Supplementary Material for: Reclassification of Giant Viruses Composing a Fourth Domain of Life in the New Order <b><i>Megavirales</i></b>

Interest in giant viruses has risen sharply since 2003, following the discovery of the Mimivirus and four other protist-infecting giant viruses that are linked to the nucleocytoplasmic large DNA viruses (NCLDVs). Despite considerable heterogeneity in hosts and genome sizes, the NCLDVs have been shown to be monophyletic based on analyses of their sequences and gene repertoires and recent studies have proposed that these viruses share a common ancient ancestor and compose a fourth domain of life. In addition, several characteristics of these giant viruses contradict or do not match the criteria used for the canonical definition of viruses, and the NCLDV denomination is not completely appropriate. We propose here to define a new viral order named <i>Megavirales</i>

Supplementary Material for: Codon Usage, Amino Acid Usage, Transfer RNA and Amino-Acyl-tRNA Synthetases in Mimiviruses

Mimiviruses are giant viruses that infect phagocytic protists, including <i>Acanthamoebae</i> spp., which were discovered during the past decade. They are the current record holder among viruses for their large particle and genome sizes. One group is composed of three lineages, referred to as A, B and C, which include the vast majority of the Mimiviridae members. <i>Cafeteria roenbergensis</i> virus represents a second group, though the Mimiviridae family is still expanding. We analyzed the codon and amino acid usages in mimiviruses, as well as both the transfer RNA (tRNA) and amino acyl-tRNA synthetases. We confirmed that the codon and amino acid usages of these giant viruses are highly dissimilar to those in their amoebal host <i>Acanthamoeba castellanii</i> and are instead correlated with the high adenine and thymine (AT) content of Mimivirus genomes. We further describe that the set of tRNAs and amino acyl-tRNA synthetases in mimiviruses is globally not adapted to the codon and amino acid usages of these viruses. Notwithstanding, Leu(TAA)tRNA, present in several Mimivirus genomes and in multiple copies in some viral genomes, may complement the amoebal tRNA pool and may contribute to accommodate the viral AT-rich codons. In addition, we found that the genes most highly expressed at the beginning of the Mimivirus replicative cycle have a nucleotide content more adapted to the codon usage in <i>A.</i><i>castellanii</i>

The virophage as a unique parasite of the giant mimivirus

Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus ( APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185- kilobase double- stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea(1-3). Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co- infected with APMV(4). Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343- kilobase circular double- stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein- coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA- binding subunit, and a Zn- ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set(5), suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses

Microbial culturomics : paradigm shift in the human gut microbiome study

Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches

Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of “Marseille” virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are “melting pots” of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins

Mimivirus shows dramatic genome reduction after intraamoebal culture

Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-μm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors