The rapidly expanding universe of giant viruses: Mimivirus, Pandoravirus, Pithovirus and Mollivirus

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1 FEMS Microbiology Reviews, fuv037, 39, 2015, doi: /femsre/fuv037 Advance Access Publication Date: 29 September 2015 Review Article REVIEW ARTICLE The rapidly expanding universe of giant viruses: Mimivirus, Pandoravirus, Pithovirus and Mollivirus Chantal Abergel 1,, Matthieu Legendre 1 and Jean-Michel Claverie 1,2 1 Structural and Genomic Information Laboratory, UMR 7256 (IMM FR 3479) Centre National de la Recherche Scientifique & Aix-Marseille University, Marseille, France and 2 Assistance Publique des Hôpitaux de Marseille, La Timone, Marseille, France Corresponding author. Structural and Genomic Information Laboratory, UMR 7256 (IMM FR 3479) Centre National de la Recherche Scientifique & Aix-Marseille University, Marseille, France. Tel: ; Chantal.Abergel@igs.cnrs-mrs.fr One sentence summary: The discovery of four families of giant viruses and how they impacted the concept of virus. Editor: Alain Filloux ABSTRACT More than a century ago, the term virus was introduced to describe infectious agents that are invisible by light microscopy and capable of passing through sterilizing filters. In addition to their extremely small size, most viruses have minimal genomes and gene contents, and rely almost entirely on host cell-encoded functions to multiply. Unexpectedly, four different families of eukaryotic giant viruses have been discovered over the past 10 years with genome sizes, gene contents and particle dimensions overlapping with that of cellular microbes. Their ongoing analyses are challenging accepted ideas about the diversity, evolution and origin of DNA viruses. Keywords: DNA virus; giant virus; Mimiviridae; Pandoraviridae; Pithoviridae; Mollivirus HISTORICAL PERSPECTIVE Since the historical experiment by Ivanovsky (1892) revealing that the causative agent of tobacco mosaic disease could pass through the Chamberland sterilizing filter, the notion that viruses are by definition much smaller than bacteria has remained central in virology. From 1898, when Beijerinck introduced the word virus (Beijerinck 1898) to 1939 when they were first visualized using the newly invented electron microscope (Kausche, Pfankuch and Ruska 1939), their nature remained mysterious and their relationship with the living/cellular world was hotly debated. Lwoff (1957) proposed the first set of formal criteria to discriminate between viruses and cells. These criteria that were further refined in a subsequent article (Lwoff and Tournier 1966) can be summarized as follows: viruses possess a DNA (or RNA) genome but, in contrast to cells (including obligatory intracellular parasites), they do not divide, do not encode a protein translation apparatus and do not synthetize the ATP they consume for their replication. Thus, despite its historical importance, small size was not part of Lwoff s criteria, even though it was still mentioned as an important characteristic correlating with some essential properties which are responsible for some fundamental differences (Lwoff 1957). Yet, the unique property of viruses to pass through sterilizing filters with a porosity of μm remained central to most isolation procedures until today, especially in most studies seeking to isolate viruses from the environment (Breitbart et al. 2002; Mizuno et al. 2013). Thus, during the 20th century, a microorganism that was retained by a sterilizing filter or that could be seen under a light microscope could not possibly be a virus. This epistemological barrier delayed the discovery of the viral nature of Mimivirus until 2003, 10 years after it was first isolated (La Scola et al. 2003). Giant virus, defined as those exhibiting viral particles easily visible by light microscopy (i.e. smallest dimension >0.3 μm), now encompass four distinct families: the Mimiviridae (with a dozen different isolates/strains) (Raoult et al. 2004; La Scola et al. 2010; Received: 30 June 2015; Accepted: 15 August 2015 C FEMS All rights reserved. For permissions, please journals.permissions@oup.com 779

2 780 FEMS Microbiology Reviews, 2015, Vol. 39, No. 6 Arslan et al. 2011;Colsonet al. 2011b; Legendre et al. 2012;Yoosuf et al. 2012, 2014a,b; Boughalmi et al. 2013a,b; Saadi et al. 2013a,b), the Pandoraviridae (three known members) (Philippe et al. 2013; Antwerpen et al. 2015), Pithovirus (possibly two isolates) (Michel et al. 2003b; Legendre et al. 2014; Claverie and Abergel 2015) and Mollivirus (Legendre et al. 2015). All of them infect species of the amoebozoan genus Acanthamoeba, one of the most common protozoa in soil (Geisen et al. 2014), ubiquitous in natural aquatic environments and sediments (Sawyer, Visvesvara and Harke 1977), as well as in man-made water systems (domestic networks, cooling towers or wastewater treatment plants) (Thomas et al. 2008). Although Mimivirus was isolated in the context of a pneumonia outbreak (Raoult, La Scola and Birtles 2007), their pathogenicity to humans remains uncertain (Vanspauwen et al. 2013). Box 1: Giant viruses and NCLDV Originally, nucleocytoplasmic large DNA viruses (NCLDVs) were regrouping the four known families of large DNA viruses infecting eukaryotes, the Poxviruses, Asfarviruses, Iridoviruses (including Ascoviruses) and Phycodnaviruses, postulated to have a common origin (Iyer, Aravind and Koonin 2001). They have dsdna genomes with sizes ranging from 100 to 400 kb and can either be exclusively cytoplasmic or recruit functions from the cell nucleus for the earliest phases of their infectious cycles. The list of NCLDVs expanded with the discovery of Mimivirus (Raoult et al. 2004; Iyer et al. 2006), then of close relatives infecting Acanthamoeba (Raoult et al. 2004;La Scola et al. 2010; Arslan et al. 2011; Colson et al. 2011b; Legendre et al. 2012; Yoosuf et al. 2012, 2014a,b; Boughalmi et al. 2013a,b; Saadi et al. 2013a,b) and related large DNA viruses infecting other unicellular protists (Fischer et al. 2010;Yauet al. 2011;Santiniet al. 2013). Together they form the newly defined family Mimiviridae. In parallel, the Marseilleviridae, a distinct family (although related to the Iridoviridae) of Acanthamoeba-infecting ds- DNA viruses is rapidly growing (Boyer et al. 2009; Thomas et al. 2011; Boughalmi et al. 2013b; Colsonet al. 2013b; Doutre et al. 2014). Comparison of the Mimiviridae and Marseilleviridae with the previous families constituting the NCLDVs places them within this group, that was proposed to form the new order Megavirales (Colson et al. 2013a) (not yet accepted by ICTV). A set of 47 (almost) conserved genes was proposed to originate from their postulated common ancestor (Yutin et al. 2009). The proposed inclusion of the Pandoraviruses in the NCLDV on the basis of even less shared conserved genes (Yutin and Koonin 2013) is much more questionable. THE STRUCTURES OF GIANT VIRUS PARTICLES Mimiviridae: hairy pseudo-icosahedrons Historical context In 1992, Rowbotham (1983) investigated the origin of a pneumonia outbreak in Bradford (UK) using an Acanthamoeba coculture protocol. From the water of a cooling tower, he isolated what light microscopy observations suggested to be a Gram-positive coccoid bacterium that he called Bradford coccus. However, this microorganism resisted cultivation attempts in various axenic media and characterization using universal 16S rdna bacterial Figure 1. Four distinct virions. Light microscopy images (scale bar = 2 μm) and scanning EM images (scale bar = 100 nm) of Mimivirus (A, C), Mollivirus (B, D), Pandoravirus (E, G) and Pithovirus (F, H). primers (La Scola et al. 2003; Raoult, La Scola and Birtles 2007). Ten years later, the viral nature of this mysterious microorganism was finally elucidated through an electron microscopy (EM) study of its replication cycle and partial genome sequencing (La Scola et al. 2003). This was the first example of a giant virus, large enough to be confused with a bacterium. It was officially renamed Acanthamoeba polyphaga Mimivirus (short for MIcrobe MImicking Virus ) (La Scola et al. 2003; Raoult et al. 2004) Mimiviridae virions morphologies Mimivirus virions exhibit an icosahedral protein capsid 440 nm in diameter, making them easily visible without staining under the light microscope (Fig. 1A). The perfect icosahedral symmetry is broken by a five-pronged star structure, called the stargate (Zauberman et al. 2008) that is present at a single vertex of the particle. Except above the stargate, the capsid surface is covered by a 150-nm-thick fibril layer with a composition thought to be related to peptidoglycan (Piacente et al. 2012, 2014b) (Fig. 2A). This reticulated layer, probably responsible for the Gram colouration of Mimivirus particles, also makes them extremely difficult to break open, except using a powerful chemical lysis protocol (Renesto et al. 2006). The stargate is the portal through which the internal nucleoid of the particle is unloaded (Zauberman et al. 2008). Its opening enables the internal virion lipid membrane layering the protein capsid to deploy and fuse with the phagosomal membrane of Acanthamoeba, resulting in the delivery of the virion

3 Abergel et al. 781 nucleoid into the cytoplasm (Figs 2A2 and 3A). The nucleoid is a membrane-bound spherical compartment approximately 320 nm in diameter containing a linear double-stranded (ds) DNA genome of up to 1.26 Mb (Arslan et al. 2011) as well as associated proteins necessary to initiate the infection cycle, such as the machinery responsible for early gene transcription. The DNA genome of Mimivirus is not tightly compacted as confirmed by the diffraction pattern of individual virions produced by a hard X-ray free electron laser beam (Seibert et al. 2011) and the consecutive 3D reconstruction (Ekeberg et al. 2015). A cryo-em 3D reconstruction of a Mimivirus particle (lacking fibres) has been achieved at 65 A resolution, which confirmed the large interior volume enclosed by two 70-A -thick layers (Xiao et al. 2009). The overall structure of the particle is well conserved among the various known species of Acanthamoeba-infecting Mimiviridae, except for a reduced thickness of the fibre layer in Megavirus chilensis (75 ± 5 nm) (Arslan et al. 2011) and Moumouvirus (100 ± 5 nm) (Yoosuf et al. 2012). These differences may be linked to the presence of different acetamido sugars in their composition (Piacente et al. 2014b). Morphogenesis of the particle inside infected amoeba has been analysed using a variety of sophisticated techniques, which have shown that lipid membranes are Figure 2. Four distinct giant virus particles and replication cycles. Thin-section EM images of Acanthamoeba infected with purified virions. (A) Mimivirus replication cycle: (1) phagocytosis (bacterial mimicry); (2) opening of the stargate and delivery of the nucleoid into the cytoplasm; (3) early transcription and protein translation leading to the building of a large electron-dense virion factory from the periphery of which a large number of new particles will emerge first empty, then filled up with the nucleoid, then covered with a thick fibre layer. The nucleus remains intact during the whole replication cycle. (B) Pandoravirus replication cycle: (1) phagocytosis; (2) opening of the apical pore and fusion of the virus internal membrane with the vacuole membrane; during the eclipse phase the virus genome is transferred to the host s nucleus for transcription and replication; (3) the nucleus membrane is progressively recycled into virus membranes and multiple new virions are synthesized at its periphery. (C) Pithovirus replication cycle: (1) phagocytosis; (2) removal of the cork and fusion of the virus internal membrane with the vacuole membrane; there is an eclipse phase while the virus genome is transferred to the host s cytoplasm and presumably transcribed by the virion-imported machinery; (3) buildup of a faint cytoplasmic virion factory recognizable by the exclusion of the cell organelles at its periphery; translation of the viral transcripts in the cytoplasm; synthesis of new particles from membrane vesicles of unknown origin; early exit of neo-formed particles through exocytosis prior to massive release following the complete lysis of the host cell. The nucleus (N) remains intact during the whole replication cycle. (D) Mollivirus replication cycle: (1) phagocytosis; (2) opening of the apical pore and fusion of the virus internal membrane with the vacuole membrane; there is an eclipse phase while the virus genome is transferred to the host s nucleus and transcribed by the cellular machinery; (3) the nucleus membrane is progressively recycled into virus membranes and multiple new virions are synthesized; early exit of neo-formed particles through exocytosis prior lysis of the host cell. Inset: enlarged view of the virion factory where fibres of unknown composition accumulate and seems to contribute to the virion synthesis.

4 782 FEMS Microbiology Reviews, 2015, Vol. 39, No. 6 Figure 3. Schematic representation of the four giant virus infectious cycle. (A) Mimivirus,(B) Pithovirus cytoplasmic infectious cycles, (C) Pandoraviruses and (D) Mollivirus nucleocytoplasmic infectious cycle. While all virions keep their integrity, Mollivirus particles lose their spherical morphologies when in vacuoles. recruited from the endoplasmic reticulum and served as scaffolds to initiate and guide the assembly of the protein capsid, the vertex bearing the stargate budding first from the virion factory (Klose et al. 2010; Kuznetsov et al. 2010, 2013; Mutsafi et al. 2013; Suárez et al. 2013). Pandoravirus: amphora-shaped particles Historical context The systematic sampling of a variety of aquatic environments (and their sediments) in search for additional Acanthamoebainfecting Mimiviridae recently led to the unexpected discovery of the Pandoraviridae family of giant viruses, of which two representatives have been studied in detail (Philippe et al. 2013). Like for Mimivirus, they were identified in samples exhibiting strong lytic activity during cocultivation with Acanthamoeba. The first one named Pandoravirus salinus originated from a sample of superficial marine sediment layer from the coast of central Chile, whereas P. dulcis wasisolatedfrommudatthebottomofafreshwater pond near Melbourne, Australia. Pandoraviridae virions morphologies After several rounds of amplification in Acanthamoeba, the two Pandoraviruses were readily observable by optical

5 Abergel et al. 783 Figure 4. Open membranes intermediates involved in virion assembly. (A) In Mimivirus early virion factory, (B) Pandoravirus, (C) Pithovirus, (D) Mollivirus virion factories. The scale bar corresponds to 500 nm. Black arrows point on open membrane intermediates, the white arrow points on the external layer of Mollivirus particles. The black arrow head points on the 20 nm spaced rings corresponding to the layers of variable lengths fibres surrounding the particles. The white arrow head points to one of the fibres accumulating in the virion factory reproducibly seen during the virion assembly process. microscopy as a lawn of ovoid particles μm in length and 0.5 μm in diameter (Figs 1E and 2B). Another Pandoravirus sharing the Pandoravirions (Pandoravirus virions) morphology, P. inopinatum, was isolated from a patient infected by Acanthamoeba keratitis and recently sequenced (Scheid, Hauröder and Michel 2010; Scheid, Balczun and Schaub 2014; Antwerpen et al. 2015). Electron microscopy imaging revealed ultrastructural features unique among previously described viruses. Thin sections of mature virions revealed a membrane-bound emptylooking compartment encased in a 70-nm-thick tegument-like envelope consisting of three layers: an internal layer of light density ( 20 nm), a dark layer composed of a dense mesh of parallel fibrils ( 25 nm) and an external layer of medium density ( 25 nm) (Fig. 2B 2 and 4B). One extremity of the particle exhibits an apical pore, the opening of which allows the uncharacterized internal content of the particle to be delivered into the host s cytoplasm, through a channel formed by the fusion of the internal membrane with that of the vacuole. In contrast with the Mimiviridae, the Pandoravirions lack an electron-dense central core, which typically corresponds to the location of the compacted genome. The DNA extracted from the purified particles was a linear double-stranded molecule of 2.77 Mbp for P. salinus, 1.93MbpforP. dulcis and 2.24 Mbp for P. inopinatum. Like for Mimivirus, the astonishing size of these particles (also combined with their unconventional asymmetrical shape) prevented their recognition as viruses 6 years earlier when they were first spotted in Acanthamoeba isolated from the inflamed eye of a patient with keratitis (Scheid et al. 2008; Scheid, Hauröder and Michel 2010; Claverie and Abergel 2015). Pithovirus: a delusive pandoravirus look Historical context The identification of two distinct families of giant viruses immediately suggested that more remained to be discovered. Accordingly, the first representative of a third family of giant virus was isolated a year after the Pandoraviruses using the same Acanthamoeba coculture protocol, this time starting from a year-old sample of Siberian permafrost (Legendre et al. 2014). Initially, this new virus, Pithovirus sibericum, seemed to resemble Pandoraviruses by its dimensions (1.5 μm in length and 0.5 μm in diameter) and particle shape. However, further analyses of its gene content, replication cycle and particle fine structure indicated that it was markedly different from the two previously characterized families of giant viruses. Pithovirus virion morphology Imaging of thin sections by TEM revealed that Pithovirus particles are enclosed by a 60-nm-thick electron-dense envelope underlined by a lipid membrane. This internal membrane delimits a compartment mostly devoid of discernible substructures (Figs 1F and 2C), with the exception of an electron-dense sphere (50 nm in diameter) and a tubular structure (observed episodically but in reproducible fashion) parallel to the long axis of the particle. In contrast with Pandoraviruses, the envelope consists of 10-nm-spaced stripes perpendicular to the surface. Mature Pithovirus particles also have an apical orifice that is plugged by a cork, composed of a highly regular hexagonal honeycomb-like grid. At the initial stage of infection, this cork is expelled in the amoeba vacuole, allowing the uncharacterized content of the particle to be delivered to the host cytoplasm after fusion of the virion internal membrane with that of the phagosome. Following this initial step, similar to that of Pandoraviruses and Mimiviruses, Pithovirus exhibits a markedly different replication cycle (Fig. 3B, see below). However, as for the Pandoraviruses, there is no recognizable structure suggesting the location of the 610-kb dsdna genome within the Pithovirus virion. Interestingly, as for the two other giant virus families, a modern relative of Pithovirus was first spotted in Acanthamoeba more than 15 years ago, but misinterpreted as an archaeal endocytobiont (Michel et al. 2003a,b). Mollivirus: a soft mollicute-like virion Mollivirus virion morphology Mollivirus was initially spotted using light microscopy as rounded particles multiplying in a culture of A. castellanii inoculated with the same sample of year-old Siberian permafrost that produced Pi. sibericum. The particles are roughly 600 nm in diameter and enclosed in a hairy tegument (Fig. 1D and 2D). At least one internal membrane is layering the particle interior. The virions appear crowned by two to four 20-nm-spaced rings corresponding to layers of fibres of different lengths surrounding the particles (Fig. 2D 2 ). The tegument is made of two layers of different densities, including an external layer nm thick made of nm interspaced parallel strips only visible on EM sections tangent to the tegument (Fig. 4D). A 12 to 14-nm-thick intermediate layer is made of a mesh of fibrils resembling those composing the central layer of the Pandoravirus tegument. At variance with the particles of Pandoraviruses and Pithovirus, the apex aperture of Mollivirus consists of a funnel, nm in diameter. Once internalized in host vacuoles,

6 784 FEMS Microbiology Reviews, 2015, Vol. 39, No. 6 the virions seem to lose their spherical appearance and take a soft shape evoking giant clams (Fig. 2D 1 ). As for the other nonicosahedral virions, there is no electron-dense structure suggesting how and where the 650 kb dsdna Mollivirus genome is packed in the particle. REPLICATION CYCLE OF GIANT VIRUSES Despite the considerable differences in the structure of their particles, the four families of giant viruses use the same phagocytosis-dependent entry mechanism to infect their common host (Figs 2A 1 D 1 and 3). This strategy is directly linked to the heterotrophic nature of Acanthamoeba, for which giant viral particles mimic the bacteria on which they naturally feed. It has long been known that individual particles must be larger than 0.6 μm in diameter to efficiently trigger phagocytosis; otherwise, they must aggregate prior to internalization (Korn and Weisman 1967; Claverie and Abergel 2009). There is thus an evolutionary incentive for viruses that infect Acanthamoeba to maintain their size above this threshold, particularly in diluted aquatic environments where the probability of an encounter between viruses and hosts is low. After their capture into phagocytic vacuoles, all four giant virus types use the same mechanism to reach the cytoplasm. In response to an unknown biochemical signal, the delivery portal of the particle opens, allowing an internal lipid membrane to unfold, protrude and fuse with that of the vacuole membrane (Figs 2A 2 D 2 and 3). This creates a channel through which the inner content of the particle is released into the cytoplasm, where the replication cycle can begin. Following this initial step, the fates of the four giant viruses differ (Fig. 2A 3 D 3 and 3). Mimiviridae: a giant cytoplasmic virion factory Initial ultrastructural studies of the replication cycle of Mimivirus focused either on the delivery of the nucleoid to the cytoplasm (to document the opening of the particle and subsequent membrane fusion) (Zauberman et al. 2008; Claverie and Abergel 2009) or on the late replication phase (6 12 h post-infection (P.I.)), which involves the formation of an electron-dense virion factory up to several micrometers in diameter surrounded by a large number of neo-synthetized particles budding from its periphery, stargate first (Suzan-Monti et al. 2007; Kuznetsov et al. 2013; Mutsafi et al. 2013). Open membrane intermediates, derived from endoplasmic reticulum membranes, form crescents structures (Fig. 4A) initiating the formation of the virions internal membranes. The four capsid proteins (one major capsid protein and three conserved homologues) are then seeding the protein array by shaping the icosahedral structure through a complex process where the major capsid protein fills the icosahedron faces (Kuznetsov et al. 2010; Mutsafi et al. 2013; Suárez et al. 2013). The first 4 5 h of the cycle were initially thought to correspond to an eclipse phase, during which initial rounds of viral genome replication were thought to occur in the host nucleus (Suzan-Monti, La Scola and Raoult 2006; Suzan-Monti et al. 2007). It is now clear that there is no eclipse phase (Claverie and Abergel 2009; Mutsafi et al. 2010, 2014) and that the replication cycle of Mimiviridae entirely proceeds outside of the host nucleus, like for the Poxviruses (Carter et al. 2005). Indeed, EM studies have indicated that the inner dense nucleoid of the Mimivirus (or Megavirus) particle remains detectable in the cytoplasm following its extrusion from the particle. This nucleoid (enclosing the viral genome and many associated proteins) initiates early viral gene transcription and DNA replication, behaving as a seed developing into a full blown virion factory (Fig. 2A 3 ) isolated from the cytoplasm by a filamentous mesh (Fig. 4A) (Claverie and Abergel 2009; Mutsafi et al. 2010; Arslan et al. 2011). In contrast with other known icosahedral double-stranded DNA viruses, packaging of the genome into Mimivirus particles proceeds through a transient aperture in the centre of an icosahedral face, which is distinct from the vertex portal (the stargate) used for genome delivery to the cytoplasm (Zauberman et al. 2008). The final stage of maturation in the cell cytoplasm is the assembly of the peripheral layer of fibrils (Fig. 2A 3 ). The replication cycle terminates 8 12 h P.I. (depending on the species of Acanthamoeba and virus tested), resulting in complete host cell lysis and the release of approximately 1000 mature particles. Pandoraviridae: eclipse phase, then nucleus disruption In the case of the Pandoraviruses, the delivery of the emptylooking inner compartment of the particle leads to a bona fide eclipse phase without noticeable change in the infected host cell during the subsequent 2 4 h. Following this initial phase, the host nucleus undergoes a major reorganization, involving a progressive loss of its normal spherical shape and the progressive fading of the electron-dense nucleolus. The nuclear membrane then develops multiple invaginations, resulting in the formation of numerous vesicles (Fig. 4B). Uncharacterized crystalline structures appear at the periphery of the deliquescent nucleus and progressively disappear as the cycle progresses. Approximately 8 10 h P.I., the infected cells lose their adherence and become rounded while new virions start to appear at the periphery of the region formerly occupied by the nucleus (Philippe et al. 2013) (Figs 2B 3, 3C and4b). Unlike Mimiviridae, for which empty particles are first formed then packed with internal components, the peripheral tegument and the inner compartment of Pandoravirus particles seem to be synthesized simultaneously in a continuous process initiated at the apical pore region and ending with mature virions fully closed at the opposite apex. Open membrane intermediates, probably derived from the nuclear membrane, form crescents structures (Fig. 3B) that seem to contribute to the virion formation via a process reminiscent of the assembly of the Poxvirus particles (Suárez et al. 2013). The replication cycles of P. salinus and P. dulcis exhibit the same stages and characteristics (Philippe et al. 2013)(Figs2B, 3Cand4B) and lead to cell lysis and the release of about thousand particles h P.I. At variance with the Mimiviridae, only 1% of the produced virions appear to be infectious. Pithovirus: electron-lucent virion factories Like for the pandoraviruses, the delivery of the uncharacterized inner content of the Pithovirus particle into Acanthamoeba is followed by an eclipse phase lasting from 4 to 6 h P.I. The progression into the infectious cycle coincides with the appearance of a roughly circular electron-lucent cytoplasmic area up to 4 μm in diameter. This presumed cytoplasmic virion factory is made visible by its lack of cytosolic substructures and the exclusion of cellular organelles at its periphery. Numerous electron-dense vesicles then appear at the virion factory boundary, later on followed by newly synthesized virus particles. Again, membrane intermediates of unknown origin accumulate in the virion factory (Fig. 4C). The process of virion formation is reminiscent of that of Pandoravirus, with the envelope and the interior of the Pithovirus virions being knitted simultaneously. However, two

7 Abergel et al. 785 assembly phases can be distinguished: first rectangular-shaped corked particles with a 25-nm-thick wall are made before maturing into ovoid particles enclosed in a 60-nm-thick striated electron-dense tegument (Fig. 3B) (Legendre et al. 2014). Complete lysis of infected cultures occurs approximately 15 h P.I. releasing hundreds of particles. The final burst is preceded by the continuous release of mature particles via exocytosis, starting 8 h P.I (Legendre et al. 2014). In contrast to the Pandoraviruses, the host nucleus and nucleolus appear unaffected throughout the whole Pithovirus replication cycle (Fig. 2C 3 and 3B). The differences in gene contents, virus-encoded functions and particle proteomes between Pithovirus and Pandoraviruses are consistent with their intracytoplasmic versus nucleus-dependent replication modes, as discussed below. Mollivirus: eclipse phase and fibrous virion factory During the Mollivirus infection, the cells remain adherent and keep their trophozoite shape all along the cycle, but there is a decrease in the number of visible vacuoles after 4 5 h. Like for all giant viruses infecting Acanthamoeba, the replication cycle begins with the phagocytosis of Mollivirus particles with up to 10 virions per cell, either scattered in individual vacuoles or gathered in a same vacuole (Fig. 2D 1 ). The opening of the particle was never visualized probably due to the fact that the depth of the funnel is larger than the thickness ( 70 nm) of the ultrathin section. However, the fusion between the underlying lipid membrane and the cellular vacuole membrane and the exchange of material between the virion interior and the cell cytoplasm are obvious (Fig. 2D 2 ). Like for the Pandoraviruses, the cell nucleus becomes disorganized after 3 4 h exhibiting numerous invaginations of the nuclear membrane. Neo-synthesized virions appear soon after at the periphery of the nascent virion factory incorporating the modified nucleus with no visible nucleolus. Cell organelles are excluded from the virion factory. This virion factory (and the nucleus when still visible) is uniquely filled by a unique mesh of fibrillary structures (Fig. 2D 3,inset,Fig.3D). They may correspond to viral proteins involved in the particle assembly as they seem to be progressively incorporated in the virions. The process of virion formation is reminiscent of that of Pandoravirus, the envelope and the interior of the Mollivirus particles being assembled simultaneously except that individual fibres are seen entering the newly formed virions (Figs 3D and 4D). Crescent of lipid membrane also accumulate and may be involved in the assembly of the internal membrane (Figs 3D and 4D). After 6 8 h, particles at various stages of maturation coexist in the same virion factory. New virions start to appear in the growth medium after 6 h in the absence of visible cell lysis and mature virions are often seen in vacuoles suggesting that most of them are released through exocytosis. GENE CONTENT OF GIANT VIRUSES The discovery of the giant viruses filled the gap between viruses and cellular life forms in term of physical size and genome complexity (Claverie et al. 2006; Raoult and Forterre 2008; Claverie and Abergel 2009, 2013). Thefour giantvirusfamilies also exhibit the largest genomes (with size in excess of 500 kb), a threshold that is occasionally used instead of particle dimension to distinguish giant viruses from other large DNA viruses (Yutin, Wolf and Koonin 2014). Genome sizes range from 0.99 Mbp (Boyer et al. 2011) to 1.26 Mbp (Arslan et al. 2011) for the Mimiviridae, 2.77, 2.24 and 1.93 Mbp for P. salinus, P. inopinatum and P. dulcis, respectively (Philippe et al. 2013), 0.61 Mbp for Pithovirus (Legendre et al. 2014) and 0.65 Mbp for Mollivirus. Although the viruses possessing the largest genomes are also the ones exhibiting the largest particles, there is no proportionality between the volume theoretically available for DNA packaging and the actual genome size. Interestingly, despite the ample space that would be available for the accumulation of junk DNA, the gene density of the four giant virus genomes is comparable to that of regular-sized viruses and prokaryotes, with about one predicted protein-coding gene per kb and short intergenic regions of about 200 nt in average. This compact organization has been confirmed by transcriptome studies of Mimivirus (Legendre et al. 2010, 2011) and Mollivirus (Legendre et al. 2015), and in less details for Pithovirus (Legendre et al. 2014). A striking feature shared by all four families of giant viruses is the high proportion of ORFans (i.e. predicted proteins without significant database match) encoded by their genomes. At the time of publication of the first representative of each family, the fraction of ORFans was 76% for Mimivirus (Raoult et al. 2004), 67% for Pithovirus (Legendre et al. 2014), 84% for P. salinus (Philippe et al. 2013) and 65% for Mollivirus (Fig. 5). Similar proportions of ORFans were found in the corresponding virion proteomes, suggesting that they are not the results of bioinformatic errors in gene predictions. This high proportion of ORFans in giant viruses is central to the ongoing debate on the origin and evolution of these viruses. With such divergent virus families, the number of core genes defined as those strictly shared by all viruses decreases and tends to zero as we keep expanding our knowledge of virus diversity (Claverie and Abergel 2013). This trend is best illustrated by Pandoraviruses, for which even the most abundant virion protein is an ORFan. It lacks any similarity with the major capsid protein, until now one of the few hallmarks shared by all large dsdna viruses infecting eukaryotes. Accordingly, the analysis of the ever increasing amount of metagenomic data might only reveal the presence of viruses sharing a substantial gene content and sufficient sequence similarity with members of previously described families. Finally, despite their distinct properties, it is important to note that all of the known giant viruses still obey Lwoff s original criteria discriminating viruses from cellular organisms: they do not encode ribosomal proteins or ribosomal RNAs, they do not encode an ATP-producing pathway and they do not multiply by division. It is quite remarkable that these rules, which were proposed more than 50 years ago, at a time when only a handful of viruses had been characterized, are still of operational value for determining the nature of giant viruses (Lwoff 1957; Lwoff and Tournier 1966). Genomic features of the Mimiviruses All Mimiviruses have a linear A+T rich DNA genome (Table 1). They have been classified in three groups based on their phylogenetic affinity. Group A gathers Mimivirus (Raoult et al. 2004), Mamavirus (Colson et al. 2011b), Terra2 (Yoosuf et al. 2014b) and Samba virus (Campos et al. 2014), the largest of this group. The group B corresponds to Moumouvirus (Yoosuf et al. 2012) and Moumouvirus monve, and group C corresponds to M. chilensis (Arslan et al. 2011), M. Iba (Saadi et al. 2013a), M. courdo7, M. courdo11 (Yoosuf et al. 2014a) andm. Terra1 (Yoosuf et al. 2014b) (Table 1). They were isolated from various environments including fresh water, sea water, sediments, soil, human faeces (Saadi et al. 2013b) and even from a leech (Boughalmi et al. 2013a). They were propagated using either A. polyphaga or A. castellanii. The more distant members between the three groups are Mimivirus and M. chilensis sharing about 50% of their genes with an average

8 786 FEMS Microbiology Reviews, 2015, Vol. 39, No. 6 Figure 5. Predicted proteins encoded in the genomes of giant viruses. Pie charts of the best match against the non-redundant database of all known proteins sequences at the time of the first publication for Mimivirus (Raoult et al. 2004), Pandoraviruses (Philippe et al. 2013), Pithovirus (Legendre et al. 2014) and Mollivirus (Legendre et al. 2015). Proteins are classified according to the broad taxonomic assignation of the best matches. It is worth noticing that the majority (83 out of 93) of the virus-like Mollivirus proteins have their best match in Pandoraviridae. In all cases, more than two third of the predicted proteins do not have a BlastP match with an E-value <10 5. There is no consensual theory to explain the large proportion of ORFans although they might be central to the understanding of the origin and evolution of giant (and large) dsdna viruses. sequence similarity of 50% identical residues at the protein level. About 80% of those not shared by the two viruses correspond to ORFans (Arslan et al. 2011; Legendre et al. 2012). It is becoming clear that besides the truly giant viruses comprising the Mimiviruses, the Mimiviridae family also encompasses an increasing number of smaller (genome and particle wise) representatives infecting various unicellular protists such as the marine phagotrophic flagellate Cafeteria roenbergensis (CroV) (Fischer et al. 2010), several unicellular algae (Larsen et al. 2008; Johannessen et al. 2014) including Phaeocystis globosa (PgV) (Santini et al. 2013) and even unknown hosts living in hypersaline cold environments (Yau et al. 2011). Theseviruses (with genomes ranging from 730 to 450 kb) are clearly phylogenetically distinct from other known algal viruses making their default classification as phycodnaviruses inappropriate (Santini et al. 2013) (Fig.6). Regulatory elements and transcriptional landscape of Mimiviruses Currently, Mimivirus is the only giant virus for which detailed analyses of the genome, transcriptome and particle proteome have been published (Raoult et al. 2004; Renesto et al. 2006; Legendre et al. 2010, 2011). Initially predicted to encode 911 proteins, Mimivirus was subsequently found to express 26 polyadenylated non-coding mrnas and 46 additional proteincoding transcripts (Legendre et al. 2010). Ultradeep transcriptome sequencing throughout the entire replication cycle of Mimivirus indicated that its 1.18-Mb genome generates 1018 transcripts, among which 979 encode proteins, 6 encode trnas (as sporadically found in other large DNA viruses) and 33 correspond to non-coding mrnas of unknown functions (Legendre et al. 2011). The transcriptomic study of infected A. castellanii cells by Mimivirus revealed a number of features which are shared by the member of the A, B and C Mimivirus groups. All predicted genes are transcribed at same point during the infectious cycle and belong to three classes of expression: early, intermediate and late. Regulatory elements have been identified; a strictly conserved promoter element (AAATTGA) is found 5 of nearly all early expressed genes (Suhre, Audic and Claverie 2005), and a less conserved promoter was identified upstream of the late genes (Legendre et al. 2010). Transcription arrest and polyadenylation are governed by what was named the hairpin rule (Byrne et al. 2009; Arslan et al. 2011) alongwhich polyadenylation occurs in a palindromic sequence made of a stem of at least 13 paired nucleotides and a loop of at most 5 nucleotides. This structure, not conserved at the sequence level, is located at the 3 end of the vast majority of the Mimiviruses genes. The molecular machinery responsible for transcript maturation and polyadenylation corresponds to two conserved clusters of genes responsible for the 3 end and 5 end maturation of the viral transcripts (Priet et al. 2015). One cluster encodes a protein with tandem RNAse III-like domains recognizing and cleaving inside the hairpin structure, as well as a self-processive polya polymerase able to add up to 700 nt long polya tails. The second cluster encodes a trifunctional mrna capping enzyme, capping and N7-methylating the caps in the 5 end of the transcripts (Benarroch, Smith and Shuman 2008), as well as a methyltransferase

9 Abergel et al. 787 Table 1. Characteristics of giant viruses (grey rows) compared to other large eukaryotic viruses. Particle largest Genome max. Nuclear phase Family or genus Name Virion shape dimension (nm) size (kb) G+C% Genes aars Pandoraviridae a Pandoravirus salinus Ovoid Ø Tyr Trp Pithovirus b Pithovirus sibericum Ovoid Ø Mollivirus c Mollivirus sibericum Ovoid 600 Ø Mimiviridae group C d Megavirus chilensis Icosahedral 610 Ø (440 Ø) Tyr Cys Arg Met Asn Trp Ile Mimiviridae group B e Moumouvirus Icosahedral 600 Ø (420 Ø) Tyr Cys Arg Asn Ile Mimiviridae group A f Mimivirus Icosahedral 630 Ø (390 Ø) Tyr Cys Arg Met CroV g CroV Icosahedral 300 Ø 730 kb IleRS PgV h PgV Icosahedral 460 kb Coccolithoviruses EhV 86 i Icosahedral 180 Ø 407 kb Marseilleviridae j Marseillevirus Icosahedral 220 Ø 368 kb Poxviridae Canarypox k virus Ovoid enveloped nm 365 kb Chloroviruses PBCV-NY2A l Icosahedral 200 Ø 370 kb Note: a Pandoraviridae (Philippe et al. 2013; Antwerpen et al. 2015); b Pithovirus sibericum (Legendre et al. 2014); c Mollivirus (Legendre et al. 2015), Mimiviridae ( d group C (Arslan et al. 2011; Saadi et al. 2013b; Yoosuf et al. 2014a,b), e group B (Yoosuf et al. 2012), f group A (Raoult et al. 2004; Colson et al. 2011a; Legendre et al. 2011; Campos et al. 2014; Yoosuf et al. 2014b); g Cafeteria roenbergensis virus (Fischer et al. 2010; Fischer and Suttle 2011); h Phaeocystis globosa virus (Santini et al. 2013); i Emiliania huxleyi virus 86 (Wilson et al. 2005); j Marseilleviridae (Boyer et al. 2009; Thomaset al. 2011; Boughalmi et al. 2013b; Aherfi et al. 2014; Doutre et al. 2014); k Canarypox (Tulman et al. 2004; Roberts and Smith 2008); l Paramecium bursaria chlorella virus NYA (Fitzgerald et al. 2007; Van Etten and Dunigan 2012; Jeanniard et al. 2013). Giant viruses visible by light microscopy are marked in grey. Viruses with a known virophage are marked by a red asterisk. performing the 2 -O cap methylation. The two clusters belong to the class of late expressed genes. The corresponding enzymes are loaded in the virions and readily available for action after the nucleoid is released in the cytoplasm, together with the transcription machinery and DNA repair enzymes initiating transcription. A cap-specific guanine-n2 methyltransferase is forming a 2,7-dimethylguanosine DMG cap which could also favour viral protein synthesis (Benarroch et al. 2009). Mimiviruses are cytoplasmic viruses All the sequenced Mimiviridae genomes (small and large) encode transcription machinery (Raoult et al. 2004; Fischer et al. 2010; Colson et al. 2011a; Santiniet al. 2013) consistent with the use of promoter sequences that differ from those of Acanthamoeba (Suhre, Audic and Claverie 2005; Legendre et al. 2010). In the case of Mimivirus, this machinery has been shown to be packaged in the particles as proteins (Renesto et al. 2006; Claverie, Abergel and Ogata 2009), allowing the infectious cycle to be initiated from within the cytoplasm. As the cycle proceeds, the Mimivirus/Megavirus virion factories grow from the size of 0.4 μm (the size of the nucleoid) to 10 μm in diameter (Raoult et al. 2004; Suzan-Monti et al. 2007; Claverie and Abergel 2009; Mutsafi et al. 2010; Arslan et al. 2011). The functional resemblance of these virion factories with (transitory) parasitic microorganisms is such that they can be infected by their own viruses, called virophages (La Scola et al. 2008; Claverieand Abergel 2009) (Box 2). Accordingly, the virophages use the same regulatory elements than their Mimivirus hosts (a late promoter and the terminal mrna hairpin structure), relying on the giant virus transcription apparatus to express their genes (Claverie and Abergel 2009; Ruiz-Saenz and Rodas 2010; Desnues, Boyer and Raoult 2012; Tiwari et al. 2014). Mimiviruses specific features In addition to their own DNA replication and transcription machineries, the Mimiviridae encode a large complement of DNA repair enzymes including remote homologues of Escherichia coli MutS, a component of the mismatch repair system. The sequences of the two viral versions of MutS (MutS7 and MutS8) are sufficiently distinct from their cellular counterparts to serve as specific markers in environmental studies (Ogata et al. 2011; Hingamp et al. 2013; Wilson et al. 2014). For no clear reason, the Mimiviridae also encode their own distinct version of the glutamine-hydrolyzing asparagine synthetase, the sequence of which can serve the same purpose (Mozar and Claverie 2014). By far the most unexpected finding in the genomes of the largest Mimiviridae is the recurrent presence of up to seven different amino-acyl trna synthetases (aars); these enzymes are involved in the charging of trnas with their cognate amino acids. Four aars (ArgRS, CysRS, MetRS, TyrRS) are encoded by Mimivirus (Raoult et al. 2004) to which IleRS is added in Moumouvirus (Yoosuf et al. 2012), and a further two enzymes (TrpRS and AsnRS) in M. chilensis (Arslan et al. 2011) (Table 1). The Mimivirus aars are active and the structure of the TyrRS

10 788 FEMS Microbiology Reviews, 2015, Vol. 39, No. 6 Figure 6. Gene-content cladistic clustering of the large and giant DNA viruses infecting eukaryotes (from Legendre et al. 2015). suggested that the enzyme recognizes a two-letter codon due to a shorter anticodon-binding loop (Abergel et al. 2007), conserved in all Mimiviridae endowed with this aars. There are also several other enzymes involved in translation such as translation initiation and elongation factors and even an autoregulated termination factor the translation of which proceeds through two stop codons via two distinct recoding events, a frameshift and a readthrough (Jeudy et al. 2012). The presence of these central components of the translation machinery, normally a trademark of cellular organisms, is central to the ongoing debate about the origin and evolution of giant viruses (see below). As expected from a viral genome entirely expressed outside of the host nucleus, the genes are not interrupted by spliceosomal introns. A small fraction of genes are interrupted by inteins (Ogata, Raoult and Claverie 2005) and group I self-excising introns including a major capsid protein presenting two type I introns (Azza et al. 2009). Among the 10% of Mimiviridae proteins that are similar to proteins with known functions, in addition to the proteins involved in transcription and translation, the following enzymes have also been characterized in detail: a DNA topoisomerase IB (Benarroch et al. 2006), an NAD + -dependent DNA ligase (Benarroch and Shuman 2006), a DNA glycosylase (Imamura et al. 2012), the first virally encoded nucleoside diphosphate kinase with a unique affinity for pyrimidine nucleotides due to a shorter Kpn loop conserved in all Mimiviruses (Jeudy et al. 2009), a cyclophilin (Thai et al. 2008), a lysyl hydroxylase (Luther et al. 2011), a sulfhydryl oxidase (Hakim and Fass 2009) and a

11 Abergel et al. 789 copper chaperone-independent superoxide dismutase (Lartigue et al. 2015). Surprisingly, they encode pathways for biosynthesis of the unusual sugar composing their fibres capsids combining enzymes encountered either in bacteria or eukaryotes and a number of carbohydrate-processing enzymes (Parakkottil Chothi et al. 2010; Piacenteet al. 2012, 2014a,b). Recently, the structure of one of the major protein components of Mimivirus fibres suggested that it could be involved in the viral entry by enzymatically digesting its host cell wall (Klose et al. 2015). In most cases, the Mimivirus-encoded enzymes have structural and/or functional features similar to that of their cellular homologues. The puzzling number of additional ORFan genes in each newly sequenced genome, which cannot be explained by lateral gene transfer from the host, suggests that the ancestor of Mimiviridae may have been more complex than its descendants. The presence of protein translation-related enzymes also suggested that the viruses may originate from an ancestral cell belonging to an extinct fourth domain of the tree of life (Raoult et al. 2004; Claverie et al. 2006; Colson et al. 2011a; Desnues, Boyer and Raoult 2012; Legendre et al. 2012). The hot debate on alternative evolutionary scenarios (Yutin, Wolf and Koonin 2014) will be presented in a separate section. As additional Mimiviridae were isolated with genomes in the same Mbp-size range, the feeling was growing that an asymptote of maximal genetic complexity had been reached for giant DNA viruses. This belief was soon to be proven wrong by the discovery of the Pandoraviruses. Genomic features of the Pandoraviridae In contrast to most large DNA viruses, the nucleotide composition of Pandoraviruses genomes is G+C rich( 61%). There are at least six copies of a terminal fragment of 50 kb for P. salinus and 1 repeat of a 20-kb terminal fragment for dulcis. A total of 2556 putative protein-coding sequences (CDSs) were identified in the P. salinus 2.47-Mb unique genome sequence (considering a single terminal repeat), 1502 for P. dulcis and 1839 for P. inopinatum. However, the sequence comparison of these predicted genes with those in public databases revealed that 84% were ORFans. The three Pandoraviruses share around 650 genes with an average of 60% sequence identity at the protein level, but surprisingly, one half of their genes are unique to one or the other. The vast majority of Pandoravirus-encoded proteins do not resemble anything even in metagenomics databases, thus ruling out that they might originate from lateral gene transfers from their cellular host (Fig. 5). More than half of the remaining genes with homologues in databases correspond to noninformative structural motifs (such as Morn, ankyrin and F-box) or are homologous to uncharacterized proteins. Despite lacking a recognizable major capsid protein gene, as well as many of the other core genes presumed to be conserved among large dsdna viruses (Iyer et al. 2006), their genomes remain typically viruslike, with the largest fraction of the genes with a predicted function (54 out of the 136) devoted to DNA replication, transcription, repair and nucleotide synthesis (Philippe et al. 2013). Pandoraviruses are nucleocytoplasmic viruses The Pandoraviruses encode four RNA polymerase subunits (RPB1, RPB2, RPB5, RPB10), together with their own mrna capping enzyme and at least three other transcription factors. However, the virally encoded transcription machinery is not a component of mature virions, implying that the host nucleus must be actively involved in the early stage of the Pandoravirus infection before it decays, as observed by EM. Furthermore, although they encode their own DNA polymerase, the Pandoraviruses lack other essential components of the DNA replication machinery such as a DNA ligase, topoisomerases and a DNA sliding clamp. This strongly suggests that these viruses could not replicate without the help of host enzymes that are normally found in the nucleus. Finally, the presence of spliceosomal introns in about 10% of the viral genes further suggests that at least part of the Pandoravirus genome is transcribed in the host nucleus. Another remarkable feature of the Pandoravirus genome is the presence of two amino-acyl-trna synthetase (TyrRS and TrpRS) genes, which are also found in M. chilensis. However,these TyrRS and TrpRS are more similar to their Acanthamoeba homologues (57 and 58% identical residues, respectively) than to their Mimiviridae counterparts, suggesting that they might have been acquired from an Acanthamoeba-related host. The proteomic study of the purified P. salinus particles confirmed that the ORFans corresponded to bona fide proteins, 80% of which without homologues in the public databases. Given this unusual proportion of ORFans, the absence of a major capsid protein homologue, and their unique particle morphology, the Pandoraviruses appear to define a new dsdna virus family, the Pandoviridae. Genomic features of Pithovirus In contrast to Pandoravirus, Pithovirus has a G+C poor (36%) genome that is only 610 kb in size. This genome size is surprisingly small given a particle internal volume that is larger than that of Pandoraviruses (Table 1). Furthermore, the Pithovirus 610-kb genome only encodes 467 proteins. Compared with other large DNA viruses, the low coding density (68%) of its genome is due to the presence of a large number of regularly interspersed palindromic motifs. These motifs (about 150 nt in length) are most often found in 2-kb long arrays of tandem repeats. They are not transcribed and do no resemble previously described mobile elements or repeats found in other viral genomes (Allen, Schroeder and Wilson 2006; Delhon et al. 2006; Legendre et al. 2014). More Pithovirus relatives will need to be studied to determine if this is a general characteristic of the family and how it might be linked to its evolution. The topology of the Pithovirus genome, a terminally redundant circularly permutated linear dsdna molecule (or a closed circle), is also unique among giant viruses. Such topology is encountered in the Iridoviruses, a family of large icosahedral DNA viruses. Pithovirus exhibits some affinity with Iridoviruses and Marseilleviruses Compared to other giant viruses, the gene content of Pithovirus is globally more similar to those of previously described large icosahedral eukaryotic DNA viruses (Legendre et al. 2014). This is illustrated by the phylogenetic clustering of the Pithovirus DNA polymerase (Fig. 6) within a clade comprising the Iridoviruses and Marseilleviruses, two distantly related families of eukaryotic large dsdna viruses. The Pithovirus RNA polymerase subunits RPB1 and RPB2 display a similar clustering pattern, although their sequences remain fairly distant from those of their closest homologues in Marseilleviruses (about 30% identical residues). However, only a third of the 467 predicted Pithovirus proteins (i.e. 152, 32.5%) have recognizable databases homologues, including only 19, 15 and 10 best matching their counterparts in Marseilleviridae, Mimiviridae and Iridoviridae, respectively (Fig. 5). Given the very partial overlap of its gene