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1.  Evolution of DNA ligases of Nucleo-Cytoplasmic Large DNA viruses of eukaryotes: a case of hidden complexity 
Biology Direct  2009;4:51.
Background
Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood.
Results
Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III.
Conclusions
Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different stages of subsequent evolution. These findings emphasize complex routes of viral evolution that become apparent through detailed phylogenomic analysis but not necessarily in reconstructions based on phyletic patterns of genes.
Reviewers
This article was reviewed by: Patrick Forterre, George V. Shpakovski, and Igor B. Zhulin.
doi:10.1186/1745-6150-4-51
PMCID: PMC2806865  PMID: 20021668
2.  Hidden evolutionary complexity of Nucleo-Cytoplasmic Large DNA viruses of eukaryotes 
Virology Journal  2012;9:161.
Background
The Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) constitute an apparently monophyletic group that consists of at least 6 families of viruses infecting a broad variety of eukaryotic hosts. A comprehensive genome comparison and maximum-likelihood reconstruction of the NCLDV evolution revealed a set of approximately 50 conserved, core genes that could be mapped to the genome of the common ancestor of this class of eukaryotic viruses.
Results
We performed a detailed phylogenetic analysis of these core NCLDV genes and applied the constrained tree approach to show that the majority of the core genes are unlikely to be monophyletic. Several of the core genes have been independently acquired from different sources by different NCLDV lineages whereas for the majority of these genes displacement by homologs from cellular organisms in one or more groups of the NCLDV was demonstrated.
Conclusions
A detailed study of the evolution of the genomic core of the NCLDV reveals substantial complexity and diversity of evolutionary scenarios that was largely unsuspected previously. The phylogenetic coherence between the core genes is sufficient to validate the hypothesis on the evolution of all NCLDV from a common ancestral virus although the set of ancestral genes might be smaller than previously inferred from patterns of gene presence-absence.
doi:10.1186/1743-422X-9-161
PMCID: PMC3493329  PMID: 22891861
3.  Eukaryotic large nucleo-cytoplasmic DNA viruses: Clusters of orthologous genes and reconstruction of viral genome evolution 
Virology Journal  2009;6:223.
Background
The Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) comprise an apparently monophyletic class of viruses that infect a broad variety of eukaryotic hosts. Recent progress in isolation of new viruses and genome sequencing resulted in a substantial expansion of the NCLDV diversity, resulting in additional opportunities for comparative genomic analysis, and a demand for a comprehensive classification of viral genes.
Results
A comprehensive comparison of the protein sequences encoded in the genomes of 45 NCLDV belonging to 6 families was performed in order to delineate cluster of orthologous viral genes. Using previously developed computational methods for orthology identification, 1445 Nucleo-Cytoplasmic Virus Orthologous Groups (NCVOGs) were identified of which 177 are represented in more than one NCLDV family. The NCVOGs were manually curated and annotated and can be used as a computational platform for functional annotation and evolutionary analysis of new NCLDV genomes. A maximum-likelihood reconstruction of the NCLDV evolution yielded a set of 47 conserved genes that were probably present in the genome of the common ancestor of this class of eukaryotic viruses. This reconstructed ancestral gene set is robust to the parameters of the reconstruction procedure and so is likely to accurately reflect the gene core of the ancestral NCLDV, indicating that this virus encoded a complex machinery of replication, expression and morphogenesis that made it relatively independent from host cell functions.
Conclusions
The NCVOGs are a flexible and expandable platform for genome analysis and functional annotation of newly characterized NCLDV. Evolutionary reconstructions employing NCVOGs point to complex ancestral viruses.
doi:10.1186/1743-422X-6-223
PMCID: PMC2806869  PMID: 20017929
4.  Phylogenetic evidence for extensive lateral acquisition of cellular genes by Nucleocytoplasmic large DNA viruses 
Background
Nucleo-Cytoplasmic Large DNA viruses (NCLDV), a diverse group that infects a wide range of eukaryotic hosts, exhibit a large heterogeneity in genome size (between 100 kb and 1.2 Mb) but have been suggested to form a monophyletic group on the basis of a small subset of approximately 30 conserved genes. NCLDV were proposed to have evolved by simplification from cellular organism although some of the giant NCLDV have clearly grown by gene accretion from a bacterial origin.
Results
We demonstrate here that many NCLDV lineages appear to have undergone frequent gene exchange in two different ways. Viruses which infect protists directly (Mimivirus) or algae which exist as intracellular protists symbionts (Phycodnaviruses) acquire genes from a bacterial source. Metazoan viruses such as the Poxviruses show a predominant acquisition of host genes. In both cases, the laterally acquired genes show a strong tendency to be positioned at the tip of the genome. Surprisingly, several core genes believed to be ancestral in the family appear to have undergone lateral gene transfers, suggesting that the NCLDV ancestor might have had a smaller genome than previously believed. Moreover, our data show that the larger the genome, the higher is the number of laterally acquired genes. This pattern is incompatible with a genome reduction from a cellular ancestor.
Conclusion
We propose that the NCLDV viruses have evolved by significant growth of a simple DNA virus by gene acquisition from cellular sources.
doi:10.1186/1471-2148-8-320
PMCID: PMC2607284  PMID: 19036122
5.  Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses 
Bioinformatics  2012;28(24):3186-3190.
Motivation: Eukaryote-infecting nucleo-cytoplasmic large DNA viruses (NCLDVs) feature some of the largest genomes in the viral world. These viruses typically do not strongly depend on the host DNA replication systems. In line with this observation, a number of essential DNA replication proteins, such as DNA polymerases, primases, helicases and ligases, have been identified in the NCLDVs. One other ubiquitous component of DNA replisomes is the single-stranded DNA-binding (SSB) protein. Intriguingly, no NCLDV homologs of canonical OB-fold-containing SSB proteins had previously been detected. Only in poxviruses, one of seven NCLDV families, I3 was identified as the SSB protein. However, whether I3 is related to any known protein structure has not yet been established.
Results: Here, we addressed the case of ‘missing’ canonical SSB proteins in the NCLDVs and also probed evolutionary origins of the I3 family. Using advanced computational methods, in four NCLDV families, we detected homologs of the bacteriophage T7 SSB protein (gp2.5). We found the properties of these homologs to be consistent with the SSB function. Moreover, we implicated specific residues in single-stranded DNA binding. At the same time, we found no evolutionary link between the T7 gp2.5-like NCLDV SSB homologs and the poxviral SSB protein (I3). Instead, we identified a distant relationship between I3 and small protein B (SmpB), a bacterial RNA-binding protein. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins, respectively.
Contact: venclovas@ibt.lt
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts626
PMCID: PMC3519460  PMID: 23097418
6.  “Megavirales”, a proposed new order for eukaryotic nucleocytoplasmic large DNA viruses 
Archives of virology  2013;158(12):2517-2521.
The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family “Marseilleviridae”. The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order “Megavirales”, in reference to the large size of the virions and genomes of these viruses.
doi:10.1007/s00705-013-1768-6
PMCID: PMC4066373  PMID: 23812617
7.  The Giant Cafeteria roenbergensis Virus That Infects a Widespread Marine Phagocytic Protist Is a New Member of the Fourth Domain of Life 
PLoS ONE  2011;6(4):e18935.
Background
A recent work has provided strong arguments in favor of a fourth domain of Life composed of nucleo-cytoplasmic large DNA viruses (NCLDVs). This hypothesis was supported by phylogenetic and phyletic analyses based on a common set of proteins conserved in Eukarya, Archaea, Bacteria, and viruses, and implicated in the functions of information storage and processing. Recently, the genome of a new NCLDV, Cafeteria roenbergensis virus (CroV), was released. The present work aimed to determine if CroV supports the fourth domain of Life hypothesis.
Methods
A consensus phylogenetic tree of NCLDVs including CroV was generated from a concatenated alignment of four universal proteins of NCLDVs. Some features of the gene complement of CroV and its distribution along the genome were further analyzed. Phylogenetic and phyletic analyses were performed using the previously identified common set of informational genes present in Eukarya, Archaea, Bacteria, and NCLDVs, including CroV.
Findings
Phylogenetic reconstructions indicated that CroV is clearly related to the Mimiviridae family. The comparison between the gene repertoires of CroV and Mimivirus showed similarities regarding the gene contents and genome organization. In addition, the phyletic clustering based on the comparison of informational gene repertoire between Eukarya, Archaea, Bacteria, and NCLDVs unambiguously classified CroV with other NCLDVs and clearly included it in a fourth domain of Life. Taken together, these data suggest that Mimiviridae, including CroV, may have inherited a common gene content probably acquired from a common Mimiviridae ancestor.
Conclusions
This further analysis of the gene repertoire of CroV consolidated the fourth domain of Life hypothesis and contributed to outline a functional pan-genome for giant viruses infecting phagocytic protistan grazers.
doi:10.1371/journal.pone.0018935
PMCID: PMC3084725  PMID: 21559486
8.  Plant genomes enclose footprints of past infections by giant virus relatives 
Nature Communications  2014;5:4268.
Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb–2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.
Nucleocytoplasmic large DNA viruses, NCLDVs, are eukaryotic viruses with large genomes, known to infect animals and diverse unicellular eukaryotes. Here, Maumus et al. find NCLDV-like sequences in two land plants and show that they are transcriptionally inactive and highly methylated.
doi:10.1038/ncomms5268
PMCID: PMC4083422  PMID: 24969138
9.  Mimiviridae: clusters of orthologous genes, reconstruction of gene repertoire evolution and proposed expansion of the giant virus family 
Virology Journal  2013;10:106.
Background
The family Mimiviridae belongs to the large monophyletic group of Nucleo-Cytoplasmic Large DNA Viruses (NCLDV; proposed order Megavirales) and encompasses giant viruses infecting amoeba and probably other unicellular eukaryotes. The recent discovery of the Cafeteria roenbergensis virus (CroV), a distant relative of the prototype mimiviruses, led to a substantial expansion of the genetic variance within the family Mimiviridae. In the light of these findings, a reassessment of the relationships between the mimiviruses and other NCLDV and reconstruction of the evolution of giant virus genomes emerge as interesting and timely goals.
Results
Database searches for the protein sequences encoded in the genomes of several viruses originally classified as members of the family Phycodnaviridae, in particular Organic Lake phycodnaviruses and Phaeocystis globosa viruses (OLPG), revealed a greater number of highly similar homologs in members of the Mimiviridae than in phycodnaviruses. We constructed a collection of 898 Clusters of Orthologous Genes for the putative expanded family Mimiviridae (MimiCOGs) and used these clusters for a comprehensive phylogenetic analysis of the genes that are conserved in most of the NCLDV. The topologies of the phylogenetic trees for these conserved viral genes strongly support the monophyly of the OLPG and the mimiviruses. The same tree topology was obtained by analysis of the phyletic patterns of conserved viral genes. We further employed the mimiCOGs to obtain a maximum likelihood reconstruction of the history of genes losses and gains among the giant viruses. The results reveal massive gene gain in the mimivirus branch and modest gene gain in the OLPG branch.
Conclusions
These phylogenomic results reported here suggest a substantial expansion of the family Mimiviridae. The proposed expanded family encompasses a greater diversity of viruses including a group of viruses with much smaller genomes than those of the original members of the Mimiviridae. If the OLPG group is included in an expanded family Mimiviridae, it becomes the only family of giant viruses currently shown to host virophages. The mimiCOGs are expected to become a key resource for phylogenomics of giant viruses.
doi:10.1186/1743-422X-10-106
PMCID: PMC3620924  PMID: 23557328
10.  Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes 
The ISME Journal  2013;7(9):1678-1695.
Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2–1.6 μm size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 104–105 genomes ml−1 for the samples from the photic zone and 102–103 genomes ml−1 for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.
doi:10.1038/ismej.2013.59
PMCID: PMC3749498  PMID: 23575371
eukaryotic viruses; marine NCLDVs; taxon co-occurrence; oomycetes
11.  Open membranes are the precursors for assembly of large DNA viruses 
Cellular microbiology  2013;15(11):1883-1895.
Summary
Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convex side of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open-eight’ conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.
doi:10.1111/cmi.12156
PMCID: PMC4189181  PMID: 23751082
12.  Informational Gene Phylogenies Do Not Support a Fourth Domain of Life for Nucleocytoplasmic Large DNA Viruses 
PLoS ONE  2011;6(6):e21080.
Mimivirus is a nucleocytoplasmic large DNA virus (NCLDV) with a genome size (1.2 Mb) and coding capacity ( 1000 genes) comparable to that of some cellular organisms. Unlike other viruses, Mimivirus and its NCLDV relatives encode homologs of broadly conserved informational genes found in Bacteria, Archaea, and Eukaryotes, raising the possibility that they could be placed on the tree of life. A recent phylogenetic analysis of these genes showed the NCLDVs emerging as a monophyletic group branching between Eukaryotes and Archaea. These trees were interpreted as evidence for an independent “fourth domain” of life that may have contributed DNA processing genes to the ancestral eukaryote. However, the analysis of ancient evolutionary events is challenging, and tree reconstruction is susceptible to bias resulting from non-phylogenetic signals in the data. These include compositional heterogeneity and homoplasy, which can lead to the spurious grouping of compositionally-similar or fast-evolving sequences. Here, we show that these informational gene alignments contain both significant compositional heterogeneity and homoplasy, which were not adequately modelled in the original analysis. When we use more realistic evolutionary models that better fit the data, the resulting trees are unable to reject a simple null hypothesis in which these informational genes, like many other NCLDV genes, were acquired by horizontal transfer from eukaryotic hosts. Our results suggest that a fourth domain is not required to explain the available sequence data.
doi:10.1371/journal.pone.0021080
PMCID: PMC3116878  PMID: 21698163
13.  The genome of the brown alga Ectocarpus siliculosus contains a series of viral DNA pieces, suggesting an ancient association with large dsDNA viruses 
Background
Ectocarpus siliculosus virus-1 (EsV-1) is a lysogenic dsDNA virus belonging to the super family of nucleocytoplasmic large DNA viruses (NCLDV) that infect Ectocarpus siliculosus, a marine filamentous brown alga. Previous studies indicated that the viral genome is integrated into the host DNA. In order to find the integration sites of the viral genome, a genomic library from EsV-1-infected algae was screened using labelled EsV-1 DNA. Several fragments were isolated and some of them were sequenced and analyzed in detail.
Results
Analysis revealed that the algal genome is split by a copy of viral sequences that have a high identity to EsV-1 DNA sequences. These fragments are interspersed with DNA repeats, pseudogenes and genes coding for products involved in DNA replication, integration and transposition. Some of these gene products are not encoded by EsV-1 but are present in the genome of other members of the NCLDV family. Further analysis suggests that the Ectocarpus algal genome contains traces of the integration of a large dsDNA viral genome; this genome could be the ancestor of the extant NCLDV genomes. Furthermore, several lines of evidence indicate that the EsV-1 genome might have originated in these viral DNA pieces, implying the existence of a complex integration and recombination system. A protein similar to a new class of tyrosine recombinases might be a key enzyme of this system.
Conclusion
Our results support the hypothesis that some dsDNA viruses are monophyletic and evolved principally through genome reduction. Moreover, we hypothesize that phaeoviruses have probably developed an original replication system.
doi:10.1186/1471-2148-8-110
PMCID: PMC2373305  PMID: 18405387
14.  Phylogenetic and Phyletic Studies of Informational Genes in Genomes Highlight Existence of a 4th Domain of Life Including Giant Viruses 
PLoS ONE  2010;5(12):e15530.
The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.
doi:10.1371/journal.pone.0015530
PMCID: PMC2996410  PMID: 21151962
15.  Pandoraviruses are highly derived phycodnaviruses 
Biology Direct  2013;8:25.
The recently discovered Pandoraviruses are by far the largest viruses known, with their 2 megabase genomes exceeding in size the genomes of numerous bacteria and archaea. Pandoraviruses show a distant relationship with other nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes, lack some of the NCLDV core genes and in particular do not appear to be specifically related to the other, better characterized family of giant viruses, the Mimiviridae. Here we report phylogenetic analysis of 6 core NCLDV genes that confidently places Pandoraviruses within the family Phycodnaviridae, with an apparent specific affinity with Coccolithoviruses. We conclude that, despite their many unusual characteristics, Pandoraviruses are highly derived phycodnaviruses. These findings imply that giant viruses have independently evolved from smaller NCLDV on at least two occasions.
This article was reviewed by Patrick Forterre and Lakshminarayan Iyer. For the full reviews, see the Reviewers’ reports section.
doi:10.1186/1745-6150-8-25
PMCID: PMC3924356  PMID: 24148757
16.  Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus 
Virology Journal  2009;6:178.
Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a ~356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae, Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments.
doi:10.1186/1743-422X-6-178
PMCID: PMC2777158  PMID: 19860921
17.  Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus 
Journal of Virology  2014;88(11):6069-6075.
ABSTRACT
Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms.
IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.
doi:10.1128/JVI.03199-13
PMCID: PMC4093880  PMID: 24623441
18.  Unique nucleocytoplasmic dsDNA and +ssRNA viruses are associated with the dinoflagellate endosymbionts of corals 
The ISME Journal  2012;7(1):13-27.
The residence of dinoflagellate algae (genus: Symbiodinium) within scleractinian corals is critical to the construction and persistence of tropical reefs. In recent decades, however, acute and chronic environmental stressors have frequently destabilized this symbiosis, ultimately leading to coral mortality and reef decline. Viral infection has been suggested as a trigger of coral–Symbiodinium dissociation; knowledge of the diversity and hosts of coral-associated viruses is critical to evaluating this hypothesis. Here, we present the first genomic evidence of viruses associated with Symbiodinium, based on the presence of transcribed +ss (single-stranded) RNA and ds (double-stranded) DNA virus-like genes in complementary DNA viromes of the coral Montastraea cavernosa and expressed sequence tag (EST) libraries generated from Symbiodinium cultures. The M. cavernosa viromes contained divergent viral sequences similar to the major capsid protein of the dinoflagellate-infecting +ssRNA Heterocapsa circularisquama virus, suggesting a highly novel dinornavirus could infect Symbiodinium. Further, similarities to dsDNA viruses dominated (∼69%) eukaryotic viral similarities in the M. cavernosa viromes. Transcripts highly similar to eukaryotic algae-infecting phycodnaviruses were identified in the viromes, and homologs to these sequences were found in two independently generated Symbiodinium EST libraries. Phylogenetic reconstructions substantiate that these transcripts are undescribed and distinct members of the nucleocytoplasmic large DNA virus (NCLDVs) group. Based on a preponderance of evidence, we infer that the novel NCLDVs and RNA virus described here are associated with the algal endosymbionts of corals. If such viruses disrupt Symbiodinium, they are likely to impact the flexibility and/or stability of coral–algal symbioses, and thus long-term reef health and resilience.
doi:10.1038/ismej.2012.75
PMCID: PMC3526182  PMID: 22791238
coral reef; Heterocapsa circularisquama RNA virus (HcRNAV); nuclear cytoplasmic large DNA virus (NCLDV); Phycodnaviridae; Symbiodinium; virome
19.  Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool 
PLoS ONE  2013;8(4):e60595.
We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.
doi:10.1371/journal.pone.0060595
PMCID: PMC3629200  PMID: 23613733
20.  Structure of a Baculovirus Sulfhydryl Oxidase, a Highly Divergent Member of the Erv Flavoenzyme Family ▿ 
Journal of Virology  2011;85(18):9406-9413.
Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.
doi:10.1128/JVI.05149-11
PMCID: PMC3165737  PMID: 21752922
21.  Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae 
BMC Genomics  2009;10:224.
Background
Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis.
Results
We determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species.
Conclusion
This study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in the Iridoviridae family. Given virus-host co-evolution and the phylogenetic relationship among vertebrates from fish to reptiles, we propose that iridovirus might transmit between reptiles and amphibians and that STIV and FV3 are strains of the same viral species in the Ranavirus genus.
doi:10.1186/1471-2164-10-224
PMCID: PMC2689277  PMID: 19439104
22.  The ancient Virus World and evolution of cells 
Biology Direct  2006;1:29.
Background
Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones.
Results
Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data.
Conclusion
The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history.
Reviewers
W. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.
doi:10.1186/1745-6150-1-29
PMCID: PMC1594570  PMID: 16984643
23.  The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate? 
Biology Direct  2006;1:22.
Background
Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes.
Results
I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation.
Conclusion
The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution – numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis – subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views.
Reviewers
this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole.
doi:10.1186/1745-6150-1-22
PMCID: PMC1570339  PMID: 16907971
24.  Identification of an l-Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses▿  
Journal of Virology  2010;84(17):8829-8838.
Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar l-rhamnose: two UDP-d-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-d-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection.
doi:10.1128/JVI.00770-10
PMCID: PMC2918987  PMID: 20538863
25.  Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing 
Virology Journal  2011;8:99.
Background
Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes.
Findings
We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation.
Conclusions
This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.
doi:10.1186/1743-422X-8-99
PMCID: PMC3058096  PMID: 21375749

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