A new example of viral intein in Mimivirus

Hiroyuki Ogata 1 Didier Raoult 0 Jean-Michel Claverie 1 0 Unite des Rickettsies, CNRS UPRESA 6020, Faculte de Medecine , 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05 , France 1 Information Genomique et Structurale , UPR2589 CNRS, IBSM, IFR88, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20 , France Background: Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in all domains of life, but only once to date in the genome of a eukaryote-infecting virus. Results: Here we report the identification and bioinformatics characterization of an intein in the DNA polymerase PolB gene of amoeba infecting Mimivirus, the largest known double-stranded DNA virus, the origin of which has been proposed to predate the emergence of eukaryotes. Mimivirus intein exhibits canonical sequence motifs and clearly belongs to a subclass of archaeal inteins always found in the same location of PolB genes. On the other hand, the Mimivirus PolB is most similar to eukaryotic Pol sequences. Conclusions: The intriguing association of an extremophilic archaeal-type intein with a mesophilic eukaryotic-like PolB in Mimivirus is consistent with the hypothesis that DNA viruses might have been the central reservoir of inteins throughout the course of evolution. - Background Mimivirus is the largest known virus, both in particle size (>0.4 m in diameter) and genome length, recently discovered in amoeba, following the inspection of a hospital cooling tower prompted by a pneumonia outbreak [1]. Recently, its entire 1.2-Mbp genome sequence was determined [2]. Extensive phylogenetic studies and gene content analyses defined Mimivirus as a new family of nucleocytoplasmic large DNA viruses (NCLDV) besides Poxviridae, Iridoviridae, Phycodnaviridae and Asfarviridae, and suggested its early origin, probably before the individualization of the three domains of life [2]. While analyzing Mimivirus genome sequence, we noticed the unusual length of its putative DNA polymerase. A detailed analysis identified an intein in this gene. After the recent discovery of an intein in Chilo iridescent virus [3], an insect-infecting NCLDV of Iridoviridae, this is the second report of an intein sequence in a eukaryote-infecting virus. Inteins are "protein introns" that catalyze self-splicing at the protein level. The splicing is defined by the self-catalytic excision of an intervening sequence ("intein") from a precursor host protein where it is located, and the concomitant ligation of the flanking amino- and carboxy-terminal fragments ("exteins") of the precursor. Inteins often possess a homing endonuclease domain, and are considered as mobile elements. Since their first discovery in 1990 [4,5], inteins have been identified in a wide variety of organisms, including bacteria, archaea, and unicellular eukaryotes, albeit with sporadic distribution (see http:// bioinformatics.weizmann.ac.il/~pietro/inteins/ for a comprehensive list). For instance, they are relatively abundant in some hyperthermophilic archaea species (such as Methanococcus jannaschii possessing nineteen inteins), but absent in closely related species such as Methanococcus maripaludis [6]. Similarly, they are observed in many unrelated bacterial clades, but appear often limited to several species within each clade. It was suggested that viruses were potential "vectors" of inteins across species and responsible for the sporadic distribution of inteins [3]. Accordingly, inteins have been identified in many bacteriophages and prophages [7-10]. To our knowledge, the sole published account of eukaryote-infecting viruses harboring an intein concerns iridoviruses [3]. Results Eukaryotic Pol-like Mimivirus PolB Mimivirus genome sequence exhibits a putative ORF (R322, 1740 amino acid long) corresponding to a family B DNA polymerase PolB. This ORF R322 exhibits high scoring sequence homology (BLAST E-value<10-24) against eukaryotic PolBs in the public database. However, this Mimivirus PolB is much larger than its eukaryotic and viral homologues (about 1000 aa), and its optimal alignment with the other PolB sequences reveals four unmatched extraneous segments (Fig. 1A, Fig. S1). Focusing on these extra segments, we identified a 351-aa intein (position 1053 to 1403) in the Mimivirus PolB sequence. After removing those four Mimivirus specific insertions, the Mimivirus PolB sequence exhibited the highest BLAST scores (E-value = 10-125, 32% identity) against a soybean DNA polymerase Pol (SWISS-PROT: O48901) with an alignment covering both the entire Mimivirus and the target sequence. Near equivalent matches are observed with a variety of eukaryotic (from yeast to human) family B DNA polymerase sequences. The best viral homologues were found in phycodnaviruses (E-value = 10-116). Conserved carboxylate residues (aspartate and glutamate) at the exonuclease and polymerase active sites [11,12] were all identified in the Mimivirus PolB (Fig. S1). There was no other ORF encoding a putative PolB in the genome. These suggest that R322 encodes a functional PolB. Consistent with the homology search result, a phylogenetic analysis places the Mimivirus PolB near the root of eukaryotic Pols (Fig. 1B). A similar branching position is obtained for the seven universally conserved Mimivirus genes [2]. Despite low bootstrap values for some of the deep branches in the Fig. 1B, this tree clearly indicates the lack of any specific affinity between the Mimivirus PolB and the archaeal PolB sequences containing inteins (bold letters in the Fig. 1B). It should also be noted that several other large DNA viruses are known to possess PolBs with a similar phylogenetic pattern [13]. Canonical/archaeal type Mimivirus intein The Mimivirus intein sequence (351 aa) exhibits significant sequence similarities to several known inteins (Evalue<10-4), all of which are from thermophilic/halophilic archaea. The best matching intein (E-value = 3 108) is the second intein of the Thermococcus sp. PolB (InBase: Tsp-GE8 Pol-2) with 24% amino acid sequence identity. The Mimivirus sequence exhibits all the expected features required for an active intein (Fig. 2). Sequence motifs [14] characterizing the splicing domain (N1-4, C2, C1) and the dodecapeptide LAGLIDADG homing-endonuclease domain (EN1-4) were all identified in the Mimivirus sequence except N4 motif. N4 motif is occasionally absent in the previously characterized active inteins [14]. Amino acid residues providing nucleophilic groups in self-splicing reactions are all present: the first serine and the last asparagine residues of the intein, and the first threonine residue of the downstream extein. Accordingly the Mimivirus intein is a canonical "asparagine-type" intein, of which the close homologues have previously been observed only in archaea species. In contrast, the previously reported Chilo iridescent virus intein is a noncano (...truncated)