Giant Viruses and their Genomes

In: Viral Genomes: Diversity, Properties and Parameters ISBN 978-1-60741-067-6 Editors: Zhi Feng and Ming Long © 2009 Nova Science Publishers, Inc.

Chapter VI

Giant Viruses and their Genomes William H Wilson1, 2∗ and Michael J Allen2+ 1

Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, USA 2 Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, UK

Abstract The giant viruses are ancient double-stranded DNA viruses that infect a wide range of host organisms and have genomes larger than 280 kbp. Mimivirus is the largest at an incredible1.2-million base pairs, larger than some of the smallest bacterial genomes. Most of the giant viruses are members of the NCLDV (nuclear cytoplasmic large DNA virus) family and include representatives of the Phycodnaviridae, Mimiviridae, and Poxviridae families, though Herpesviridae, Nimaviridae, Myoviridae and Polydnaviruses are also represented in this elite group. It is likely they are extremely abundant in aquatic environments.

Keywords: Giant virus, Mimivirus, NCLDV, Phycodnaviridae, Poxviridae, Herpesviridae, Nimaviridae, Myoviridae, Polydnavirus

Introduction Viruses are commonly thought of as “a small bag of genes”, with no metabolism or translational machinery of their own and the bare minimum of ingredients to allow successful infection and propagation to take place. They are obligate parasites that hijack their host replication machinery to produce multiple copies of themselves. Such an apparently simple, yet successful, replication strategy can be conducted with very few genes, for example, the ∗ +

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William H Wilson and Michael J Allen

devastating effects of HIV are well documented, even though HIV only contains 9 genes. Conventional thinking would suggest there is no need for viruses to be large. However, this assumption does not take into account the enormous diversity of propagation strategies underpinned by high levels of genetic diversity and genome size range of viruses in any environment, whether it be the mammalian system or an ocean gyre. In the virus size spectrum there are a wide variety of large viruses, all of which have genomes composed of double stranded DNA. Listing the largest viruses in order of genome size (Table 1, also see www.giantvirus.org) immediately reveals their diversity. These include poxviruses, phycodnaviruses (algal viruses), herpesviruses, a bacteriophage and the only known Nimaviridae (shrimp white spot syndrome virus). The definition of a ‘large’ virus has had to be redefined following the discovery of Acanthamoeba polyphaga Mimivirus; which is 750 nm in diameter with a 1.2 million bp genome [1]. This virus is big enough to be seen easily by light microscopy (Fig. 1). This undisputable giant of the virus world was isolated from a cooling tower in Bradford, UK. When it was first observed it was mistaken for a bacterium because of its hairy appearance and, crucially, its positive Gramstaining. Consequently after it was realised it was actually a virus the name ‘mimicking microbe’ (hence ‘mi-mi’) was assigned.

Figure 1. ‘Carpet’ of Mimivirus. This subtle yet incredible image is Mimiviruses plainly photographed using a light microscope in phase contrast. Image courtesy of Jean-Michel Claverie.

Giant Viruses and their Genomes

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Blurred Boundary between Virus and Cell? Mimivirus and other giant viruses blur the boundary between viruses and cells, a boundary that was historically considered obvious. However, the Mimivirus genome (in particular) is considerably larger than a number of obligate parasitic bacteria that have been sequenced [2, 3]. With a circular high coding density genome of 160 kbp, including many overlapping genes, the endosymbiotic bacterium Carsonella ruddii has a very characteristic virus-like genome [4]. Many of the genes considered essential for life are missing and these authors suggest C. ruddii may have achieved organelle-like status. The smallest free-living bacterium to be sequenced is Mycoplasma genitalium at 580 kbp; it is a parasitic bacterium which lives on the ciliated epithelial cells of the primate genital and respiratory tracts. With only 470 coding genes the top 3 giant viruses (Mimivirus, Phage G and algal virus EhV-86 [Fig. 2]) are all ‘larger’ than M. genitalium.

Figure 2. Coccolithovirus strain EhV-86 from the family of giant algal viruses, the Phycodnaviridae, currently the 3rd largest full virus genome to be sequenced (Table 1) [7]. Scanning electron microscope image of EhV-86 (approx. 190 nm diameter) attached to a host E. huxleyi coccolith (A). Transmission electron microscope image of EhV-86 virions, each virus approx. 170-190nm in diameter (B).

So what differentiates a tiny bacterium from a giant virus? Tiny parasitic bacteria lack many genes that code for nucleotide and amino acid biosynthesis, typically they will rely on the hosts they parasitize for some of their metabolic capabilities. Other than that most tiny bacteria have a typical bacterial lifestyle. Giant viruses, like all viruses, do not divide like cells; instead they are assembled from pre-formed subunits. Certainly the most obvious difference is viruses have no protein translation apparatus (ribosomes) which are found even in the smallest bacteria.

Towards a Definition What is clear from this small but diverse collection of giant viruses is their hosts are equally diverse and include bacteria, algae, amoeba, invertebrates and vertebrates. Indeed, there is no obvious connection between the different virus-host systems in this group, making

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a definition of ‘giant virus’ surprisingly difficult to ascertain. There is no formal definition of ‘giant virus’, though the generic term has been gradually evolving since the 230 kbp human cytomegalovirus (human herpesvirus 5) was sequenced in 1990 [5]. Clearly the discovery of Mimivirus accelerated evolution of the term and the name “girus” was even proposed to encompass this select club of large double stranded DNA viruses [2]. It is difficult to define when a virus stops being large and starts being a giant. A simple genome size cut-off could be the simplest way to define a giant virus and a useful analysis was performed by Claverie et al [2] which has aided this definition. Using bacterial genomes as an example and ranking them in order of size, the researchers showed that genome size is not distributed evenly. More importantly the discontinuity is not generated by mathematical misrepresentation, but instead appears to coincide with the organism’s evolutionary constraints. For example, at the smaller genome end of the scale (6 million bases), there appears to be a pressure to acquiring more genes, possibly due to the increased requirement for the regulation of the enlarged genomes. A similar analysis can be applied to virus genomes (Fig. 3), which also shows an uneven distribution at the high end of the scale. Consequently, a useful starting definition for giant viruses is a virus with a genome larger than 280 kbp.

Figure 3. Rank versus size plot of the genome size distribution of the top 572 largest virus genomes from GenBank. The arrow indicates a gap and changed incline of the distribution for viruses with genomes greater than 280 kbp.

Not only does this analysis provide us with a convenient marker for allocating giant virus status, but it also has important evolutionary implications for the giant viruses. If viruses are thought of as obligate intracellular parasites that were once metabolically active symbionts of their hosts (or even free living), this analysis suggests that evolution has acted to reduce their


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genomes to the bare minimum in order to adapt to this parasitic life style. Presumably, if this were the case, giant viruses have not been subjected to the same evolutionary constraints and have retained their larger genomes regardless of the strong selection pressures exerted on other viruses. Alternatively, if the giant viruses are following an evolutionary process akin to that being displayed by the larger bacteria, their large genomes will presumably continue to increase in size to deal with the demands of coordinating an increasingly complex genome.

Giant Virus Characteristics One conclusion that has become increasingly apparent over the past decade is that there is no such thing as an average giant virus. Their genomes have proven to contain a plethora of genetic novelty and have shown massive variation in lifestyles and host range. However, the giant viruses isolated thus far can be characterised by having double stranded genomes greater than 280 kbp and typically have icosahedral shaped virions (Poxviruses are an exception) greater than 150 nm. What is striking about these viruses is the coding density of their genomes: viruses have a reputation for high coding densities and for making maximum use of their genomic material, but these viruses have coding densities similar to bacteria with approximately 1 gene per 1000 bases of sequence. An obvious question for these giant viruses is why so large and why so many genes? Clearly a high number of genes is indicative of complex molecular mechanisms driving their parasitic propagation. However, an unfortunate characteristic of giant viruses is that many of the putative genes are uncharacterized since they have no database matches, hence no predicted functions. For example, approximately one-third of the predicted genes in Mimivirus [6] and 86% of the predicted genes in coccolithovirus EhV-86 [7] have no database homologs. Determining the function of these unknown genes presents a clear challenge for the future. For the genes that do have homologs they can be split into 2 categories; 1) core genes that are shared between some or all of the nucleocytoplasmic large DNA viruses (NCLDVs – described below) and 2) genes with prokaryotic and/or eukaryotic homologs unique to the individual virus. The core genes of these viruses suggest they have a common ancestor and an ancient evolutionary lineage [8, 9]. Arguably the most remarkable aspect of giant viruses is their repertoire of novel and typically non-virus genes, many of which encode central cellular functions and metabolic pathways. A good example is the sphingolipid biosynthesis pathway found in coccolithovirus EhV-86 [7, 10]. It has been suggested that such a collection of genes could reflect a propagation strategy that involves deep reprogramming of host cellular functions [3]. This could simply mean making a pathway more efficient or even interfering with (for example) a host-encoded anti-virus mechanism.

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Nucleocytoplasmic Large DNA Viruses (NCLDVs) NCLDVs encompass the largest proportion of the giant virus group and many papers that discuss giant viruses are usually referring to this diverse group of large DNA viruses. The NCLDV group is composed of at least five virus families (poxviruses, iridoviruses, asfarviruses, phycodnaviruses and Mimivirus) that replicate exclusively in the cytoplasm or target the host nucleus before passage into the cytoplasm of eukaryotic host cells [8, 9, 11]. The conserved core genes, mentioned previously, characterise the group and are indicative of a common ancestry. The core genes include complex systems for DNA replication and transcription, a redox protein, a possible inhibitor of apoptosis, and virion assembly. Nine genes are found to be shared by genomes from all family members (Group I) and a further twenty two are found in at least three of the four families (Group II & III). It is thought that the ancestral NCLDV was likely to have had both nuclear and cytoplasmic phases of its life cycle. Lineage-specific gene loss and accretion within the NCLDV families is thought to contribute to the highly diverse characteristics of present day forms. It has been suggested that their large genomes may act as an ‘invention factory’ for new genes [12].

Mimivirus This special virus deserves a section of its own since it typifies the essence of giant viruses. The Mimivirus was originally discovered associated with the amoeba Acanthamoeba polyphaga, during research into a pneumonia outbreak in Bradford, UK in 1992 [1]. At first, its large size caused it to be was mistaken for a gram positive bacterium, and it was designated Bradford coccus. Following the repeated failure to determine the sequence of its ribosomal RNA, electron microscopy revealed a polyhedral structure nearly identical in morphology (though many times larger) to that of the NCLDV viruses. Following extensive characterization and the determination of its entire genome, Bradford coccus was renamed as the Mimivirus due to its microbial mimicry (full name Acanthamoeba polyphaga Mimivirus) and the term giant virus was born. The Mimivirus is currently the largest virus ever sequenced with a genome of 1,181,404 bases [6]. The size, genomic content and phylogenetic characterisation of the Mimivirus make it one of the most exciting discoveries in modern virology. Analysis of the Mimivirus’ 911 predicted genes clearly places it as a member of the NCLDV and phylogenetic analysis places the mimvirus on a lineage independent to all previously identified viruses. It is currently the sole member of the family Mimiviridae. Of the 911 Mimivirus genes, 298 are associated with functional attributes. In addition to the core genes, the Mimivirus encodes genes for eight protein translation associated proteins. One of the few clear distinctions between viruses and living organisms is the absence of core protein translational machinery in viruses. The Mimivirus was the one of the first viruses discovered which begins to blur this distinct boundary. Ten protein translation associated components are encoded on the genome (4 amino acyl tRNA synthetases, a peptide release factor, and translation elongation and initiation factors) in addition to six tRNAs. Despite its giant genome the Mimivirus has failed to display any reason why it requires so many genes. Its life cycle shows no sign of displaying any unusual


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characteristics. It is unusually hardy and can stand extreme temperatures suggesting that it is capable of surviving long periods of time outside of the host system. It is has been suggested that the large particle size (750 nm) makes it look like a small bacterium, which would be preferentially eaten by the host amoeba [2, 13]. This perhaps explains why there are so many bacterial-like genes found in Mimivirus; close proximity to other bacteria during the infection process will provide an ideal scenario for recombination events through horizontal gene transfer. Despite the mimvirus’ giant size, perhaps the best illustration of the uniqueness and sheer novelty of giant viruses is obtained when studying the phycodnaviruses. It is highly likely that members of the Mimiviridae will be just as diverse as new strains are isolated, but hitherto there is only one member for comparison. However, there are other Mimivirus-like sequences in public databases particularly from the Venter Global Ocean Survey and Sargasso Sea metagenomic databases.

Phycodnaviridae The Phycodnaviridae comprise a genetically diverse [14], yet morphologically similar, family of large icosahedral viruses that infect marine or freshwater eukaryotic algae with dsDNA genomes ranging from 180 to 560 kbp [15]. Members of the Phycodnaviridae are currently grouped into six genera (named loosely after the hosts they infect), Chlorovirus, Coccolithovirus (Fig. 2), Prasinovirus, Prymnesiovirus, Phaeovirus and Raphidovirus [16]. So far, each phycodnavirus genus appears to be unique in lifestyle, with each virus having its own peculiarities and novelties. Nearly everything that can vary, does vary within the Phycodnaviridae, from genome size, structure (circular or linear) and content, to propagation strategy and life style. Complete genome sequences have been obtained from representatives of the Chlorovirus, Coccolithovirus and Phaeovirus genera [14] and evolutionary analysis of their genomes places them within a major, monophyletic clade of the NCLDVs [9, 11]. Their common evolutionary origin is reflected in their appearance (despite their range in sizes, these viruses are structurally and morphologically similar) and through the presence of NCLDV core genes [11]. Their hosts, eukaryotic algae, are an incredibly diverse group of organisms that represent at least five distinct evolutionary lineages. They are ubiquitous in marine, freshwater and terrestrial habitats, and the number of algal species may be as high as several million. Their role in global carbon and nutrient cycling make O2-evolving eukaryotic algae arguably, the most important microorganisms for maintaining life on planet earth. It is perhaps surprising that only a few hundred (at most) phycodnaviruses have been isolated, less than a dozen have been sequenced and only one (PBCV-1) has been studied in any depth [17].

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Other Giant Viruses Nimaviridae White spot syndrome virus (also know as white spot bacilliform virus) is a major shrimp pathogen and was the first marine invertebrate virus to be sequenced. The high commercial interest in the penaeid shrimp (for which this virus is particularly virulent) has made this one of the best studied giant virus-host systems. The Chinese WSSV type species has giant circular genome of 305,107 bp contains 181 predicted genes [18], two further isolated have genomes of 293 kbp (Thailand isolate) and 307 kbp (Taiwan isolate). The variation in size is thought to be caused by several small deletions and one large deletion of 12 kbp. In common with other giant viruses, only 30% of WSSV genes have any putative function assigned to them.

Poxviridae The majority of poxviruses fall in the normal range for genome size, except two: canary pox and fowl pox with genomes of 359,853 bp and 288,539 bp respectively [19, 20]. Both the canary pox and fowl pox viruses are members of the avipoxvirus subfamily within the chordopoxviridae, which boasts examples of large viruses ranging from 134,431 bp (Bovine papular stomatitis virus) up to the largest, the canary pox virus. The common ancestor of all known poxviruses is estimated to have contained at least 66 genes [9]. The reasons for such large variation in the remainder of the genomic content is unclear at this stage, but it is likely to be due to specific adaptations to their host organisms. The phylogenies of poxviruses appear to mirror the phylogeny of their animal hosts [9], and it appears, so far, that the avipoxviruses (i.e. bird pox viruses) are for some reason larger than their ancestral relatives.

Herpesviridae Like the poxviruses, the majority of the herpes viruses fall in the normal range for genome size. Despite their icosahedral capids and large genome size, the herpesviruses are not classified as NCLDV because of prominent differences in virion assembly and DNA packaging [9]. Herpesviruses undoubtedly share a common ancestor with NCLDVs, but the evolutionary history appears to be complex. Three closely strains capable of infecting common carp and koi (Cyprinus carpio) from Japan, USA and Israel have been shown to have genomes of 295,052 bp; 295,146 bp; and 295,138 bp, respectively [21]. The genomes differ at few loci and are predicted to encode 164 genes.


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Myoviridae Bacillus megaterium bacteriophage G is a giant virus which easily disproves the notion that giant viruses are restricted to infecting eukaryotic hosts. With a predicted genome size of approximately 670 kbp, (currently a draft sequence is available for over 497 kbp from the Pittsburgh Bacteriophage Institute, http://pbi.bio.pitt.edu/). Bacteriophage G has a icosahedral capsid of 80 nm diameter and a tail extension [22]. The phiKZ-like viruses represent a distinct branch of the myoviridae, of which there are currently 21 members. Another of the members of this family, phiKZ, sneaks into giant virus status with a genome size of 280,334 bp [23]. These viruses are classified as phage, infect Pseudomonas aeruginosa and definitively prove that giant virus status is not restricted to viruses infecting eukaryotic hosts. The phiKZ virion is composed of an icosahedral capsid (120 nm in diameter) coupled to a long contractile tail. The phiKZ genome encodes 306 predicted genes which are organised into clusters and usually have clockwise direction. Only 20% of phiKZ genes have an assigned putative function.

Polydnaviruses Polydnaviruses (PDVs) are exploited by parasitoid wasps to facilitate development of their progeny within the body of immunocompetent insect hosts [24]. Cotesia congregata bracovirus (CcBV) exhibits a large genome (567,670 bp) that contains relatively few genes (156). In contrast to all the other giant viruses genomes described here, CcBV has a segmented genome composed of 30 circular segments ranging in size from 5 to 40 kbp and encodes a mere 156 predicted genes. More than 70% of the genomic material is noncoding. Another polydnavirus of interest is the Glypta fumiferanae virus (GfV) [25]. This wasp virus has an estimated genome size of 290.4 kbp which is segmented into an amazing 105 portions which is far higher than that seen with other segmented viruses. Segments range in size from approximately 1.5 kb to just under 5.2 kbp, and prove that it is possible to achieve giant virus status by piecemeal accretion.

Conclusion As of December 6th 2007, there are 2444 virus genomes listed as complete in the GenBank database. Only a handful of these can be classified as giants (Table 1), it is highly probable they are considerably more abundant than this table suggests. One explanation for the change in incline on the plot in Fig. 3 is simply lack of isolated representatives in this size range. If numerous new giant viruses were isolated and the x-axis extended, theoretically the plot would show a single continuous incline. So why haven’t any more viruses been isolated in this giant end of the continuum? The simple answer is the sampling strategy used for isolating viruses. If the assumption is viruses are small; then passing a sample through a 0.2 µm filter and looking for viruses in the filtrate is a good procedure. However, most large


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viruses will not be filterable through such a pore size. Hence even at this early stage, researchers often introduce a size bias in their sampling strategy. With up to 100-million viruses per millilitre of seawater [26, 27], aquatic environments seem to be an ideal place to search for giant viruses. Several algal viruses (Phycodnaviridae) that fall into the giant virus category have already been isolated and characterized, though they have not yet been sequenced, the largest of which is approx. 560 kbp [28]. Giant viruses are frequently observed by electron microscopy in both freshwater and marine environments [29-32]. Analysis of fixed water samples from 2 contrasting freshwater environments revealed giant phage-like particles, though their hosts at this stage are uncertain (Fig. 4). Giant virus particles up to 750 nm (similar in size to Mimivirus) have been observed in abundance in food vacuoles of phaeodarian radiolarians, pelagic omnivorous generalists that feed on a variety of marine microscopic organisms ranging in size from bacteria to large protozoans [33]. It was perhaps no surprise then that numerous Mimivirus sequence homologs have been identified in the Venter Sargasso Sea environmental database [34]. Indeed, these authors suggest that their data is indicative of high concentrations of Mimivirus ‘relatives’ in the ocean. Only a concerted sampling effort to specifically isolate giant viruses in these environments will identify new strains and force us to re-think the boundaries of virology. A

B

Figure 4. Transmission electron micrographs of ‘Giant’ phage-like particles from fixed water samples collected from Light Lake, Signy Island, Antarctica [32] (A) and Priest Pot, a 1-hectare pond in the English Lake District (B). Scale bars are 265 nm (A) and 200 nm (B).


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Table 1. The largest fully sequenced ‘giant’ viruses listed in GenBank. Accurate to Dec 6th 2007. Note: 497,513 bp of draft sequence is available for Bacillus phage G from Pittsburgh Bacteriophage Institute (http://pbi.bio.pitt.edu/). Size (bp) 1181404

Year sequenced 2003

567670

2004

407339

2005

NC_009898

368683

2006

Chordopoxvirinae

NC_005309

359853

2004

Phycodnaviridae

NC_009899

344691

2007

Phycodnaviridae Phycodnaviridae

NC_002687 NC_000852

335593 330743

2001 1996

Phycodnaviridae

NC_008603

321240

2007

Nimaviridae

AF440570

307287

2005

Nimaviridae

NC_003225

305107

2001

Herpesviridae Herpesviridae Herpesviridae

DQ657948 DQ177346 AP008984

295146 295138 295052

2007 2007 2007

Nimaviridae

AF369029

292697

2004

Chordopoxvirinae

NC_002188

288539

2002

Phycodnaviridae

NC_008724

288047

2007

Myoviridae

AF399011

280334

2002

Virus

Family

Accession

Acanthamoeba polyphaga Mimivirus

Mimiviridae

Cotesia congregata bracovirus

Polydnaviridae

Emiliania huxleyi virus 86 Paramecium bursaria Chlorella virus NY2A Canarypox virus Paramecium bursaria Chlorella virus AR158 Ectocarpus siliculosus virus 1 Paramecium bursaria Chlorella virus 1 Paramecium bursaria Chlorella virus FR483 Shrimp white spot syndrome virus (Taiwan) Shrimp white spot syndrome virus (China) Koi herpesvirus (USA) Koi herpesvirus (Israel) Koi herpesvirus (Japan) Shrimp white spot syndrome virus (Thailand) Fowlpox virus Acanthocystis turfacea Chlorella virus 1 phiKZ

Phycodnaviridae

NC_006450 NC006633 NC006662 NC_007346

Phycodnaviridae

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