466-467 (2014) 3–14

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Virology

journal homepage: www.elsevier.com/locate/yviro

Review Infection cycles of large DNA : Emerging themes and underlying questions

Yael Mutsafi n, Yael Fridmann-Sirkis, Elad Milrot, Liron Hevroni, Abraham Minsky n

Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel article info abstract

Available online 2 July 2014 The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant fi Keywords: blurred the distinction between viruses and cells. These ndings elicited lively debates on the and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, Giant viruses however, concerned with new insights into fundamental structural and physical aspects of viral Herpesvirus replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely Membrane biogenesis cytoplasmic replication cycles of and are discussed in light of the highly limited Mimivirus trafficking of large macromolecules in the crowded of cells. The extensive spatiotemporal Nucleocytoplasmic large dsDNA viruses order revealed by cytoplasmic viral factories is described and contended to play an important role in (NCLDVs) promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis Stargate Vaccinia mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self- Viral factories assembly. Mimivirus encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral . Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. & 2014 Elsevier Inc. All rights reserved.

Contents

Introduction...... 3 Mimivirus, Pandoraviruses and other large nucleocytoplasmic DNA viruses ...... 4 Replication cycles of large dsDNA viruses: underlying questions ...... 5 and genome release ...... 5 Genome trafficking within host cells and nucleus targeting...... 5 Cytoplasmic viral factories, membrane biogenesis and generation ...... 6 Replication cycles of Mimivirus and other large DNA viruses...... 7 Viral entry and genome release ...... 7 Genome trafficking within host cells and nucleus targeting...... 8 Cytoplasmicviralfactories,membranebiogenesisandcapsidgeneration...... 10 Concluding comments: new insights and open questions ...... 12 References...... 12

Introduction

Until recently viruses were generally considered as mere side-

n products of cellular systems with little relevance to the generation Corresponding authors. and evolution of . This view has dramatically changed in the E-mail addresses: yael.mutsafi@weizmann.ac.il (Y. Mutsafi), [email protected] (A. Minsky). past decade following the discovery of the giant http://dx.doi.org/10.1016/j.virol.2014.05.037 0042-6822/& 2014 Elsevier Inc. All rights reserved. 4 Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 polyphaga Mimivirus (La Scola et al., 2003). With a particle size in the assembly of multiple, highly complex virion particles. In this comparable to that of small (750 nm) and a 1.2 Mbp essay we review recent studies concerned with the structure and double-strand DNA genome that carries 1000 open reading replication cycle of large DNA viruses with emphasis on the frames and encodes never before encountered in viruses, Mimivirus. We discuss how new insights provided by these studies such as components of the machinery, the may impinge on our understanding of fundamental processes such Mimivirus blurred the established boundaries between viruses as intracellular trafficking, membrane biogenesis and self-assembly. and cells. The discovery of additional giant viruses such as Finally, we highlight intriguing questions that are raised by infec- chilensis (Arslan et al., 2011), Pandoraviruses (Philippe tion cycles of giant viruses, to which future studies are likely to et al., 2013) and (Legender et al., 2014), which high- provide novel and exciting answers. lights the abundance and diversity of these viruses, is reviving intriguing debates on topics that for a long time were reputed as intractable and thus of limited interest. These include the origin Mimivirus, Pandoraviruses and other large nucleocytoplasmic of viruses, their position, if any, on the tree of life, the potential DNA viruses contribution of viruses to the establishment of the three domains of life (Bacteria, , and Eukarya), and their putative role in The ameba-infecting A. polyphaga Mimivirus is a member of the generation of eukaryotic nuclei (Bell, 2001, 2006; Claverie and the Megaviridae family that also includes Moumouvirus and Abergel, 2010; Forterre and Prangishvili, 2013; Koonin, 2005; M. chilensis (Arslan et al., 2011; Yoosuf et al., 2012). This family Koonin and Yutin, 2010; Raoult and Forterre, 2008; Van Etten, belongs to the nucleocytoplasmic large DNA viruses (NCLDV) 2011). A particularly lively discourse motivated by the discovery of , which comprises the diverse -infecting DNA giant viruses concerns the question how did DNA viruses emerge – families , , and Asfarviridae did giant viruses start small and get bigger by acquiring (Iyer et al., 2001). The NCLDV clade has been recently expanded by from their hosts, or get smaller by progressively losing genes the discovery of even larger viruses, the Pandoraviruses (Philippe (Claverie and Abergel, 2013; Iyer et al., 2006)? et al., 2013) and Pithovirus sibericum (Legender et al., 2014). The In addition to prompting heated debates on the nature, origin Mimivirus particle is composed of a genome-containing core that and potential roles in evolution, large DNA viruses raise intriguing is surrounded by a lipid-bilayer underlying an icosahedral capsid. questions associated with their replication process that culminates A unique feature of the Mimivirus is a 120 nm-long fiber mesh that

Fig. 1. Structural features of Mimivirus. (A) Scanning Transmission Electron Microscopy (STEM) Tomography slice of a Mimivirus particle. Scale bar: 100 nm. (B) Magnified representation of the region delineated in (A). The layers comprising the Mimivirus virion are (1) DNA genome; (2) core wall; (3) inner membrane; (4) a layer proposed to represent an additional membrane; (5) inner capsid shell; (6) outer capsid shell; (7) tightly packed fiber shell (Xiao et al., 2005; Zauberman et al., 2008). Scale bar: 20 nm. (C) Density plot of the various layers along the line drawn in (B). Digits correspond to those depicted in (B) (reproduced from Mutsafi et al., 2013). (D) Scanning Electron Microscopy (SEM) image of the star-shaped structure in a Mimivirus particle. Scale bar: 200 nm. (E) Transmission Electron Microscopy (TEM) image of a cryo-fixed extracellular Mimivirus particle revealing a star-shaped structure that is located at a single vertex and extends throughout the five icosahedral edges emanating from this vertex (Zauberman et al., 2008). Scale bar: 100 nm. Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 5 coats the capsid and forms a dense layer at their attachment site release of the Mimivirus genome occurs has been partially elucidated (Raoult et al., 2004; Xiao et al., 2005)(Fig. 1A–C). In addition, a (see below), the mechanisms that mediate entry of large DNA viruses single conspicuous modified icosahedral vertex is detected in the into their hosts, including that of the extensively studied Vaccinia virions (Xiao et al., 2009; Zauberman et al., 2008)(Fig. 1D and E). (Mercer and Helenius, 2008; Moss, 2006; Sodeik and Krijnse-Locker, The structural data available on Pandoraviruses and on P. sibericum 2002) need to be further investigated. are quite astonishing. These giant viruses lack a clear capsid layer and an icosahedral structure (which characterize Megaviridae), Genome trafficking within host cells and nucleus targeting and reveal an ovoid morphology and a unique apical pore (Legender et al., 2014; Philippe et al., 2013). While it was claimed With the exception of few viruses discussed below, all currently that genomes of these viruses are released into host known DNA viruses are assumed to carry out replication and through a portal generated by a fusion of an internal viral lipid transcription either entirely or partially within their host nuclei. membrane with host phagosome membranes (as previously Viral genomes can reach nuclei through several pathways. Follow- shown for Mimivirus (Mutsafi et al., 2010; Zauberman et al., ing ejection at the host periphery ‘free’ non-encapsidated genomes 2008), no data were provided to substantiate this claim. Thus, can be trafficked to and into the nucleus, as is believed to be the and as further discussed below, the mechanisms responsible case for the Phycodnavirus PBCV-1 (Thiel et al., 2010; Van Etten for genome release of the giant Pandoraviruses and P. sibericum et al., 2002; Wulfmeyer et al., 2012; Yamada et al., 2006). remain unknown. Alternatively, genomes can reach the nucleus while encapsidated and then ejected following docking of the to nuclear pores, as exemplified by several large DNA viruses such as Adenoviruses Replication cycles of large dsDNA viruses: underlying questions and Herpesviruses (Peng et al., 2010; Shahin et al., 2006; Sodeik et al., 1997). Yet another possible alternative is a combination of Extensive studies notwithstanding, many aspects concerning these transactions, consisting of the entry of viral particles via cycles remain poorly understood. These uncer- endocytosis that is followed by the release of the genome into the tainties, which encompass all stages of the replication cycles are cytoplasm and its subsequent targeting toward the host nucleus. specifically and dramatically underlined in large viruses due to Such a pathway was suggested for large viruses from the Iridovir- their size and complexity, their very long genomes and, as idae family (Williams et al., 2005) and, until recently, the Mimi- discussed below, their particular modes of replication. virus. Notably, the replication cycle of the huge P. salinus, which carries a 2.47 Mbp genome, was suggested to Viral entry and genome release proceed through a similar pathway (Philippe et al., 2013). The prerequisite of transporting large capsids or very long viral The initial stage in the infection cycle of bacteriophages genomes from their entry sites at the periphery toward the involves the ejection of the viral genome into the host cytoplasm. nucleus entails, however, remarkable hurdles. Specifically, the Notably, several large eukaryote-infecting DNA viruses such as the cellular milieu is highly refractory to the motion of large particles large algae-infecting Phycodnavirus PBCV-1 were also shown to and long DNA molecules due to the high viscosity of the cytosol, initiate their infection through a similar ejection step (Thiel et al., the dense molecular sieve generated by the network 2010; Van Etten et al., 2002; Wulfmeyer et al., 2012; Yamada et al., and even more so by the tight organization of the endoplasmic 2006). According to the current wisdom, in many bacteriophages reticulum (Novak et al., 2009; Pouton et al., 2007). As a result of this stage is a two-step process, whereby a first ‘push’ stage is these obstacles, complexes larger than 500 kDa or 20 nm in promoted by the large pressure generated by the tight genome diameter, and DNA segments longer than 10 kbp are practically packaging within the capsid (Ponchon et al., 2005; Tzlil et al., immobile in cellular environments in the absence of active trans- 2003), while a second necessarily involves active pulling of the port (Dauty and Verkman, 2005; Dohner et al., 2005; Minsky, genome that is stimulated by several potential processes. These 2003; Pouton et al., 2007; Weiner et al., 2009). Indeed, it has been (still widely debated) processes include transcription-based inter- estimated that the capsid of a virus (HSV) would nalization, interactions with specific DNA-binding proteins, con- need 23 years for a diffusion-mediated transport of 1 mm in densation of the released genome within the host cytoplasm axonal cytoplasms (Sodeik, 2000). In light of such impediments, (Evilevitch et al., 2003; Knobler and Gelbart, 2009; Leforestier trafficking of viral components during viral replication has been et al., 2008; Molineux, 2006; Sao-Jose et al., 2007), or re-hydration extensively studied and shown to critically depend on the host of tightly packed viral genomes (the hydrodynamic model) cytoskeleton network (Dohner et al., 2005; Greber and Way, 2006; (Grayson and Molineux, 2007; Molineux and Panja, 2013). Howard and Moss, 2012; Jouvenet et al., 2004; Leopold and Pfister, Notably, such ejection mechanisms are unlikely to be relevant 2006; Lyman and Enquist, 2009; Mallardo et al., 2001; Ploubidou to eukaryote-infecting large DNA viruses. Specifically, DNA packa- et al., 2000; Radtke et al., 2006; Rietdorf et al., 2001; Sodeik et al., ging densities in large DNA viruses such as the Phycodnavirus 1997; Ward and Moss, 2001). PBCV-1, the Mimivirus, and in particular Pandoraviruses and As depicted in Fig. 2A, even an active, -mediated P. sibericum are substantially lower than those characterizing translocation of HSV capsids within the crowded cytoplasm appears bacteriophages (Claverie and Abergel, 2010; Legender et al., as a highly demanding journey toward the nucleus (Lyman and 2014; Philippe et al., 2013; Wulfmeyer et al., 2012). In and of itself Enquist, 2009) (note that the ER network, which has been suggested this observation implies that pressure-driven mechanisms that to represent a particularly exacting hurdle for intracellular traffick- initiate ejection of genomes are unlikely to con- ing (Novak et al., 2009), is not included in the model). The HSV tribute to the release of genomes of large DNA viruses into their translocation model highlights the challenges faced by large DNA host cytoplasm. Furthermore, since (in contrast to bacteria) no viruses. Specifically, whereas the diameter of the HSV capsid is specific DNA-binding proteins are present in eukaryotic cyto- 125 nm, the diameter of the Mimivirus genome-containing core is plasms, ratchet-like genome pulling mechanisms mediated by almost 3 times larger (340 nm) (Mutsafi et al., 2010). Thus, intra- such proteins are improbable. Similarly implausible in the case cellular trafficking of the cores of giant viruses such as the of large eukaryotic viruses is the re-hydration pathway, as genomes Mimivirus or of the entire virion particles of Pandoraviruses in such viruses are not tightly condensed and therefore likely to be (1 μm in diameter) is unlikely. Similarly improbable is a hydrated to begin with. Indeed, whereas the pathway by which the nucleus-targeting of ‘free’, uncoated genomes of large viruses. This 6 Y. Mutsafi et al. / Virology 466-467 (2014) 3–14

Fig. 2. Inherent hurdles involving intracellular trafficking of viral genomes. (A) Scale drawing of a Herpes virus capsid (yellow) transported toward the nucleus along a microtubule. The highly crowded nature of the cytoplasm, including actin, intermediate filaments, and multiple other large complexes, is highlighted (reproduced with permission from (Lyman and Enquist, 2009)). Notably, the ER network that has been suggested to represent a particularly exacting hurdle for intracellular trafficking (Novak et al., 2009)isnot included in this drawing. The HSV translocation model underlines the challenges faced by large DNA viruses: whereas the diameter of the HSV capsid is 125 nm, the diameter of the Mimivirus genome-containing core is almost 3-times larger (340 nm). (B) Infection of an algae cell by the Phycodnavirus PBCV-1. The 330 kbps PBCV-1 genome ejected at the host periphery (blue arrow) may need to be translocated toward the host nucleus (Nu) through multiple photosynthetic membrane layers (green arrows) occupying a substantial volume of the host algae cells (Van Etten et al., 2002). Scale bar: 500 nm. (C) High-magnification micrograph depicting a PBCV-1 particle infecting an alga cell. The multiple photosynthetic membrane layers that may hinder the journey of viral DNA toward the host nucleus are evident. Micrograph courtesy of Dr. Nathan Zauberman. notion is underlined by the infection cycle of the Phycodnavirus necessarily entail an orderly trafficking and precise nuclear loca- PBCV-1. As demonstrated in Fig. 2B and C, the 330 kbp PBCV-1 lization of multiple viral proteins that are expressed in the host genome (Van Etten et al., 2002) ejected at the host periphery may cytoplasm. need to be translocated through multiple photosynthetic membrane These predicaments are, however, dwarfed when compared to layers occupying a substantial volume of algae cells that act as those imposed by cytoplasmic factories. Specifically, the nucleus PBCV-1 hosts. Indeed, the mechanisms by which genomes of large provides an optimal platform for viral replication as it includes the viruses such as those of PBCV-1 or Iridoviruses (Williams et al., factors required for replication and transcription and inherently 2005) reach their host nuclei are as unclear and controversial as the provides a ready-made spatial platform for these transactions. pathways responsible for the translocation of the bacterial Agro- In sharp contrast, cytoplasmic viral factories and exclusive cyto- bacteium DNA complex (T-DNA) toward and subsequently into the plasmic infection necessitate de-novo generation of platforms that host nuclei (Gelvin, 2012). provide the factors, machinery, and most significantly, the elabo- Once reaching the nucleus, viral genomes need to be translo- rate architecture required for replication of large DNA viruses. The cated into this organelle. The mechanisms that mediate this evident structural complexity of Vaccinia cytoplasmic factories process remain largely unknown (Greber and Fassati, 2003; (Condit et al., 2006; Cyrklaff et al., 2007; Husain et al., 2006; Liashkovich et al., 2011; Pouton et al., 2007; Whittaker et al., Katsafanas and Moss, 2007; Risco et al., 2002) or of the Mimivirus 2000). Several observations implied that, rather surprisingly con- (Mutsafi et al. 2010, 2013) renders the spatiotemporal features that sidering the diameter of nuclear pores, the herpes simplex virus characterize the factories – nuclear, cytoplasmic, or both – of the genome is imported into the nucleus through nuclear pores in a larger Pandoraviruses and P. sibericus into a particularly compel- condensed structure (Shahin et al., 2006). In light of the fact that ling case study. the Mimivirus genome is 8-fold larger than that of HSV-1 Equally intriguing questions concern the biogenesis of internal (152 kbp) and genomes of Pandoraviruses are 12–16 times longer membrane layers (Huiskonen and Butcher, 2007). Specifically, the (Philippe et al., 2013), the notion that such huge genomes are current leading (yet still debatable) view is that large DNA viruses translocated into nuclei is daunting. (Vaccinia, PBCV-1 and Asfarviridae) carry a single inner membrane bilayer (Chlanda et al., 2009; Hawes et al., 2008; Heuser, 2005; Cytoplasmic viral factories, membrane biogenesis and capsid Mutsafi et al., 2013; Suárez et al., 2013; Szajner et al., 2005; Van generation Etten et al., 2010; Yan et al., 2000). Whereas the generation of an outer membrane can be straightforwardly assigned to a budding Infection of a large variety of DNA and RNA viruses leads to the process and the formation of two-membrane bilayers can be formation of distinct cellular organelles, coined viral factories, in explained by wrapping of collapsed ER cisternae; a single internal which viral replication and assembly occur (Condit, 2007; bilayer requires either single-layer membrane precursors or the de Castro et al., 2013; Erickson et al., 2012; Fontana et al., 2010; loss of one ER membrane layer by yet unidentified mechanisms Netherton et al., 2007; Netherton and Wileman, 2011; Novoa et al., (Chlanda et al., 2009; Mutsafi et al., 2013). As described below, 2005; Schramm et al., 2006; Tolonen et al., 2001; Zauberman et al., recent studies provide new insights into the mechanisms by which 2008). These viral factories, which were suggested to represent the internal single-membrane layers are generated in large viruses actual living stage of viruses (while viral particles correspond to (Chichon et al., 2009; Chlanda et al., 2009; Cockburn et al., 2004; ‘mere’ seeds that mediate host to host spreading (Claverie, 2006)), Hawes et al., 2008; Heuser, 2005; Laliberte and Moss, 2010; Maruri- are generated either in the nucleus or in the cytoplasm of the host Avidal et al., 2013; Miller and Krijnse-Locker, 2008; Mutsafi et al., cells. For large and complex viruses either site entails significant 2013; Salas and Andres, 2013; Sodeik and Krijnse-Locker, 2002; hurdles. In addition to the above-mentioned quandaries related to Suárez et al., 2013; Unger et al., 2013; Yan et al., 2000). An additional genome translocation, nuclear viral factories (e.g. HSV-1) open question relates to the generation of the viral capsids – what Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 7

Fig. 3. Mimivirus entry into an ameba host cell. (A and B) SEM micrographs of the initial stage of Mimivirus infection, revealing that viral internalization proceeds through phagocytosis of entire virion particles (Suzan-Monti et al., 2007; Zauberman et al., 2008) in a process that is apparently mediated by actin protrusions (arrow in inset of panel B; scale bar: 400 nm). Scale bars in (A) and (B) 2 μm. (C) TEM micrograph showing cellular protrusions that engulf a Mimivirus virion. Scale bar: 200 nm. (D) Mimivirus internalization culminates by phagosome-enclosed virions within the cytoplasm of the host cells (TEM). A stargate vertex is evident in one of the two phagosome-enclosed virion particles (arrowhead). Micrograph courtesy of Dr. Nathan Zauberman. Scale bar: 200 nm. are the factors that dictate the precise and reproducible size and cytoplasm of the host cells (Fig. 3)(Suzan-Monti et al., 2007; morphology of viral capsids? For relatively small icosahedral viruses Zauberman et al., 2008). Whereas a phagocytosis-dependent entry a tape-measure protein has been proposed to act as the factor that pathway has been proposed for other large DNA viruses such as determines the capsid size (Abrescia et al., 2004, 2008). Such a Iridoviruses (Williams et al., 2005), M. chilensis, Pandoraviruses mechanism is, however, unlikely to be operative in giant icosahedral (Philippe et al., 2013) and Pithovirus (Legender et al., 2014); it viruses such as the Mimivirus or M. chilensis. Similarly, the factors contrasts a phage-like ejection mechanism suggested for the that dictate the unusual size and ovoid shape of the Pandoraviruses algae-infecting Phycodnavirus PBCV-1 (Thiel et al., 2010; Van and P. sibericus viruses remain unknown. Etten et al., 2002; Wulfmeyer et al., 2012; Yamada et al., 2006). Finally, an underlying question concerns the mechanisms respon- The inherent and severe hurdles associated with such a genome sible for efficient, rapid and accurate genome packaging within pre- ejection mechanism and a subsequent genome intracellular traf- assembled capsids (e.g., Baumann et al., 2006). It is generally assumed ficking were discussed above and are illustrated in Fig. 2. These that build-up of internal pressure in pre-assembled bacteriophage hurdles represent a fascinating open question. capsids during packaging is sensed by packaging motors, and thus Once internalized and localized within a phagosome, the plays a central role in ensuring encapsidation of a single genome per Mimivirus releases its genome by the opening of the virion capsid. capsid (the ‘head-full’ DNA packaging model (Legender et al., 2014; This major conformational change occurs at a particular star- Nurmemmedov et al., 2007)). Evidently, such a pressure-mediated shaped structure coined ‘stargate’ (Zauberman et al., 2008), which sensing mechanism cannot be operative in large DNA viruses such as is centered at a unique icosahedral vertex and extends along the PBCV-1, Mimivirus or Pandoraviruses, in which no or very limited whole length of the five icosahedral edges emanating from this internal pressure is generated during genome packaging (Claverie and vertex (Fig. 1D and E). The stargate opening enables protrusion of Abergel, 2010; Philippe et al., 2013; Wulfmeyer et al., 2012). This point the Mimivirus internal membrane toward the phagosomal mem- is highlighted by a recent report demonstrating that even following brane and a subsequent membrane fusion. Such a fusion leads to substantial size reduction, Mimivirus genomes are correctly encapsi- the generation of a large portal (Fig. 4)(Zauberman et al., 2008) dated within pre-assembled capsids (Boyer et al., 2011). through which the whole genome-containing internal core of the Mimivirus is released into the host cytoplasm (Fig. 5)(Mutsafi et al., 2010). It should be noted that a conceptually similar genome Replication cycles of Mimivirus and other large DNA viruses delivery through a membrane tube generated by an internal membrane has been suggested for the bacteriophage PRD1 Viral entry and genome release (Grahn et al., 2002). For this to occur, the PRD1 capsid must open up in a yet uncharacterized process that might be similar to the Mimivirus infection is initiated by the internalization of entire genome release process occurring in Mimivirus. High-resolution virion particles in a process that is apparently mediated by actin structural studies of PRD1 life cycle will be required to address this protrusions and results in phagosome-enclosed virions within the intriguing scenario. 8 Y. Mutsafi et al. / Virology 466-467 (2014) 3–14

Fig. 4. Mimivirus stargate opening within phagosomes of an ameba host. (A) Tomographic slice of a late phagosome enclosing three Mimivirus particles at early, advanced, and final uncoating stages (particles 1, 2, and 3, respectively). At the early uncoating stage, a partial opening of the inner protein shell at the stargate assembly is observed. The opening of the stargate allows extrusion of the viral membrane toward the phagosome membrane, a stage demonstrated by particle 2. In the final uncoating stage, fusion between viral and phagosome membranes occurs, as revealed in particle 3 (Zauberman et al., 2008). (B) Schematic representation of a Mimivirus particle at its final un-coating stage. The capsid (red) is opened at the stargate, allowing for fusion of the viral and phagosome membranes (light and dark blue, respectively), thus generating a star-shaped membrane conduit. (C) Extracellular Mimivirus particle revealing a 5-fold opening. Such open stargates were detected in a small population of extracellular particles, and may represent virions that initiated infection and were released upon viral-induced lysis of host cells. Scale bars: 100 nm.

While the cues that trigger stargate opening remain unknown, (Fig. 5G and H; YM and AM, unpublished results), but the role of detection of multiple lysosomes undergoing fusion with the this engulfment requires further studies. The similarity between Mimivirus-containing phagosomes (Zauberman et al., 2008) might Vaccinia and Mimivirus is further highlighted by the involvement imply that lysosomal activity contributes to this process. Notably, of mitochondria in the infection cycles of these viruses. Mitochon- while the M. chilensis was shown to contain a stargate (Arslan dria were demonstrated to be closely associated with Vaccinia et al., 2011) and hence is likely to initiate its replication cycle in a replication sites (Tolonen et al., 2001). Our studies reveal that process similar to that revealed by Mimivirus, the ovoid-shaped early, membrane-enclosed Mimivirus replication sites include Pandoraviruses (Philippe et al., 2013) and Pithovirus (Legender mitochondria (Fig. 5G and H). While spatial proximity of mito- et al., 2014) reveal apical ‘cork-like’ morphologies. The mechan- chondria and viral replication factories is straightforwardly isms by which genomes of these giant viruses are released into the assigned to the need of extensive and continuous energy supply cytoplasm of their host cells await to be elucidated. to these factories, the mechanisms that mediate such spatial proximity remain unclear. Genome trafficking within host cells and nucleus targeting Our observations that indicate an entirely cytoplasmic Mimi- virus replication cycle resolve the quandaries associated with Realizing that the internal Mimivirus core is released into the transport of large cores or very long DNA molecules within host cytoplasm through the opening of the stargate, and in light of crowded cytoplasms as well as with nuclear import; simply, no the fact that, with the single exception of Poxviruses (Broyles, such processes are required during the Mimivirus replication 2003; Condit et al., 2006; Katsafanas and Moss, 2007; Mallardo cycle. However, issues pertaining to trafficking and nuclear import et al., 2002; Moss, 2006; Schramm and Krijnse-Locker, 2005; during viral infection remain unclear. Specifically, Pandoraviruses Tolonen et al., 2001), all DNA viruses are believed to replicate in lack genes that are essential for DNA replication and therefore host nuclei, we turned to elucidate the ensuing stage of the proposed to entail a nuclear-replication stage (Philippe et al., Mimivirus replication cycle. As discussed above, genome traffick- 2013). This claim, supported by phylogenetic analyses that imply ing toward nuclei in the highly crowded host cytoplasms, either as that Pandoraviruses represent a close derivative of Phycodnavirus genome-encapsidated large cores (see Fig. 2A) or as ‘free’ DNA (Yutin and Koonin, 2013), which are assumed to involve a nucleus- molecules, is unlikely. Indeed, our studies indicate that Mimivirus dependent replication stage (Thiel et al., 2010; Wulfmeyer et al., genome-containing cores deliver their content at or near to the 2012; Yamada et al., 2006), underlines the fact that our current cytoplasmic sites at which they were released (Mutsafi et al., 2010). understanding of intracellular transport processes of viral compo- Shortly thereafter, massive replication of viral DNA at the nents during infection cycles is lacking. As is the case for the release site is initiated (Fig. 5D–F), leading to the generation of extensively studied Vaccinia, the cues that trigger the release of early replication centers (Mutsafi et al., 2010). These early centers core contents into the host cytoplasm remain unknown. Interest- subsequently coalesce to form a single replication factory in which ingly, however, in both Vaccinia (Cyrklaff et al., 2007; Mallardo all transactions required for the production of viral progeny occur et al., 2001, 2002) and Mimivirus (Mutsafi et al., 2010), early (Howard and Moss, 2012; Mutsafi et al., 2010, 2013; Zauberman transcription occurs already within intact cores (Fig. 5B and C). et al., 2008), as previously reported for the Vaccinia virus (Cyrklaff Such intra-core synthesis of mRNA might trigger core opening and et al., 2007; Katsafanas and Moss, 2007). An additional similarity subsequent genome release into the cytoplasm through the gen- between Vaccinia and Mimivirus is concerned with the role of eration of internal pressure, but this conjecture needs to be further membranes in early viral replication processes. Specifically, it has examined. been shown that shortly after the generation of Vaccinia replica- The profoundly similar patterns that characterize the replica- tion centers these centers are engulfed by an endoplasmic reticu- tion cycles of Vaccinia and Mimivirus, which include the presence lum (ER) membrane layer and that this engulfment is essential for of cytoplasmic genome-containing cores, intra-core early tran- viral DNA replication (Tolonen et al., 2001). We detect a similar scription, membrane engulfment of early replication centers, membrane engulfment of early Mimivirus replication centers fusion of multiple early centers into a single viral factory, as well Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 9

Fig. 5. Early Mimivirus replication centers: the Vaccinia connection. (A) Multiple Mimivirus internal cores (white arrowheads) are present in the host cytoplasm at an early (2 h) post-infection stage. Scale bar: 200 nm. (B) TEM micrograph of a genome-containing core. Scale bar: 100 nm. (C) 3-Dimensional reconstruction derived from electron tomography of the core shown in (B). Yellow: core; blue: viral DNA. The non-uniform substance distribution within the core is consistent with initiation of intra-core transcription that in turn may promote genome release (Mutsafi et al., 2010). (D and E) Viral genome release into host cytoplasm and massive replication of viral DNA, respectively, resulting in the generation of early cytoplasmic replication centers. Two empty cores surrounded by replicating viral DNA are shown in (E) (Mutsafi et al., 2010). Dark regions represent viral DNA, as indicated by fluorescence studies (results not shown). Scale bars: 100 nm. (F) 3-Dimensional reconstruction derived from electron tomography of the square delineated in (E). (G) Tomogram slice revealing an (ER) membrane layer surrounding an early Mimivirus replication center. Scale bar: 200 nm. Similar membrane engulfment of early replication centers has been demonstrated during the infection cycle of Vaccinia viruses (Tolonen et al., 2001). Notably, the membrane layer is decorated by ribosomes (inset), implying that membrane engulfment of early viral replication centers is derived from cellular rough endoplasmic reticulum (RER). Our finding that early Mimivirus replication centers include mitochondria (white arrowhead) further highlights the similarity between the infection cycles of Mimivirus and Vaccinia (Tolonen et al., 2001). (H) 3-Dimensional reconstruction of the replication center shown in (G). The within the replication center is highlighted by an arrowhead. as the highly organized morphology of these factories (further Iridoviridae and Phycodnaviridae (Claverie and Abergel, 2009), discussed below), are notable. On the basis of this similarity, which Mimivirus replication cycle reveals a profound Poxviridae-like is underscored by the finding that both viruses maintain an physiology (Mutsafi et al., 2010, 2013). This physiological classifi- entirely cytoplasmic infection cycle, we have suggested that cation highlights the need for detailed structural studies of the whereas phylogenetic analyses position the Mimivirus in between replication cycles of Phycodnaviridae and Pandoraviruses that, as 10 Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 mentioned above, appear to be closely related on the basis of Indeed, the involvement of nuclear enzymes in the cytoplasmic phylogenetic analyses (Yutin and Koonin, 2013). Specifically, we replication of the Vaccinia virus has been demonstrated (Katsafanas claim that phylogenetic analyses and structural studies of mature and Moss, 2007; Oh and Broyles, 2005). extracellular virion particles must be complemented by extensive The assembly process of cytoplasmic factories of large viruses high-resolution structural studies of replication cycles in order to as well as the structure–function patterns that characterize these enable a reliable classification of viruses. organelles is enthralling. Mimivirus factories, which are generated within a short time (4–5 h) following infection, represent an elaborate de-novo generated intracellular organelle that acts as Cytoplasmic viral factories, membrane biogenesis and capsid generation an efficient ‘production line’ where all stages of viral reproduction, including DNA replication, membrane biogenesis, capsid assembly As is the case for Vaccinia, early Mimivirus replication centers and genome-encapsidation, are occurring concomitantly.Asfurther eventually fuse to produce a large cytoplasmic viral factory in discussed below, we claim that the efficacy of the viral factories that which the multiple transactions required to produce virus progeny promote rapid assembly of several hundreds viral progeny derives take place. As described above, infection of diverse DNA and RNA from a strict and well-defined compartmentalization of the multiple viruses leads to the formation of viral factories (de Castro et al., transactions within these production lines (Fig. 6A–C). Notably, our 2013; Fontana et al., 2010; Netherton et al., 2007; Netherton and structural studies of the Mimivirus factories reveal that the stargate Wileman, 2011; Novoa et al., 2005; Schramm et al., 2006; structure is consistently pointing away from the factory center Zauberman et al., 2008), yet Vaccinia, Mimivirus and presumably (Fig. 6D and E) (Zauberman et al., 2008). These observations imply members of the Megaviridae family are unique in their entirely that the formation of the stargate occurs at an early stage of the cytoplasmic replication cycles (Condit et al., 2006; Cyrklaff et al., viral assembly, and that in addition to acting as a DNA release 2007; Husain et al., 2006; Moss, 2006; Mutsafi et al., 2010, 2013; portal, the stargate might be involved in the initiation of Mimivirus Schramm and Krijnse-Locker, 2005; Zauberman et al., 2008). assembly as its generation represents a symmetry-breaking event. It should be emphasized, however, that an entirely cytoplasmic Indeed, previous studies have indicated that portals are involved in replication cycle does not imply a nucleus-independent process, as theinitiationofcapsidassemblyofHerpesvirus(Newcomb et al., nuclear factors may still be delivered to cytoplasmic factories. 2005).

Fig. 6. Mimivirus cytoplasmic factories. (A) Confocal fluorescent microscopy image showing a Mimivirus factory in the cytoplasm of an 8-h post-infected cell revealing viral DNA (DAPI, blue) surrounded by assembling virions (anti-fibril , red) (Mutsafi et al., 2013). Scale bar: 5 μm. (Β) TEM of a cytoplasmic Mimivirus factory showing icosahedral capsids generated at the factory periphery (Zauberman et al., 2008). Scale bar: 2 μm. (C) Magnification of the delineated region in (B), depicting viral assembly zones overlying TEM image. Three distinct zones are detected – viral replication, membrane biogenesis and capsid assembly zones. Such a well-defined compartmentaliza- tion of the multiple transactions within is proposed to promote the efficacy of Mimivirus factories within which rapid assembly of several hundreds viral progeny occurs. (D) SEM micrograph of a viral factory isolated 8 h post-infection. The factory is studded with viral particles at various assembly stages. (E) TEM micrograph of an intracellular factory. Notably, both imaging techniques reveal that stargates are pointing away from the factory center, implying that stargate formation occurs at an early stage of viral assembly (Zauberman et al., 2008). Scale bars in (C–E): 500 nm. Y. Mutsafi et al. / Virology 466-467 (2014) 3–14 11

Fig. 7. Mimivirus factories represent efficient ‘production lines’ that enable continuous viral membrane and capsid biogenesis. (A and B) A tomogram slice (A) and a 3-D reconstruction (B) of multivesicular membrane structures that are generated at the periphery of the Mimivirus factory and act as a continuous source for the internal viral membrane layer (Mutsafi et al., 2013). Scale bar: 50 nm. (C and D) A tomogram slice (C) and a 3D reconstruction (D) revealing an icosahedral capsid (yellow) that is progressively assembled on top of the membrane sheet (blue) and molding it into an icosahedral shape. Immunolabeling studies demonstrated that the Mimivirus protein L425 acts as a capsid scaffolding protein (Mutsafi et al., 2013). Scale bar: 100 nm. (E) Model of the generation of Mimivirus membrane and capsid biogenesis. Small membrane vesicles (blue spheres) that bud-out from host ER cisternae (elongated blue structures) diffuse toward the viral factory (VF) and form multivesicular bodies (A and B) that eventually rupture into open membrane sheets (blue lines). Icosahedral capsids (yellow) are assembled on top of these sheets (C and D) (Mutsafi et al., 2013).

The strict structural and functional compartmentalization that The last topic considered here concerns genome packaging in characterizes viral factories is clearly manifested by the pathways pre-assembled capsids of large DNA viruses. This largely uncharted that mediate the formation of the Vaccinia and Mimivirus internal territory is of interest due to the huge amount of DNA that needs membrane layer during their replication cycles (Chichon et al., to be encapsidated, as well as the fact that the ‘head-full’ packa- 2009; Chlanda et al., 2009; Condit et al., 2006; Huiskonen and ging pathway that is thought to be responsible for the accuracy of Butcher, 2007; Laliberte and Moss, 2010; Maruri-Avidal et al., genome packaging in many bacteriophages (Nurmemmedov et al., 2011; Miller and Krijnse-Locker, 2008; Mutsafi et al., 2013; Salas 2007), is unlikely to be operative in large DNA viruses. Specifically, and Andres, 2013; Suárez et al., 2013; Unger et al., 2013). as the internal space of such viruses is large enough to accom- By combining diverse imaging techniques we demonstrated that modate multiple genome copies (Claverie and Abergel, 2010; Mimivirus membrane biogenesis entails a multistage process that Legender et al., 2014; Philippe et al., 2013; Wulfmeyer et al., occurs at a well-defined zone localized at the periphery of the viral 2012), the mechanisms ensuring that only one copy is encapsi- factories. Membrane biogenesis is initiated by fusion of multiple dated are intriguing. Moreover, while packaging motors appar- small vesicles that derive from the host ER network and enable ently do not need to function against an internal pressure, they continuous supply of lipids to the membrane-assembly site. The still must mediate a unidirectional genome translocation from resulting multivesicular bodies subsequently rupture to form large viral factories toward and into viral capsids. open single-layered membrane sheets from which viral mem- Our electron tomography studies demonstrated that DNA branes are generated (Fig. 7A and B) (Mutsafi et al., 2013). Such a packaging in Mimivirus proceeds through a transient aperture rather unexpected formation of open single-layered membrane located at a distal site of the stargate site, thus revealing that, in sheets that serve as precursor for the internal membrane layer has contrast to all heretofore-characterized viruses, genome delivery also been demonstrated for Vaccinia (Chlanda et al., 2009; Suárez into the host cell and genome packaging process occur at two et al., 2013). The mechanisms that mediate multivesicular rupture distinct sites (Fig. 8)(Zauberman et al., 2008). Such a ‘division of into open membrane sheets in both Mimivirus and Vaccinia, the labor’ between genome release and encapsidation is likely to factors that stabilize these open structures in cellular aqueous contribute to the efficiency of these distinct processes. Moreover, environments, as well as the enzymatic machinery that mediates whereas the diameter of putative bacteriophage packaging portals these processes remain to be identified. was estimated to be 3 nm (barely larger than the diameter of Mimivirus membrane production is accompanied by a progres- dsDNA helix) (Bernal et al., 2003; Uchiyama and Fane, 2005), the sive assembly of icosahedral viral capsids (Fig. 7C and D) in a aperture through which DNA is packaged in Mimivirus reveals a process involving the hypothetical major capsid protein L425, diameter of 18–20 nm, thus capable of concomitantly accommo- which apparently acts as a scaffolding protein (Mutsafi et al., dating multiple DNA duplexes (Fig. 8)(Zauberman et al., 2008). 2013). The similarity between protein L425 and the Vaccinia This finding implies that DNA packaging into pre-assembled scaffolding protein D13 (Heuser, 2005; Hyun et al., 2011; Szajner capsids of large DNA viruses proceeds through condensed DNA et al., 2005) is noteworthy. Significantly, the assembly model structures, as suggested for genome delivery of the herpes simplex reveals how multiple Mimivirus progeny can be continuously virus (Shahin et al., 2006). The mechanisms that promote and efficiently generated, and further underscores the similarity DNA packaging in Phycodnaviruses, Pandoraviruses and Vaccinia between the infection cycles of Mimivirus and Vaccinia. (for which protein A32 was suggested to be involved in genome 12 Y. Mutsafi et al. / Virology 466-467 (2014) 3–14

Fig. 8. Mimivirus genome release and genome packaging occur through distinct portals. (A) Tomographic slice of a capsid undergoing DNA packaging. The DNA packaging portal and the stargate are highlighted (green and red arrowheads, respectively). Scale bar, 100 nm. (B) Volume reconstruction of the particle depicted in (A), showing the orifice through which DNA (green) is packaged, which spans the outer and inner capsid shells, as well as the internal membrane (red, yellow, and blue, respectively). The protein core underlying the membrane is shown in gray. The unprecedented presence of distinct portals for genome release and packaging was proposed to enhance the efficiency of these processes (Zauberman et al., 2008). encapsidation (Koonin et al., 1993), yet leaving the actual packaging infection cycles of diverse RNA viruses, regardless of the initial – pathway unclear (Condit et al., 2006)) remain to be elucidated. nuclear or cytoplasmic – replication site. The profound spatiotem- A related open question concerns the structural features revealed poral order revealed by these assemblies was proposed to act during genome encapsidation as well as in mature virions – do such as a central determinant that fosters their ability to rapidly features resemble those (still extensively debated) patterns char- and continuously generate multiple virus progeny (Mutsafi et al., acterizing bacteriophages (Leforestier, 2013), or do they adopt the 2013). Indeed, replication factories generated by large viruses were highly ordered morphology characterizing genomes of stationary- suggested to constitute the actual living stage of viruses (Claverie, state bacteria (Frenkiel-Krispin et al., 2001)? 2006). The mechanisms that lead to the formation of the elaborate viral factories and determine their spatiotemporal patterns thus Concluding comments: new insights and open questions represent an enticing research topic. In addition, the pathways by which huge amounts of , proteins and lipid compo- Structural and biochemical studies conducted on giant DNA nents are targeted toward viral factories need to be elucidated. viruses provided novel insights into viral replication cycles in Equally unclear are issues related to the generation of large and general, and highlighted their diversity. The initial stage of genome well defined, in terms of size and shape, capsids and the mechan- entry into host cells was suggested to proceed through phage-like isms that mediate genome packaging within pre-assembled ejection in Phycodnaviruses (Thiel et al., 2010; Wulfmeyer et al., capsids. Finally, phylogenetic analyses position Mimivirus in 2012; Yamada et al., 2006), apoptotic mimicry in Vaccinia (Mercer between Iridoviridae and Phycodnaviridae (Claverie and Abergel, and Helenius, 2008), and phagocytosis in Mimivirus (Zauberman 2009). However, observations reported throughout this essay et al., 2008), Iridoviruses (Williams et al., 2005), M. chilensis highlight the extensive similarity between the replication cycles (Arslan et al., 2011), Pandoraviruses (Philippe et al., 2013) and of Mimivirus and Vaccinia. Further analysis of the Poxviridae-like Pithovirus (Legender et al., 2014). The factors that regulate the physiology of Mimivirus may provide novel insights into the particular entry pathway need to be further elucidated. Replication evolution of giant viruses. cycles of Poxviruses and Mimivirus occur exclusively in cytoplas- High-resolution imaging studies using cutting-edge methodol- mic factories (Condit et al., 2006; Cyrklaff et al., 2007; Husain et al., ogies such as Scanning Transmission Electron (STEM) Tomography, 2006; Moss, 2006; Mutsafi et al., 2010, 2013; Schramm and Focused Ion Beam (FIB) and super-resolution light microscopy will Krijnse-Locker, 2005; Zauberman et al., 2008), whereas those of certainly continue to provide thought-provoking answers to the Phycodnaviruses and Pandoraviruses (Philippe et al., 2013)are open questions indicated above. Inclusive understanding of infec- initiated in host nuclei. Importantly, while a nucleus-located tion cycles of giant viruses will require, however, the establish- infection cycle of Pandoraviruses is mandated by the lack of genes ment of genetic and molecular biology tools that will produce essential for DNA replication (Philippe et al., 2013), as is the case novel biochemical insights and thus complement structural ana- for the genetically related Phycodnaviruses, the factors that lyses. For ameba cells that act as a common host of giant viruses a-priori determine the actual site of viral replication remain such tools are not yet available. unclear. The mechanisms responsible for the translocation of the huge genomes of these viruses toward and into host nuclei similarly need to be resolved. 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