of the established families of large eukaryotic DNA viruses (2). We identified 21 Mimivirus proteins with known functional attributes and clear homologs in at least one of these virus families, as follows: nine inPhycodnaviridae, six in Poxviridae, five inIridoviridae, and one in Baculoviridae. Some of the genes also exhibited lower similarity to Baculoviridaeor Asfarviridae homologs. These results suggest that Mimivirus occupies an intermediary position betweenPoxviridae, Iridoviridae, andPhycodnaviridae, with which Mimivirus appears to share the Vp54 capsid protein and a glucosamine synthetase unique to theParamecium bursaria Chlorella virus. Mimivirus appears as a deep branch in the phylogenetic tree (Fig. 1C), suggesting early divergence from other virus families.

Although further characterization is needed, Mimivirus's icosahedral ultrastructure and the typical eclipse phase in its life cycle support its viral nature. Furthermore, Mimivirus lacks universal bacterial genes, such as those encoding ribosomal RNA or proteins, as well as other ubiquitous bacterial proteins involved in protein translation. The high fraction (80%, P value < 10–6) of ORFs without significant similarity to other organisms is also typical of viruses. Finally, the Mimivirus genome has 21 genes encoding homologs to proteins highly conserved in most NCLDVs (8). We propose that Mimivirus is a member of a new family of giant virus, the Mimiviridae, that represents a divergent taxon within the NCLDV group.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5615/2033/DC1

Materials and Methods

Figs. S1 and S2

  • * To whom correspondence should be addressed. E-mail: didier.raoult{at}medecine.univ-mrs.fr; Jean-Michel.Claverie{at}igs.cnrs-mrs.fr

REFERENCES AND NOTES

Navigate This Article