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Papers in Pathology Plant Pathology Department

5-2010

DNA : The Really Big Ones (Giruses)

James L. Van Etten University of Nebraska-Lincoln, [email protected]

Leslie C. Lane University of Nebraska-Lincoln, [email protected]

David Dunigan University of Nebraska-Lincoln, [email protected]

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Van Etten, James L.; Lane, Leslie C.; and Dunigan, David, "DNA Viruses: The Really Big Ones (Giruses)" (2010). Papers in Plant Pathology. 203. https://digitalcommons.unl.edu/plantpathpapers/203

This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in Annual Review of Microbiology 64 (2010), pp. 83–99; doi: 10.1146/annurev.micro.112408.134338 Copyright © 2010 by Annual Reviews. Used by permission. http://micro.annualreviews.org

Published online May 12, 2010.

DNA Viruses: The Really Big Ones

(Giruses)

James L. Van Etten,1,2 Leslie C. Lane,1 and David D. Dunigan 1,2

1. Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, Nebraska 68583 2. Nebraska Center for Virology, University of Nebraska–Lincoln, Lincoln, Nebraska 68583 Corresponding author — J. L. Van Etten, email [email protected]

Abstract Viruses with greater than 300 kb and up to 1200 kb are being discovered with increas- ing frequency. These large viruses (often called giruses) can encode up to 900 and also many tRNAs. Consequently, these viruses have more -encoding genes than many bac- teria, and the concept of small particle/small that once defined viruses is no longer valid. Giruses infect and although most of the recently discovered ones in- fect . Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and ) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseil- levirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, - style, and genome structure. This review focuses on some of these differences rather than pro- vides extensive details about individual viruses. Keywords: algal , phycodnavirus, , White spot virus, jumbo phage, NCLDVs

83 84 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010)

Contents genomes ranging from 100 to 280 kb, includ- ing herpesviruses, asfarviruses, baculovi- Introduction...... 84 ruses, iridoviruses, and ascoviruses, and also are not discussed in this review. Discovery of Large Viruses ...... 86 To put the size of these large viral ge- Large DNA Virus Families ...... 86 nomes into perspective, the smallest free- Brief Descriptions of Some Large Viruses ...88 living bacterium, , ...... 88 encodes 470 proteins (18). Although esti- mates of the minimum genome size required ...... 90 to support life are ~250 protein-encoding Nimaviridae ...... 94 genes (45), a symbiotic bacterium (Candida- ...... 95 tus Hodgkinia cicadicola) in the cicada Dici- ceroprocta semicincta has a 145-kb genome Concluding Remarks...... 95 (37). Thus, giruses have more protein-en- coding genes (CDSs) than some single-celled organisms. Given the coding capacity of these large viruses, it is not surprising that they encode many proteins that are atypical of or novel for a virus. However, the majority of their Introduction CDSs do not match anything in the data- bases. Some of these viruses also encode in- Typically, one views viruses as small par- trons and inteins, which are uncommon in ticles that readily pass through 0.2-μm filters viruses. A type IB exists in several and contain small genomes with a few pro- phycodnaviruses (66). Mimivirus has six tein-encoding genes. However, huge viruses self-excising (9). Because introns are with large dsDNA genomes that encode hun- often detected when they interrupt coding dreds of proteins, often called giruses, are sequences of known proteins, additional in- now being discovered with increasing fre- trons located within anonymous virus CDSs quency. This review concentrates on viruses will probably be discovered. Inteins are pro- with genomes in excess of 300 kb and focuses tein-splicing domains encoded by mobile in- on partially characterized viruses with anno- tervening sequences, and they catalyze their tated genomes (Table 1; annotated genomes own excision from the host protein. Although in the public domain have an accession num- found in all domains of life, their distribution ber). Most of these viruses inhabit aquatic en- is sporadic. Mimivirus and phycodnaviruses vironments and infect protists. Examples in- NY-2A and CeV01 are among the few intein- clude Mimivirus, which infects amoebae and containing viruses. has the largest genome (~1.2 Mb); viruses that The morphogenesis of large viruses is infect algae (phycodnaviruses) and have ge- also interesting because presumably they nomes up to ~560 kb; viruses that infect bac- are too large to self-assemble. Furthermore, teria and have genomes up to ~670 kb; and structures of giruses vary significantly Fig ( - White spot shrimp virus (WSSV), which has a ure 1). Therefore, these viruses must encode genome of ~305 kb. At least one member of proteins that measure size and other proteins the poxvirus family has a genome larger than that serve as assembly catalysts or scaffolds. 300 kb (canarypox virus – 360 kb); however, One issue that is perhaps not appreciated most poxviruses have genomes ranging from by the microbiology community is that large 180 to 290 kb and therefore we have not dis- viruses, even ones classified in the same fam- cussed canarypox virus in this review. The ily, can differ significantly in morphology, are enigmatic with respect to lifestyle, and genome structure. This review WSSV: White spot genes and particle structure, means of repli- focuses on some of these differences and shrimp virus cation, and transmission (see sidebar, Polyd- only provides brief details about individual CDS: protein-encoding naviruses); these are not considered further. viruses. See noted comprehensive reviews gene Other large, dsDNA-containing viruses have for more information about specific viruses. DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 85 Plantae Plantae Plantae Plantae Plantae Plantae Plantae Animalia Animalia Animalia Kingdom Protozoa Protozoa Protozoa Chromista Chromista Chromista Chromista Chromista Chromista Chromista Proteobacteria a Yes Yes Yes Yes Yes Yes Yes Yes N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A None Lipids Yes (I) Yes Yes (I) Yes (I) Yes Yes (I) Yes Yes (I) Yes Yes (I) Yes Yes (I) Yes Yes (I) Yes Protozoa

Yes Yes 122 500 160 220 197 250 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 190 (nm) Virion Host 160–190 150–190 300 N/A 222×180 diameter Nucleocapsid

N/A Helical Helical Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric Isometric symmetry Nucleocapsid Nucleocapsid 7 6 9 1 1 6 7 11 10 N/A N/A N/A N/A N/A N/A N/A N/A N/A genes None None tRNA None 22 N/A Isometric multipartite 366 404 360 335 328 461 911 240 472 331 531 156 457 N/A N/A N/A N/A N/A N/A N/A N/A N/A CDS 543 N/A Predicted Predicted

c N/A N/A N/A N/A N/A N/A N/A N/A N/A Shape Shape Linear Linear Linear Linear Linear Linear Linear Linear Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular % 90 92 86 70 27 92 93 90 93 90 89 92 N/A N/A N/A N/A N/A N/A N/A N/A N/A coding 90 N/A Circular Circular % 39 40 27 51 34 40 44 30 23 45 40 45 41 45 N/A N/A N/A N/A N/A N/A N/A N/A G+C 40–52

28 331 369 336 568 345 321 360 618 317 340 466 560 407 510 485 321 340 356 670 368 Size (kb) 305 1181

b

2 6 28 53 53 69 28 47 13 1200 number number personal Accession Reference Reference M. Fischer, M. (NCBI) or DQ491001 NC_000852 NC_009898 NC_006450 NC_003225 NC_002687 NC_009899 NC_008603 NC_005309 NC_010821 NC_007346 NC_006633 communication

virus virus

1 virus 1 virus a virus 1 virus 01 phage G virus 1 2-1 φ 34 PBCV-1 PBCV-1 NY2A MT325 ) ) EsV-1 EhV86 ( Canarypox virus (CNPV) member Type Acanthamoeba polyphaga Mimivirus (APMV) Pseudomonas chlororaphis phage 201 Bacillus megaterium White spot shrimp virus (WSSV1) Chlorovirus AR158 Chlorovirus FR483 Chlorovirus Chlorovirus Ectocarpus siliculosus virus 1 ( virus 86 Chrysochromulina ericina 1(CeV01) orientalisPyramimonas (PoV-01) Phaeocystis globosa 1) (PgV group Phaeocystis pouchetii (PpV-01) Cafeteria roenbergensis 1) (CroV circularisquamaHeterocapsa virus (HcDNAV) Ectocarpus fasciculatus (EfasV) Myriotrichia clavaeformis a (MclaV) (CcBv) Giruses and their properties Table 1. Table Classification Genome Virus family Mimiviridae Nimaviridae Phycodnaviridae Polydnaviridae N/A with the virion and internal to nucleocapsid. found lipids are Yes, (I), Yes a. NC_006633 to NC_006662. from inclusive are CcBV genome accessions numbers b. segments of dsDNA. The CcBV genome exists as 30 circular c. 86 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010)

through 0.2-μm-pore filters to remove bac- Polydnaviruses teria and protists. However, these filters also often exclude large viruses. Standard meth- Polydnaviruses that infect thousands of species of endoparasitic ods for plaquing bacteriophage missed large have complex lifestyles and large, multipartite genomes. phages because the high soft agar concentra- Two genera (Bracovirus and ) represent this family, tions prevented formation of visible plaques and these viruses manipulate the defenses, development, and (54). In addition, large viruses may grow physiology of the parasitized lepidopteran larval hosts, where more slowly than smaller viruses and have the virus facilitates a symbiotic or mutualistic condition of the lower burst sizes. Large viruses have larger larvae with an otherwise resistant lepidopteran host. The surface areas and are thus more likely to ag- gregate and/or adsorb to extraneous mate- viruses are evolutionarily linked to the family (3). The rial. None of these issues is a problem as long life cycle of polydnaviruses life cycle may be the most complex as one is aware of them. Second, the hosts known in virology. Upon the virus integrates into the for some of these large viruses were not ex- genome of specialized cells of the wasp’s ovaries and is carried amined for virus until recently. Fi- in the parasitic eggs and larvae as a , as well as circular nally, the discovery of some of these large vi- episomal DNA within virions. In both wasp and lepidopteran cells, ruses was serendipitous; e.g., Mimivirus was the virus has a closed circular DNA that replicates in the nucleus. initially believed to be a parasitic bacterium In the case of , replication and particle production (9). occur only in the ovaries of the wasp and the virus is transmitted It is now obvious that many large vi- ruses await discovery. For example, Monier vertically, yet the virions contain no bracovirus structural proteins. et al. (41) recently conducted a metagenomic Rather, the structural components are derived from a baculovirus. study using samples collected on the Sor- Another distinctive feature of the polydnaviruses is their low cerer II Global Ocean Sampling Expedition coding density (~27%) compared to other giruses, which are to determine the relative abundance of DNA typically ~90%. In addition, their protein-encoding genes have polymerase fragments that could be assigned little relationship to free replicating viruses. Thus, polydnaviruses to virus groups. In 86% of sample sites, Mim- blur the distinction of viruses as obligate parasites and challenge ivirus relatives were the most abundant, af- virologists’ understanding of the role of viruses in nature, where ter . This high abundance a virus appears to be domesticated by a cellular organism for the suggests that Mimivirus-like viruses may infect other marine protists (11). In another sake of obligate mutualism (4). metagenomics study using three other pro- teins as the queries, phycodnaviruses were commonly found (31).

Large DNA Virus Families Discovery of Large Viruses Many of the viruses listed in Table 1 probably have a common evolutionary an- Most large viruses have been discov- cestor, perhaps arising before the diver- ered and characterized in the last few years. gence of the major eukaryotic kingdoms (26, Two exceptions are bacteriophage G, ini- 51, 64, 71). These viruses, which include the tially described about 40 years ago (13) and phycodnaviruses, poxviruses, asfarviruses, largely ignored until recently, and the chlo- iridoviruses, ascoviruses, and the Mimivi- rella virus Paramecium bursaria chlorella vi- ruses, are referred to as nucleocytoplasmic rus 1 (PBCV-1), which was first described large dsDNA viruses (NCLDVs) (25, 26). Re- in 1982 (62) and has since been studied cently, another large NCLDV, named Mar- PBCV-1: Paramecium continuously. seillevirus, that is distantly related to the iri- bursaria chlorella virus 1 Large viruses went undetected for many doviruses and ascoviruses was isolated from NCLDV: nuclear years for several reasons. First, there were an amoeba (6). NCLDVs contain 9 common cytoplasmic large DNA technical problems. For example, classical genes, and 177 additional genes are present virus virus isolation procedures include filtration in at least two of the virus families (71). DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 87

Figure 1. (a) Transmission electron micrograph of Mimivirus. (b) Atomic force microscopy of defibered Mimivirus. The unique star-faced ver- tex is clearly visible. (c) Fivefold averaged cryo-electron micrographs of virus PBCV-1 reveal a long, thin, cylindrical spike structure at one vertex and protrusions (fibers) extending from one unique capsomer per trisymmetron. (d) Central cross section of panel c. Note the gap between the unique vertex and the membrane enclosing the DNA. Also the unique vertex contains a portal-like protein. (e) PBCV-1 attached to the cell wall as viewed by the quick-freeze, deep-etch procedure. Note fibers attach the virus to the wall. ( f ) Transmission electron micrograph of EhV. (g) Schematic of freshly isolated EhV (left) and stored EhV (right). Note the external membrane swells with age. (h, i) Morphology of the White spot shrimp virus (WSSV) virion. (h) Negative contrast electron micrograph of intact WSSV virion with its tail-like extension. (i ) Schematic based on panel h showing the layered structures of a WSSV virion, i.e., envelope, tegument, and nucleocapsid. ( j ) Electron micrograph of bacteriophage G. The insert shows coliphage lambda to the same scale. Panel a from Reference 10, b from Reference 72, c and d from Reference 7, e from Refer- ence 61, f and g from Reference 36, h and i from Reference 35, and j from Reference 24—all published with permission.

Although the hypothesis of a common suggested that NCLDVs should be consid- ancestor for the NCLDVs is generally ac- ered the fourth kingdom of life (12, 51), oth- cepted, there is disagreement on the size ers have suggested that NCLDV genes arose and morphology of its ancestral virus and from the original gene pool that led to pro- how it diverged into the different virus fam- karyotes and (26), and still others ilies. A recent maximum-likelihood recon- have suggested that horizontal gene trans- struction of NCLDV evolution produced a fer has driven the evolution of their genomes set of 47 conserved genes, which are consid- (39). These suggestions have stimulated con- ered the minimum genome for the common troversy over whether the tree of life should ancestor; NCLDVs then evolved by losing include these viruses.1 Another interesting some of these common genes and acquiring hypothesis is that primitive NCLDVs gave new genes from their hosts and bacterial en- rise to the eukaryotic nucleus or vice versa dosymbionts as well as by gene duplications (5). Regardless, some viruses, including (71). Another scenario suggests the ancestral the NCLDVs, have a long evolutionary his- NCLDV was a huge virus or even a cellular tory, and viruses probably contributed to the organism that evolved primarily via genome emergence and subsequent structure of mod- contraction (51). Finally, Filee et al. (15) pro- ern cellular life forms (29). posed that NCLDVs evolved from a small DNA virus by gene acquisition from cells. The origin of NCLDVs is controver- 1. For example, References 8, 52, 40, and comments sial. For example, some researchers have by seven others in Nat. Rev. Microbiol. 2009. 7:614–27 relate to this discussion. 88 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010)

It is becoming difficult to classify some of Brief Descriptions of Some Large MCP: major these large viruses into distinct families. Re- Viruses protein cent phylogenetic analysis of the DNA poly- LPS: lipopolysaccharide merase protein from four putative phycodna- Mimiviridae viruses illustrates this problem. (a) The DNA polymerase from three phycodnaviruses, Mimivirus, Mamavirus, and Marseillevi- CeV01, PpV01, and PoV01 (Table 1), is more rus all infect amoebae; the first two are the similar to Mimivirus than to the other phy- largest viruses ever reported (9). Mamavi- codnaviruses (41). (b) The HcDNAV poly- rus has an 18.3-kb DNA virus (called merase indicates its closest relative is African Sputnik) that can only replicate in the pres- swine fever virus (50). Therefore, it is clear ence of Mamavirus (34). that giruses, like the DNA phages (23), have Mimivirus virions have an icosahedral exchanged genes for eons. core capsid with a diameter of ~500 nm. The Contributing to the uncertainty about capsid is uniformly covered with a 140-nm- NCLDV evolution is the discovery that the thick layer of closely packed fibers, forming structure of the PBCV-1 major capsid pro- an ~750-nm spherical object (Figure 1a) (67). tein (MCP) resembles MCPs from other This peripheral fiber layer, which is absent in smaller dsDNA viruses with hosts in all other NCLDVs, might be linked to the het- three domains of life, including human ade- erotrophic nature of the host amoeba; i.e., noviruses, bacteriophage PRD1, and a virus Mimivirus particles might mimic the bacteria infecting an archaeon, Sulfolobus solfataricus. on which amoebae prey. For amoebae to ini- This similarity suggests that these three vi- tiate phagocytosis, individual particles have ruses might have a common evolutionary to be larger than 600 nm in diameter or ag- ancestor with the NCLDVs, despite the lack gregate before being engulfed (30). The ex- of sequence similarity among ternal fiber layer might also serve another their MCPs (32). role. After engulfing bacteria, bacterial sur- All NCLDVs are assembled in virus fac- face lipopolysaccharides (LPSs) stimulate en- tories located in the cytoplasm. The role of docytic vesicle formation in amoebae. There- the nucleus in the replication of NCLDVs fore, the outer layer of Mimivirus particles, varies. For example, poxviruses (43) and which stains gram positive (51), might re- Mimivirus (46) carry out their entire life semble the gram-positive bacterial surface cycle in the cytoplasm. In contrast, the nu- LPSs. Consistent with this hypothesis, Mim- cleus is probably essential for replication of ivirus encodes several sugar-manipulating the phycodnaviruses and other NCLDVs. enzymes and some of them are directly re- However, the nuclear role in virus repli- lated to surface LPS-specific sugars, such as cation probably differs, even among the perosamine (9). phycodnaviruses. Electron tomography and volume-re- The four viruses in Table 1 that are not construction analyses of viral particles in- NCLDVs are a , WSSV, and side infected amoeba cells at final infection two bacteriophages, PhageG and 201φ2-1. stages establish that the Mimivirus capsid WSSV, which causes huge economic losses is composed of two superimposed shells to the shrimp industry, is an enigma because with different densities (72). The inside it is not obviously related to known viruses. of the virus particle has a membranous Large bacteriophages, referred to as jumbo sac enveloping the viral genome. In addi- phages, resemble smaller phages that may tion to the two shells, a prominent fivefold have acquired increased genome functions star-shaped structure is located at one ico- over evolutionary time. sahedral vertex and extends along the en- tire length of the five icosahedral edges that center around this unique vertex (Fig- ure 1b) (46, 56, 67, 72). Mimivirus initiates infection by phagocytosis followed by lys- DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 89 osome fusion with the phagosomes. This separated by an average of 157 bp. In con- p.i.: post infection lysosomal activity is predicted to open the trast to some other NCLDV members, Mim- ORF: open reading viral capsid at the stargate portal. The fu- ivirus genomic termini lack large inverted frame sion of the particle’s internal membrane repeats. Instead the Mimivirus genome has with the endocytic vacuole membrane a 617-bp inverted repeat beginning at nu- forms a large membrane conduit through cleotide position 22,515; its unique comple- which the genome-containing Mimivirus mentary counterpart begins at nucleotide core enters the cytoplasm. Although initial position 1,180,529 (9). The extreme conser- studies suggested the Mimivirus genome vation of these intergenic regions suggests moved into the nucleus and shuttled back they serve an important role in Mimivirus to the cytoplasm following a few rounds of replication. Pairing these regions produces replication (56), a more recent study indi- a putative Q-like form in the genome, with cates its genome remains in the cytoplasm a long (22,514 bp) and a short (259 bp) tail. and that, like the poxviruses, the entire The short tail region has no CDSs. The long replication cycle takes place in the cyto- tail region has a lower coding density than plasm (9, 46). Like poxviruses, Mimivirus the rest of the genome (75% versus 90.5%) possesses its own machinery and larger intergenic spacers (435 bp versus and it packages 12 transcription proteins in 157 bp on average). This region encodes 12 Mimivirus particles. Transcription of early proteins, 7 of which are involved in DNA Mimivirus genes, in conjunction with a replication. conserved promoter element AAAATTGA, Thirty-three percent of the Mimivirus is believed to occur in the core particles. genes are related to at least one other Mim- The cores release virus DNA, forming cy- ivirus gene because of gene duplications toplasmic replication factories where virus (55). Thirty-six of the Mimivirus 910 CDSs DNA replication begins, followed by tran- are associated with functions not previously scription of late genes. The replication fac- found in a virus (9). For instance, Mimivi- tories form around the viral cores and ex- rus possesses a complete set of DNA re- pand until they occupy a large fraction of pair enzymes capable of correcting nucleo- the amoeba cell volume at 6 h post infec- tide mismatches as well as errors induced tion (p.i.). by oxidation, UV irradiation, and alkylat- Later stages of the Mimivirus replication ing agents. Mimivirus is also the only virus cycle occur from 6 to 9 h p.i., when empty fi- to encode three topoisomerases. In addition, berless procapsids, which are only partially Mimivirus encodes a variety of polysaccha- assembled, as well as icosahedral procap- ride-, amino acid–, and lipid-manipulat- sids undergoing DNA packaging, appear at ing enzymes. Such metabolic capabilities, the periphery of the large replication facto- although covering a broader biochemical ries. A statistical survey of particles under- spectrum in Mimivirus, also exist in other going DNA packaging indicated that 60% of NCLDVs, especially the phycodnaviruses the particles package DNA through a face- (66), where they often vary among isolates. centered rather than a vertex-centered aper- Probably the most unexpected discovery in ture. Thus, Mimivirus DNA exit and pack- the Mimivirus genome was finding homo- aging proceeds through different portals, logs to 10 translation-related proteins (9). Fi- which is a unique feature among viruses nally, Mimivirus, like the chlorella viruses, (72). encodes several putative glycosyltransfer- The Mimivirus linear ~1.2-Mb ge- ases that might help glycosylate its MCP. nome encodes ~910 predicted CDSs and Proteomic analysis of the Mimivirus vi- six tRNA genes. Despite its much larger ge- rion identified 114 virus-encoded proteins, nome, Mimivirus has a high coding density including the transcription proteins men- (90.5%) similar to that of other NCLDVs. tioned above. Adjacent open reading frames (ORFs) are 90 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010)

PFU: plaque forming Phycodnaviridae Cryo-electron microscopy and 3D im- unit Three viruses, PBCV-1, EhV, and EsV, age reconstruction of PBCV-1 indicate the are selected to represent the Phycodnaviridae outer capsid is icosahedral and covers a sin- family. These three viruses group into a sin- gle lipid bilayered membrane, which is re- gle family (14, 66) and at first glance appear quired for infection. The capsid shell consists to be more similar than they actually are. As of 1680 donut-shaped trimeric capsomers noted below, the apparent long evolutionary plus 12 pentameric capsomers, one at each history of these three viruses has led to ma- icosahedral vertex. The trimeric capsomers jor differences in propagation strategies (lytic are arranged into 20 triangular facets (tri- versus lysogenic), virus release (lytic versus symmetrons, each containing 66 trimers) and budding), and virus structure (unique ver- 12 pentagonal facets (pentasymmetrons, each tex with a spike versus probably no spike). containing 30 trimers and one pentamer at The fact that these three viruses only have 14 the icosahedral vertices) (Figure 1c). PBCV- common genes provides additional evidence 1 has a triangulation number of 169d quasi- of their long evolutionary history. Thus, over equivalent lattice (66). 1000 different genes exist just among these Recent fivefold symmetry averaging 3D three phycodnaviruses! reconstruction experiments revealed that one of the PBCV-1 vertices has a cylindrical spike, 250 ˚A long and 50 ˚A wide (Figure 1c) (7). A pocket exists between the inside of Chlorella viruses. The chlorella viruses the unique vertex and the enveloped nucleo- (genus Chlorovirus) infect symbiotic chlo- capsid; i.e., the internal virus membrane de- rella, often called zoochlorellae, which are parts from icosahedral symmetry adjacent to associated with the protozoan Paramecium the unique vertex (Figure 1d). Consequently, bursaria, the coelenterate Hydra viridis, and the virus DNA located inside the envelope the heliozoon Acanthocystis turfacea (58, 68). is packaged nonuniformly in the particle. Paramecium bursaria chlorella virus (PBCV- The PBCV-1 MCP is a glycoprotein and com- 1) is the type member of the genus (61). prises ~40% of the total virus protein. The The zoochlorellae are resistant to virus in- MCP consists of two eight-stranded, antipar- fection in the symbiotic state. Fortunately, allel β-barrel jelly-roll domains related by a some zoochlorellae can be grown indepen- pseudo-sixfold rotation (48). dently of their hosts, permitting plaque as- External fibers extend from some of the say of the viruses and synchronous infec- trisymmetron capsomers (probably one per tion of their hosts. Therefore, one can study trisymmetron) and may facilitate attachment the virus replication cycle in detail. The to the host (Figure 1c, e). The spike at the 46.2-Mb genome of the PBCV-1 host Chlo- unique vertex is too thin to deliver DNA and rella NC64A was sequenced recently by the so it probably aids in penetration of the wall. Department of Energy Joint Genome Insti- PBCV-1 initiates infection by attaching rap- tute and its genome annotation is publicly idly and specifically to the Chlorella NC64A available. Availability of both host and vi- cell wall (58), probably by the fibers men- rus sequences makes chlorella viruses a fa- tioned above (7). Following host cell wall vorable model system. degradation by virus-packaged enzyme(s), Freshwater throughout the world con- the viral internal membrane presumably tains chlorella viruses with titers as high as fuses with the host membrane, facilitating 100,000 plaque-forming units (PFUs) per mil- entry of the viral DNA and virion-associated liliter of native water, although typically, vi- −1 proteins into the cell, leaving an empty cap- rus titers are 1–100 PFU ml . The titers fluc- sid attached to the surface (57). This fusion tuate during the year, with the highest titers process initiates rapid depolarization of the occurring in the spring. Although chlorella host membrane and the rapid release of K+ viruses are ubiquitous in freshwater, little is from the cell. The rapid loss of K+ and associ- known about their natural history. For exam- ated water fluxes from the host reduce its tur- ple, do they have another host? DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 91 gor pressure, which may aid ejection of viral The PBCV-1 genome is a linear, ~334-kb, 5 mC: 5-methylcytosine DNA and virion-associated proteins into the nonpermuted dsDNA molecule with cova- 6 mA: N6- host. Depolarization may also prevent infec- lently closed hairpin termini. Identical ~2.2- methyladenine tion by a second virus (22). kb inverted repeats flank each hairpin end. PBCV-1 lacks a recognizable RNA poly- The remainder of the PBCV-1 genome con- merase gene, and so circumstantial evidence tains primarily single-copy DNA. Of the 365 suggests PBCV-1 DNA and DNA-associ- predicted PBCV-1 CDSs, ~35% resemble pro- ated proteins quickly move to the nucleus, teins of known function, including many that where early transcription begins 5 to 10 are novel for a virus (e.g., hyaluronan syn- min p.i. (66). In this immediate-early phase thase, K+ channel protein, and four poly- of infection (5– 10 min p.i.), host transcrip- amine biosynthetic enzymes). PBCV-1 CDSs tion machinery is reprogrammed to tran- are evenly distributed on both DNA strands scribe viral DNA. Details of reprogramming with minimal intergenic spaces. Exceptions are unknown, but host chromatin remodel- to this rule include a 1788-nucleotide se- ing is probably involved. PBCV- 1 encodes quence in the middle of the PBCV-1 genome a SET domain–containing protein (referred that encodes 11 tRNAs [cotranscribed as a to as vSET) that methylates Lys-27 in his- large precursor and then processed to ma- tone 3. vSET is packaged in the PBCV- 1 vi- ture tRNAs (68)]. rion, and circumstantial evidence indicates Most chlorella virus genomes contain vSET helps to repress host transcription fol- methylated bases. For example, genomes lowing PBCV-1 infection (44). In addition, from 37 sampled chlorella viruses have 5- host chromosomal DNA degradation begins methylcytosine (5 mC) in amounts ranging within minutes after infection, presumably from 0.12 to 47.5% of the total cytosine. In by PBCV- 1-encoded and packaged DNA addition, 24 of the 37 viral contain N6- restriction endonuclease( s) (1). This degra- methyladenine (6 mA) in amounts ranging dation also inhibits host transcription and from 1.5 to 37% of the total adenine (61). The facilitates recycling of nucleotides for viral methylated bases occur in specific DNA - se DNA replication. quences, which led to the discovery that the Viral DNA replication begins 60 to 90 chlorella viruses encode multiple 5 mC and 6 min p.i. and is followed by transcription of mA DNA methyltransferases. About 25% of late genes (58). Approximately 2 to 3 h p.i., the virus-encoded DNA methyltransferases assembly of virus begins in local- have companion DNA site-specific (restric- ized regions in the cytoplasm, which become tion) endonucleases, including some with prominent 3 to 4 h p.i. Five to 6 h p.i. the cy- unique cleavage specificities (61).2 toplasm fills with infectious progeny virus Five additional chlorella viruses have particles, and localized lysis of the host cell been sequenced, including two more viruses releases progeny at 6–8 h p.i. Each cell re- (NY-2A and AR158) that infect the same host leases ~1000 particles, of which ~30% are as PBCV- 1, Chlorella NC64A; two (MT325 infectious. and FR483) that infect Chlorella Pbi; and one Global transcription of PBCV-1 genes (ATCV-1) that infects Chlorella SAG 3.83 (66). during virus replication (70) indicate that Approximately 80% of the genes are com- (a) 98% of the 365 PBCV-1 protein-encoding mon to all six sequenced chlorella viruses, genes are expressed in laboratory conditions, suggesting they are essential for virus repli- (b) 63% of the genes are expressed before 60 cation. However, the number of chlorella-vi- min p.i. (classified as early genes), (c)37% rus-encoded genes is much larger than those of the genes are expressed after 60 min p.i. present in any one virus. Not surprisingly, (classified as late genes), and (d) 43% of the early gene transcripts are also detected at late times following infection (classified as early/ 2. The chlorella viruses were the first nonbacterial late genes). source of DNA restriction endonucleases. 92 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010) orthologs from viruses infecting the same sulfur cycles, as well as influencing the cli- host are the most similar; the average amino mate (20). It is now generally accepted that acid identity between orthologs from PBCV- the Emiliania huxleyi virus (EhV) contributes 1 and NY-2A or AR158 is ~73%. PBCV-1 and to the collapse of these blooms (66). MT325 or FR483 orthologs have ~50% amino E. huxleyi has two phenotypes in its hap- acid identity, and PBCV-1 and ATCV-1 or- lodiploid lifestyle. The diploid calcified phase thologs have ~49% amino acid identity. Us- forms algal blooms; this form is infected by ing PBCV-1 as a model, there is high synteny EhV (genus ). In contrast, the between the three viruses that infect Chlorella ecological status of the noncalcified haploid NC64A. In contrast, PBCV-1 has only slight phase is largely unknown. However, haploid synteny with the two Pbi viruses and the cells are resistant to EhV (17). SAG virus (16). Currently, no detailed structural studies Many PBCV-1-encoded enzymes are ei- exist for the icosahedral EhV virion (Figure ther the smallest or among the smallest pro- 1f, g). However, the initial assumption that teins in their family. Phylogenetic analyses it is structurally similar to PBCV-1 is proba- indicate some of these minimalist proteins bly incorrect because the EhV capsid is sur- are potential evolutionary precursors of rounded by an external lipid membrane more complex cellular proteins. Despite their and it infects its host by fusion with the host small size, the virus enzymes typically have plasma membrane and enters by endocytosis all the catalytic properties of larger enzymes. (36). In contrast, PBCV-1 uncoats at the sur- Their small size and the fact that they are of- face of the cell wall. ten laboratory friendly have made them ex- EhV has a different propagation strategy cellent models for mechanistic and structural than either the lytic chlorella viruses or the studies (66). latent EsV-1 viruses. The host alga E. huxleyi The are also unusual be- is covered with a calcium carbonate shell that cause they encode enzymes involved in would appear to create a physical barrier to sugar metabolism. For example, two PBCV- virus adsorption. However, despite this bar- 1-encoded enzymes synthesize GDP-L-fu- rier, virus adsorption to the host membrane cose from GDP-D-mannose (19), and three is rapid and intrinsically linked to the host enzymes contribute to the synthesis of hy- cell cycle (36). Real-time fluorescence micros- aluronan, a linear polysaccharide typi- copy revealed that EhV-86 rapidly enters its cally found in (68). All three host intact via either an endocytotic or an en- genes are transcribed early during PBCV- velope fusion mechanism where it rapidly 1 infection and hyaluronan accumulates disassembles. on the external surface of the infected chlo- Whereas both the chlorella viruses and rella cells. In addition, PBCV-1 encodes at EsV- 1 depend on host transcription machin- least five putative glycosyltransferases that ery, EhV is unique among the phycodnavi- likely participate in glycosylating the virus ruses because it has six RNA polymerase-en- MCP (60).3 coding genes (65). These genes suggest some virus independence from the host nucleus. Viral transcription begins immediately after Emiliania huxleyi virus. The infection, but it is limited to a specific 100- Emiliania huxleyi is a globally important uni- kb region, containing ~150 CDSs, of the vi- cellular marine phytoplankton. The alga rus genome. This 100- kb region contains a forms huge blooms that extend over 100,000 unique promoter element, and only the genes km2 and it is important in ocean carbon and transcribed during the first hour p.i. contain

this element. Thus, these CDSs undoubtedly

play a crucial and integral role early during

3. PBCV-1 was the first virus reported to encode virus infection. However, none of these CDSs

most, if not all, of the machinery to glycosylate matches anything in the databases. its MCP. DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 93

Intriguingly, proteomic analysis did not gal derived GSLs. The viral GSLs accumulate GSL: glycosphingolipids detect any transcriptional proteins in mature in the , and it is hypothesized PCD: programmed cell EhV-86 virions; therefore, host nuclear RNA that this is a mechanism to activate virus re- death polymerase(s) is presumably responsible for lease and subsequently induce PCD in sur- early transcription (66). Between 1 and 2 h rounding algal cells during natural blooms p.i., a second transcription phase begins with as a type of quorum-sensing device, termi- gene expression occurring from the remain- nating the bloom (63). This example of cell der of the genome. Because viral RNA poly- signaling by the E. huxleyi/EhV interaction merase components are expressed in this sec- suggests that aquatic viruses are very much ond phase, viral replication may no longer in control of their environment in ways virol- be nuclear dependent and transcription may ogists and ecologists are only just beginning occur in the cytoplasm. At ~4.5 h p.i., virus to fathom. progeny begin to be released via budding, during which EhV-86 virions become envel- Ectocarpus siliculosus virus. Ectocarpus silic- oped with host plasma membrane. Therefore, ulosus virus 1 (EsV-1) is the type species for unlike chlorella viruses, for which nascent in- the genus and its infection strat- fectious virions accumulate in the cytoplasm egy is regarded as typical for the genus (59, prior to release by cell lysis, EhV virions are 66). Collectively, Phaeovirus members infect released gradually (36). freeswimming, wall-less gametes or spores The EhV-86 407-kb genome, encoding 472 of filamentous marine brown macroalgae (or- CDSs, was originally thought to be linear. der Ectocapales, class Phaeophyceae) by fus- PCR amplification over the termini revealed ing with the host plasma membrane. Their a random A/T single nucleotide overhang hosts are members of benthic communities (50% A, 50% T), suggesting the virus genome in near-shore coastal environments in all the has both linear and circular phases. EhV-86 world’s oceans. Phaeovirus DNAs are inte- has three repeat families (none of which is lo- grated into the host genome and are passed cated at the ends of the genome); one repeat to daughter cells during cell division. The family is postulated to act as a replication or- EsV-1 genome persists as a latent infection igin (suggesting a circular form of DNA rep- in vegetative cells, and infected algae show lication), another family is postulated to con- no obvious growth or developmental de- tain immediate-early promoter elements, and fects, except for partial or total inhibition of the last family has a large repetitive proline- their reproductive organs. The viral genome rich domain that may bind calcium (66). is only expressed in sporangia and gametan- EhV-86 also has some unusual CDSs, in- gia cells, where the cellular organelles disin- cluding an entire metabolic pathway of seven tegrate and are replaced with densely packed genes encoding sphingolipid metabolic en- viral particles. Environmental stimuli, such zymes (65). The host also contains genes en- as temperature and light, cause lysis of re- coding this entire pathway and it is clear that productive organs, synchronously releasing occurred between spores or gametes as well as viruses. Pha- EhV and E. huxleyi (42). However, the direc- eoviruses are the only known phycodnavi- tion of the transfer is not obvious. This bio- ruses to infect members of more than one al- synthetic pathway appears to function dur- gal family. ing lytic infection and the glycosphingolipids EsV-1 has a linear dsDNA genome with (GSLs) produced induce programmed cell almost perfect inverted repeats at each end death (PCD) with corresponding activation that allows circularization. Indeed, before se- of an algal metacaspase, an essential activ- quencing, EsV-1 was thought to have a cir- ity for EhV-86 replication. Susceptible hosts cular genome. The inverted repeats are pro- accumulate both algal and viral derived posed to anneal with each other to form a GSLs that may coordinate virus maturation, cruciform structure that effectively circular- whereas resistant cells accumulate only al- izes the genome. In addition to the terminal 94 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010) repeats, tandem repeats are located through- shape (Figure 1h). They measure 80–120 nm out the EsV-1 genome and comprise ~12% of wide and 250–380 nm long. Some purified the total genome size. The genome also con- virions contain a 279- to 310-nm filamentous tains several single-stranded regions ran- tail-like appendage at one end. The nucleo- domly distributed over its length whose capsid has a segmented appearance, with functions are unknown. Another characteris- ring-like segments running perpendicular tic of the EsV-1 genome is its low gene den- to the longitudinal axis of the nucleocapsid sity, compared with the other phycodnavi- (Figure 1). Each segment (or ring) is com- ruses. The 231 CDSs only occupy 70% of the posed of two parallel rows of 12–14 globu- EsV-1 genome; they are located in islands of lar subunits, each of which is approximately densely packed genes that are separated by 8–10 nm in diameter. At least 40 structural large regions of DNA repeats and noncoding proteins have been identified in the virus sequences. particles (35). EsV-1 also encodes some unusual CDSs, WSSV infection studies have been ham- including six putative hybrid histidine ki- pered by the lack of a system, al- nases (two-component systems that form though this may be changing (27). Currently, part of a stimulus-responsive transduction researchers agree that WSSV replicates and pathway) that are widespread in and assembles in the nucleus, and in an acute in- bacteria. The relevance of these genes to EsV- fection, its life cycle is completed within 24 h. 1 infection is unknown. However, there are conflicting reports on the events associated with morphogenesis. One report suggests that nuclear protein is pack- aged into a partially enveloped empty cap- Nimaviridae sid, whereas another report suggests that the electron-dense nucleocapsid is assembled The first reported appearance of WSSV first and then enveloped by a viral mem- occurred in 1992–1993 in shrimp farms in brane (35). southern provinces of mainland China and The ~305-kb WSSV genome is circular. also in northern Taiwan. The virus quickly Most of the WSSV genome sequences are spread to shrimp farming regions all over unique and only 3% of the genome consists the world, including North and South Amer- of repetitive sequences. These repetitive se- ica, Europe, and the Middle East. WSSV is le- quences are organized into nine homologous thal to most commercially cultivated penaeid regions containing 47 repeated mini-seg- shrimp species, causing serious economic ments, which are distributed throughout the damage. For example, an acute outbreak of genome, mainly in intergenic regions. white spot disease in cultured shrimp can re- Annotation of the WSSV genome iden- sult in 100% fatality in 3 to 10 days (35). tified 531 ORFs that consist of at least 60 Unlike many viruses, WSSV infects a codons (35). One hundred and eighty-one wide range of marine, brackish water, and ORFs are non-overlapping and are clas- freshwater crustaceans in addition to penaeid sified as CDSs. About 80% of these CDSs shrimp, including crayfishes, crabs, spiny have a potential 3′- polyadenylation site lobsters, and hermit crabs. However, WSSV (AATAAA). The sizes of the proteins en- infection is usually not lethal to these other coded by these CDSs range from 60 to 6077 crustaceans; consequently, these other crus- amino acids. The 6077-amino-acid CDS en- taceans may serve as virus reservoirs. codes the extraordinarily large MCP. Only Structurally WSSV virions resemble bac- 45 of the CDSs resemble known proteins uloviruses and WSSV was initially classi- (>20% amino acid identity) or contain rec- fied as a baculovirus. However, WSSV has ognizable motifs. Twenty-seven CDSs now been assigned to its own family called are classified into 10 WSSV gene families; Nimaviridae (genus Whispovirus). WSSV viri- these families probably arose from gene ons are enveloped, cylindrical to elliptical in duplications. DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 95

The few WSSV identifiable CDSs primar- The phage G genome sequence is 498 kb, ily encode gene products involved in nucleo- but the chromosome is ~670 kb. This means tide metabolism (35). Surprisingly, only one terminal redundancy is about 35%. Phage WSSV CDS, a DNA polymerase, is related to G is predicted to have 682 CDSs and about DNA replication. WSSV also encodes a col- 10% of these are families of paralogs. Phage lagen-like protein, which is the first collagen G, like some other large viruses, encodes sev- gene to be identified in a virus genome. eral translation system components, e.g., 17 tRNAs covering 14 codon specificities and a homolog of a serine aminoacyl-tRNA synthe- Bacteriophage tase. The phage 201φ2-1 genome is also cir- cularly permuted and terminally redundant Large dsDNA bacteriophages are be- and is predicted to encode 468 CDSs. ing discovered with increasing frequency (33). However, when this review was written only two phages, 670-kb Phage G and 317- Concluding Remarks kb 201φ2- 1, had genomes larger than 300 kb (Table 1). Both viruses are members of the Although giruses are probably ancient, tailed family Myoviridae. PhageG infects Ba- they are relatively new to virologists. Even cillus megaterium and phage 201φ2-1 infects with our limited knowledge, research efforts Pseudomonas chlororaphis. Although both ge- on large viruses are contributing scientific nomes have been sequenced, annotation of and economic benefits. For example, chlo- only 201φ2-1 is in the public domain. rella viruses, which encode as many as 400 The majority of the proteins predicted CDSs, are sources of new and unexpected from the genome sequences of these phages genes. The genes not only encode commer- have no database matches, and the genomes cially important enzymes such as DNA re- themselves are diverse enough to preclude striction endonucleases, but many viral the detailed comparative analysis that has proteins are the smallest in their class. Con- occurred with smaller phages, for which sequently, these proteins serve as biochem- hundreds of genome sequences are avail- ical models for mechanistic and structural able. However, one can extrapolate the bet- studies (21). The viruses are also a source of ter-known genome organizations and mech- genetic elements for genetically engineering anisms of evolution seen in the smaller other organisms. Examples include (a) pro- phages to the jumbo phages. Typically, larger moter elements from chlorella viruses that phages contain the same core genes (struc- function well in both monocots and dicots tural and DNA replication genes), plus many of higher , as well as bacteria (38); and additional, generally smaller genes that do (b) a translational enhancer element from a not match anything in the databases and can chlorella virus that functions well in Arabi- usually be deleted without affecting repli- dopsis (49). cation. It is possible that the jumbo phages The hosts for some of these viruses either evolved from smaller-tailed phages, possibly have been sequenced recently or are in the in a process mediated by constraints on ge- process of being sequenced. Annotation of nome size by capsid size (24).4 these sequences will certainly contribute to studies on giruses. However, one obstacle to 4. In the construction of the virion of all the tailed studying these viruses is that currently none phages, an empty protein capsid is assembled of the eukaryotic viruses described in this re- first and then DNA is pumped into the capsid, view can be genetically modified by molec- presumably by a head-full packaging mech- ular techniques. The development of suc- anism. This puts an upper size limit on the ge- nome, and in fact the DNA is usually packed as cessful and reproducible host transformation tightly as physically possible. This agrees with procedures should lead to the genetic analy- the circularly permuted and terminally redun- sis of these viruses, which would be a major dant structure of the phage DNAs. achievement. 96 V a n E t t e n , L a n e , & D u n i g a n i n A n n u a l R e v i e w o f M i c r o b i o l o g y 64 (2010)

It is obvious that the discovery and char- Acknowledgments acterization of giruses are in their infancy and that many more interesting and un- Research in the Van Etten laboratory was usual members await discovery. For exam- supported in part by Public Health Service ple, metagenomic studies on environmen- grant GM32441 and NIH grant P21RR15635 tal microbial DNA sequences collected in from the COBRE program of the National the Sargasso Sea revealed many homologs Center for Research Resources. We thank Mi- of Mimivirus genes. Thus, many Mimivirus chele Malchow for help with the figures. relatives certainly exist in nature, some of which probably infect novel protists. Classi- Literature Cited fying these newly discovered large viruses will be complicated because of horizontal 1. Agarkova IV, Dunigan DD, Van Etten JL. gene swapping. 2006. Virion-associated restriction endonu- The origin of giruses is controversial. One cleases of chloroviruses. J. Virol. 80:8114–23 interesting suggestion is that amoebae, which 2. Baudoux AC, Brussaard CP. 2005. Character- harbor many diverse microorganisms, such ization of different viruses infecting the ma- as viruses, are melting pots for gene mixing, rine harmful algal bloom species Phaeocystis leading to new viruses, including large vi- globosa. Virology 341:80–90 ruses with complex gene repertoires of vari- ous origins (6). 3. Bézier A, Annaheim M, Herbinière J, Wetter- wald C, Gyapay G, et al. 2009. Polydnavi- ruses of braconid wasps derive from an an- cestral . Science 323:926–30 Summary Points 4. Bézier A, Herbinière J, Lanzrein B, Drezen 1. Really big dsDNA viruses (giruses), with JM. 2009. Polydnavirus hidden face: The genomes up to 1.2 Mb in size, are rapidly genes producing virus particles of parasitic being discovered. wasps. J. Invertebr. Pathol. 101:194–203 2. Giruses infect a variety of hosts, bacteria, 5. Bell PJL. 2006. Sex and the eukaryotic cell protists, and animals. Thus, their sizes are cycles is consistent with a viral ancestry not restricted to a specific host or phylo- for the eukaryotic nucleus. J. Theoret. Biol. genetic clade. 243:54–63 3. Giruses are much more diverse than might 6. Boyer M, Yutin N, Pagnier I, Barrassi L, be expected. For example, they have di- Fournous G, et al. 2009. Giant Marseille- verse capsid structures, lifestyles, and ge- virus highlights the role of amoebae as a nome structures. melting pot in emergence of chimeric mi- croorganisms. Proc. Natl. Acad. Sci. USA 4. Giruses that infect the same hosts and are 106:21848–53 members of the same family, e.g., phy- 7. Describes a spike struc- codnaviruses, can differ significantly. 7. Cherrier MV, Kostyuchenko VA, Xiao C, ture at a unique vertex in Bowman VD, Battisti AJ, et al. 2009. An ico- a girus. 5. Many giruses are evolutionarily old, pos- sahedral algal virus has a complex unique sibly going back to the time prokaryotes vertex decorated by a spike. Proc. Natl. Acad. and eukaryotes diverged. Sci. USA 106:11085– 89 6. The majority of the predicted CDSs in gi- 8. Claverie JM. 2006. Viruses take center stage ruses do not match anything in gene da- in cellular evolution. Genome Biol. 7:110 tabases, indicating these viruses are a rich source of novel biochemical functions yet 9. Claverie JM, Abergel C. 2009. Mimivirus and to be discovered. its . Annu. Rev. Genet. 43:49–66 10. Claverie JM, Abergel C, Ogata H. 2009. Mim- ivirus. See Ref. 58a, pp. 89–121 DNA V i r u s e s : T h e R e a ll y B i g O n e s (G i r u s e s ) 97

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