Perspectives

for all cellular organisms. Carl Woese17,18 o p i n i o n discovered the existence of three different ribosomes in the living world, which Redefining viruses: lessons from replaced the old prokaryote–eukaryote dichotomy with a trinity — archaea, bacteria Mimivirus and eukarya. All cellular organisms could thus be placed together in a universal tree of life. Viruses, however, were missing from Didier Raoult and Patrick Forterre this picture. Abstract | Viruses are the most abundant living entities and probably had a major role Unlike most other microorganisms, viruses are obligate intracellular parasites that in the evolution of life, but are still defined using negative criteria. Here, we propose cannot replicate independently. They can to divide biological entities into two groups of organisms: ribosome-encoding infect organisms from all three domains of organisms, which include eukaryotic, archaeal and bacterial organisms, and capsid- life, and can even parasitize other viruses; encoding organisms, which include viruses. Other replicons (for example, plasmids for example, the delta agent (with the 19 and viroids) can be termed ‘orphan replicons’. Based on this suggested classification hepatitis B virus ) and satellite viruses (with an adenovirus or tobacco mosaic system, we propose a new definition for a virus — a capsid-encoding organism that is virus (TMV)20,21). Despite their ubiquity composed of proteins and nucleic acids, self-assembles in a nucleocapsid and uses a and enormous importance to human ribosome-encoding organism for the completion of its life cycle. health, viruses have long been neglected by evolutionary biologists, and are thought to The Darwinian revolution created a new bacteria9, and the recent discovery of the be derived from cells. Indeed, as a direct approach to classification by proposing a largest known virus, Mimivirus10–13 (FIG. 1), consequence of the cellular theory that common origin for living organisms. Since we propose a new definition for the virus life was established in the nineteenth century, then, scientists have grouped animals and form. Of course, any attempt to redefine an living organisms and cellular organisms are plants phylogenetically, rather than by gross entire field will be controversial; however, a synonymous to most scientists. appearance. The genetic revolution and our debate of this issue, using all of the currently Viruses were initially thought to be ability to build trees based on genetic simi- available data, is needed. We propose a defi- infectious agents that are not visible under larities provided support for this method nition of viruses (and cells) that is based on a microscope and can be filtered through of classification. Over the past 30 years, the the hypothesis that viruses are more than just 0.22 µm ultrafilters (hence the name development of more efficient sequencing parasitic nucleic acids and that the presence ‘ultravirus’)22,23. During the twentieth strategies has led to the reclassification of of either capsids or ribosomes forms the century, researchers developed two theories organisms into a universal tree of life based basis of the principal classification system in about viruses. The bacteriologists Felix on ribosomal RNA sequences1. Viruses, the living world. d’Herelle, who discovered bacteriophages, however, lack ribosomes and have not yet and Macfarlane Burnet, who received the been incorporated into this universal tree Defining viruses — a history Nobel Prize in medicine in 1960, believed of life. According to Karl Popper14, definitions that viruses were organisms24,25 (as did Louis Until now, the genetic information that are based on the data and tools that are Pasteur), whereas Wendell Stanley26, who is encoded by viruses was not thought to available at a specific moment in time. In crystallized TMV and received the Nobel contain sufficient information to allow their the nineteenth century, the word ‘microbes’ Prize in chemistry in 1946, believed that general phylogenetic classification, and was coined by Sedillot15 to define cellular viruses were biomolecules. Later, while pro- consequently no clear definition of viruses microorganisms that were only visible using moting the eukaryote–prokaryote dichotomy, is currently available. This is unfortunate, as a microscope. In the middle of the twentieth Andre Lwoff 22 defined viruses as small (one viruses are the most abundant living entities century, microorganisms were divided into dimension smaller than 0.2 µm), infectious, on the planet2 and metagenomic studies from two groups, eukaryotes and prokaryotes, but not autonomous, agents that cannot randomly sequenced environmental samples based on cellular structural features16. divide by binary fission, and consist of pro- have revealed that viral genes constitute Eukaryotic cells have a nucleus and a nuclear teins and a single type of nucleic acid. Lwoff the largest part of the genosphere2,3. Recent membrane, whereas prokaryotic cells do not insisted that viruses are not organisms and research has revealed an important role for (although Planctomycetes, such as Gemmata maintained that the infectious element of viruses in various evolutionary scenarios, obscuriglobus, are bacteria that have a nucleus the virus is the nucleic acid, unlike bacteria including the origin of DNA and mam- and a nuclear membrane)16. In the last part of or other pathogens, in which the infectious mals4–7. Here, based on our knowledge of the twentieth century, molecular-biology tools agent is the organism itself (although this archaea, archaeal viruses8 and intracellular opened the way for a new classification system theory has been contradicted recently27).

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as “An individual living system such as animal, plant, fungus or microorganism” by Wikipedia, “An individual animal, plant or single-celled life form” by the Oxford English Dictionary Online and “Any living structure capable of growth and reproduc- MV tion” by Chambers Reference Online (see Further information). The definitions N from Wikipedia and the Oxford English Dictionary Online exclude intracellular VF parasites, symbionts, organelles and viruses. The definition from Chambers Reference Online, however, includes viruses and nucleic acids, as it does not retain the word cell. Mimivirus changed the perception of viruses and could also change the definition of an organism. Mimivirus virions are assembled at the periphery of a large membrane-bound nucleus-like structure — the viral factory — within the host cell (FIG. 1). Although viral factories have been described for most eukaryotic viruses 2 µm (both RNA and DNA)33, the viral factory 12 Figure 1 | Mimivirus infecting Acanthamoeba polyphaga. Transmission electron microscope image of the Mimivirus is especially spectacular that shows A. polyphaga infected with Mimivirus. Note the giant virus factory. N, nucleus; VF, virus and, when first observed using an electron factory; MV, Mimivirus virions. Nature Reviews | Microbiology microscope, was initially thought to be the nucleus of its giant amoebae host. Jean- Michel Claverie34 proposed that viruses Viruses were thus tacitly defined by most Defining organisms and living entities are entities that are associated with an molecular biologists as molecular genetic The general consensus of what constitutes intracellular viral factory, and should not be parasites that use cellular systems for their life can be sampled by consulting a global confused with virions. Interestingly, from own replication22. Indeed, with such a broad resource such as Wikipedia (the largest this view, a virus is similar to an intracellu- definition, many types of selfish genetic free online encyclopaedia; see Further lar organism, which therefore further blurs elements (such as plasmids, transposons, information), which defines life as ‘‘a the boundary between cellular organisms retroposons, viroids and virusoids) were condition that distinguishes organisms and viruses. determined to be viruses. For example, from inorganic objects’’. However, there The virus definition can also be modi- Koonin et al.28,29 recently grouped all infec- is no universal definition of life. The fied by the distinction between a virus tious-material-containing nucleic acids as frequently used ‘reproduction’ criterion and a virion. A virus can be generated either selfish elements and/or viruses and does not apply to sterile organisms. The from synthetic oligonucleotides by whole- used these terms synonymously. distinction between parasites (replicators) genome assembly to produce infectious Each of the definitions for viruses has and free-living organisms cannot be used virions35. Therefore, we believe that a recently been challenged by the discovery to distinguish between organisms. There is virus can be entirely defined by its coding of viruses that are larger than cellular now no clear-cut limit between mitochon- capacity. As for bacteria, it was recently organisms10,30. Indeed, both the particle and dria, small symbionts, intracellular bacteria shown that genome transplantation from genome sizes of viruses now overlap sig- (such as Rickettsia spp. and Candidatus one species to another is possible, and nificantly with those of bacteria, eukaryotes Carsonella spp.) and free-living bacteria that cells which were transplanted with and archaea. Mimivirus, the largest known in size or phylogenetically31. Some recent the genome of Mycoplasma mycoides were virus, is visible with an optical microscope, definitions of life, such as another from phenotypically identical to M. mycoides27; contains a 1.2 megabase chromosome that Wikipedia — “life is a characteristic of self here, the genome defined the whole bac- encodes nearly 1,000 putative genes and organizing, self recycling systems consist- terium. Experiments which showed that harbours both RNA and DNA11. Moreover, ing of populations of replicators that are synthesized or purified nucleic acids from if exposed to a Gram-stain procedure, capable of mutation, around most of which either viruses or bacteria can infect hosts Mimivirus stains Gram-positive, and was homeostatic, metabolizing organisms and be replicated, show that there are no thought for a period to be a ‘Legionella-like evolve” — clearly include viruses. We can fundamental differences between these liv- organism’. Interestingly, Mimivirus was also paraphrase Engels32 and define life as ing entities. Based on these recent data, we identified as a virus only a few years ago, “the mode of existence of living organisms”, believe that organisms and living entities when its icosahedral capsid was observed which brings us to the problem of organism can be defined by genome analysis. using an electron microscope13. The size of definition. Finally, we retain the more liberal defini- Mimivirus challenges the definition of a virus The definition of an organism is a dif- tion of organisms and living entities as it and even the definition of a microorganism ficult problem in itself and is subject to applies to viruses, and thus reclassify viruses as a living entity. controversy. An organism has been defined according to their genome content.

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Ribosome-encoding organisms J To determine a natural classification for all living organisms, we suggest that it is neces- K sary to redefine what is meant by a cellular A organism. We can now compare the genetic L information that is encoded by cellular organ- D isms from the three domains of life as well Mimivirus 30 as that of a virus of similar proportions . As O Nanoarchaeum equitans illustrated in FIG. 2, the only significant differ- Candidatus Carsonella ruddii M ences in the distribution of clusters of orthol- Encephalitozoon cuniculi ogous groups (COGs) of gene categories N between the smallest cellular organisms — P J Translation 36 K Transcription

Candidatus Carsonella ruddii (a bacterium) , ry T A RNA processing and modification Nanoarchaeum equitans (an archaeon), go L Replication, recombination and repair B Encephalitozoon cuniculi (a eukaryote) and cate D Cell-cycle control, mitosis and meiosis Mimivirus — are the number of genes that U O Post-translational modification, protein turnover and chaperones M Cell-wall and membrane biogenesis are involved in translation, which is much Z N Cell motility lower in Mimivirus owing to the lack of P Inorganic ion transport and metabolism V T Signal-transduction mechanisms ribosomal proteins, and the lack of any COGs gene nctional B Chromatin structure and dynamics that are involved in energy production and Fu C U Intracellular trafficking and secretion conversion in the virus. Although the absence Z Cytoskeleton G of these genes is a negative characteristic that V Defence mechanisms E C Energy production and conversion cannot be used to group viruses together, it is G Carbohydrate transport and metabolism also a positive feature that groups all cellular F E Amino acid transport and metabolism organisms. These genes might have been lost F Nucleotide transport and metabolism H H Coenzyme transport and metabolism independently many times during a parasitic I Lipid transport and metabolism mode of life by convergent evolution. Such I Q Secondary metabolite biosynthesis, transport and catabolism convergent evolution was noted for intracel- Q R General function (prediction only) lular bacteria that had lost most of the genes S Function unknown R that encode metabolic pathways31, and this is also the case for mitochondria, chloroplasts S and symbionts. In particular, the genes that 0 20 40 60 80 100 120 140 160 are involved in protein synthesis, specifically Number of COGs those that encode ribosomal proteins and Figure 2 | Clusters of orthologous groups (COGs) in Mimivirus and traditional cellular organisms. ribosomal RNA, are among the few genes Distribution by categories of cluster of orthologous groups’ homologues in Mimivirus, compared with cellular organisms from the three domains of life that have the smallest currently known that are conserved in all cellular organisms, Nature Reviews | Microbiology including the smallest intracellular parasites37. genomes: Nanoarchaeum equitans (archaea), Candidatus Carsonella ruddii (bacteria) and Encephalitozoon cuniculi (eukarya). This is because the last universal common ancestor (LUCA) probably possessed a sophisticated ribosome that contained at least genetically, between mitochondria, symbionts Mimiviruses, could be classified based on a 34 ribosomal proteins that are shared by all and intracellular bacteria such as Candidatus set of conserved proteins that are involved in archaeal, bacterial and eukaryotic organisms. Carsonella spp. and Rickettsia spp. viral DNA replication and transcription11,38. The descendants of the LUCA (or some of Although the viral-factory structure can its predecessors) have superseded all other Capsid-encoding organisms be viewed as an ‘organismal’ form of the virus, cellular life forms that could have used other By analysing all infectious materials other it cannot be used to define viruses because, mechanisms to synthesize their proteins. than REOs, which range from a few hundred first, the presence of viral factories has not yet Although some RNA viruses (for example, base pairs, such as the single-stranded RNA been demonstrated in archaeal and bacterial arenaviruses) do contain ribosomes within molecule that is carried by capsid borrowed viruses (possibly for methodological reasons their capsids, these ribosomes are native from a helper virus (virusoid and satellite or because the whole cell is transformed into to their hosts, and the absence of genes that RNA)19, to the giant Mimivirus, it is clear a viral factory) and, second, it is not known encode ribosomal proteins is common to that no single common protein exists in the if all viral factories share a common feature, all viruses. Thus, we suggest that all cellular virosphere. There is no genetic equivalent although they do disseminate their genetic organisms can be adequately defined as ribos- in this group to the ribosomal-RNA or information in the same way (through the ome-encoding organisms (REOs), as opposed universal proteins that are common to linear or exponential multiplication of nucleic to viruses. Interestingly, in contrast to the REOs. Furthermore, virus-specific proteins acids and massive production of virions). view that is advocated by Lwoff 22, mitochon- are only found in subsets of viral groups. We propose that the expression of a capsid dria and chloroplasts would be classified as Consequently, protein phylogenies have only is the only positive determinant that can be REOs based on this definition (instead of as been useful to tentatively establish a classifi- considered to define viruses. The viral capsid cellular organelles) because they contain their cation for selected virus groups. For example, is a necessary structure that is used by the own translation apparatus1. There is indeed according to Iyer et al.38, nucleo–cytoplasmic viral factory to disseminate the virus outside no clear difference, either morphologically or large DNA viruses (NCLDVs), such as of the REO host and infect new hosts. Indeed,

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PBCV1 Vp54 PRD1 P3 STIV In addition to capsids, analyses of the genomes of viruses and related elements, such as plasmids, have revealed the existence of replication proteins, such as the superfamily DG I G III helicases, protein-primed DNA polymer- DG C F′ D I F′ B F C′ I ases and rolling-circle initiator proteins, that G′ C′ H E G′ E′ C H′ G′ D′ H′E I′ D′ C C′ have no cellular homologues, but are present HHE F I′ ′ B′ B H E F H′E′ B B′ D′ F′ in viruses that infect organisms in different B′I′ C terminus domains. This suggests that these proteins C terminus N terminus N terminus were never encoded by cellular genomes or N terminus C terminus that they originated in ancient cellular lineages that were wiped out by the descendants B B′ B B′ B B′ of the LUCA38. Figure 3 | Capsid proteins from viruses that infect organisms from all three domains of life. The diversity of viral RNA- and DNA- Nature Reviews | Microbiology Comparison of the major capsid-protein structures from viruses that can infect the three domains of replication mechanisms and their associated life: the major capsid protein of virus Paramecium bursaria Chlorella virus 1 (PBCV1) (Vp54; Protein Data proteins indicates that various types of Bank (PDB) code 1M4X), which infects Chlorella-like eukaryotes; the bacteriophage PRD1 coat protein replicons originated in an ancient virosphere P3 subunit (PDB code 1cjd), which infects the bacterium Escherichia coli; and the capsid protein of virus and, possibly, even predate the LUCA4,29. Sulfolobus turreted icosahedral virus (STIV) (PDB code 2BBD), which infects the hyperthermophilic Consequently, the origin of viruses could archaeon Sulfolobus solfataricus P2. These three capsid proteins contain the typical double-jelly-roll stem from an association between cassettes fold (shown in red) that is absent from cellular proteins, which confirms that these structures originated from a common ancestral protein. Other features shared by these viruses suggest that this protein was of capsid-encoding genes and particular rep- already a capsid protein of an ancestral virus that was present at the time of the last universal common licons (including an origin of replication and ancestor, or even earlier. C terminus, carboxyl terminus; N terminus, amino terminus. genes that encoded replication-machinery proteins which could have used this origin for self-replication). the viral capsid has been called the ‘virus have multiple origins, as different unique Interestingly, most genes that are encoded self’ by Dennis Bamford and colleagues39, folds are present in the capsid proteins of by viruses which infect organisms in all three who first identified clear homologous traits otherwise apparently unrelated viruses (and domains of life have no cellular homologues, between capsid proteins and the capsid archi- again are absent from any cellular proteins) which is in contrast to the traditional view tecture of viruses that were infecting bacteria which links head-and-tailed bacterial viruses that viruses are derived from genetic ele- (enterobacteria phage PRD1) and eukarya (Caudovirales) and eukaryotic herpesvi- ments that escaped from cells and became (an adenovirus)40. Later, it was shown that ruses44. Future work should compare the coat infectious. The persistence of two different the double-jelly-roll fold (which has not been proteins of viruses that have non-icosahedral names for viruses — those that are associated found in any cellular protein) is also present morphologies (filamentous, rod-shaped or in the capsid proteins of Paramecium bursaria pleomorphic) to identify additional folds that Chlorella virus 1 (PBCV1), an NCLDV that are unique to viral capsids. infects eukaryotic algae41, and Sulfolobus tur- Capsids might originally have been stor- reted icosahedral virus (STIV), an archaeal age devices that were designed to protect virus that was isolated from a Yellowstone hot nucleic acids that were accidentally released Caspid-encoding organisms 42 spring (FIG. 3). Modelling experiments have from lysed cells. These structures could Viruses of Bacteria shown that the capsid protein of Mimivirus have been selected within an intracellular Viruses of Eukarya contains the same fold, which suggests that it parasite because of their ability to dissemi- Viruses of Archaea is present in all NCLDVs43. All these viruses nate multiple replication copies by uncou- are double-stranded DNA viruses and have pling replication of the parasite genome from an internal lipid layer (with the exception of that of the host genome. Capsids that possess adenoviruses). These observations favour the associated mechanisms that are used the hypothesis that an ancient form of virus to exit from one host cell and enter a new that had this type of capsid predates, or was a host are specific and complex structures Bacteria contemporary of, the LUCA43. Interestingly, that could have appeared independently Archaea Eukarya the double-jelly-roll fold, which is common several times; however, we argue that this to double-stranded DNA viruses that have event would not have occurred frequently Ribosome-encoding organisms an internal lipid layer, is also present in the over the course of evolutionary time. In capsid proteins of some single-stranded any case, the appearance of capsids was RNA viruses, and single-jelly-roll folds are a crucial event in the early evolution of observed in the capsid proteins of many other life that resulted in divergence among Figure 4 | Redefining viruses. Representation DNA and RNA viruses. It will be important all subsequent organisms. We therefore of viruses with their capsids, and the three to determine if, as suggested by Rossmann propose to define viruses as capsid encod- domains of life thatNa haveture evolvedReviews |from Microbiolog the lasty 41 and co-workers , all these jelly-roll folds are ing organisms (CEOs). The presence of universal common ancestor. The three domains evolutionarily related, which would suggest a capsid defines a group of living entities have ribosomes, but lack a capsid. The newly a common and ancient origin for some that contain nucleic acid and a capsid, and defined viruses have a capsid, but no ribosome. DNA and RNA viruses. Capsids probably overlaps one of the trivial virus definitions. Other infectious elements are not shown.

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Virus Taxonomy: mycoides | Nanoarchaeum equitans | Sulfolobus solfataricus P2 Classification and Nomenclature of Viruses. Eighth could partly explain why the prokaryote– Report of the International Committee on Taxonomy of FURTHER INFORMATION eukaryote division persists, despite the vast Viruses 1–1259 (Academic, San Diego, 2005). Didier Raoult’s homepage: http://ifr48.timone.univ-mrs.fr/ 20. Dodds, J. A. Satellite tobacco mosaic virus. Curr. Top. portail2/index.php?option=com_content&task=view&id=78 amount of molecular evidence that indicates Microbiol. Immunol. 239, 145–157 (1999). Chambers Reference Online: http://www.chambersharrap. the existence of three domains of ribosome- 21. Atchison, R. W., Casto, B. C. & Hammon, W. M. co.uk/chambers/features/chref/chref.py/main NCBI COG s database: http://www.ncbi.nlm.nih.gov/COG / 1 Adenovirus-associated defective virus particles. encoding cells . Here, we propose to reinstall Science 149, 754–756 (1965). Oxford English Dictionary Online: http://www.oed.com/ a primary dichotomy in the classification of 22. Lwoff, A. The concept of virus. J. Gen. Microbiol. 17, Protein Data Bank: http://www.rcsb.org/pdb/home/home.do Wikipedia: http://www.wikipedia.org/ the living world between REOs and CEOs. 239–253 (1957). 23. Raoult, D. The journey from Rickettsia to Mimivirus. All links are active in the online pdf We conclude that two connected natural ASM News 71, 278–284 (2005).