The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes

Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.In recent years there has been growing interest in viruses infecting hosts in the domain Archaea (43). Archaeal viruses were discovered 35 years ago (52), and today about 50 such viruses are known (43). They represent highly diverse virion morphotypes in contrast to the vast majority (96%) of head-tail virions among the over 5,000 described bacterial viruses (1). Although archaea are widespread in both moderate and extreme environments (13), viruses have been isolated only for halophiles and anaerobic methanogenes of the kingdom Euryarchaeota and hyperthermophiles of the kingdom Crenarchaeota (43).In addition to soil and marine environments, high viral abundance has also been detected in hypersaline habitats such as salterns (i.e., a multipond system where seawater is evaporated for the production of salt) (19, 37, 50). Archaea are dominant organisms at extreme salinities (36), and about 20 haloarchaeal viruses have been isolated to date (43). The majority of these are head-tail viruses, whereas electron microscopic (EM) studies of highly saline environments indicate that the two other described morphotypes, spindle-shaped and round particles, are the most abundant ones (19, 37, 43). Thus far, the morphological diversity of the isolated haloarchaeal viruses is restricted compared to viruses infecting hyperthermophilic archaea, which are classified into seven viral families (43).All of the previously described archaeal viruses have a double-stranded DNA (dsDNA) genome (44). However, a newly characterized haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), has a single-stranded DNA (ssDNA) genome (39). HRPV-1 and its host Halorubrum sp. were isolated from an Italian (Trapani, Sicily) solar saltern. Most of the studied haloarchaeal viruses lyse their host cells, but persistent infections are also typical (40, 44). HRPV-1 is a nonlytic virus that persists in the host cells. In liquid propagation, nonsynchronous infection cycles of HRPV-1 lead to continuous virus production until the growth of the host ceases, resulting in high virus titers in the growth medium (39).The pleomorphic virion of HRPV-1 represents a novel archaeal virus morphotype constituted of lipids and two major structural proteins VP3 (11 kDa) and VP4 (65 kDa). The genome of HRPV-1 is a circular ssDNA molecule (7,048 nucleotides [nt]) containing nine putative open reading frames (ORFs). Three of them are confirmed to encode structural proteins VP3, VP4, and VP8, which is a putative ATPase (39). The ORFs of the HRPV-1 genome show significant similarity, at the amino acid level, to the minimal replicon of plasmid pHK2 of Haloferax sp. (20, 39). Furthermore, an ∼4-kb region, encoding VP4- and VP8-like proteins, is found in the genomes of two haloarchaea, Haloarcula marismortui and Natronomonas pharaonis, and in the linear dsDNA genome (16 kb) of spindle-shaped haloarchaeal virus His2 (39). The possible relationship between ssDNA virus HRPV-1 and dsDNA virus His2 challenges the classification of viruses, which is based on the genome type among other criteria (15, 39).HRPV-1 is proposed to represent a new lineage of pleomorphic enveloped viruses (39). A putative representative of this lineage among bacterial viruses might be L172 of Acholeplasma laidlawii (14). The enveloped virion of L172 is pleomorphic, and the virus has a circular ssDNA genome (14 kb). In addition, the structural protein pattern of L172 with two major structural proteins, of 15 and 53 kDa, resembles that of HRPV-1.The structural approach has made it possible to reveal relationships between viruses where no sequence similarity can be detected. It has been realized that several icosahedral viruses infecting hosts in different domains of life share common virion architectures and folds of their major capsid proteins. These findings have consequences for the concept of the origin of viruses. A viral lineage hypothesis predicts that viruses within the same lineage may have a common ancestor that existed before the separation of the cellular domains of life (3, 5, 8, 26). Currently, limited information is available on the detailed structures of viruses infecting archaea. For example, the virion structures of nontailed icosahedral Sulfolobus turreted icosahedral virus (STIV) and SH1 have been determined (21, 23, 46). However, most archaeal viruses represent unusual, sometimes nonregular, morphotypes (43), which makes it difficult to apply structural methods that are based on averaging techniques.A biochemical approach, i.e., controlled virion dissociation, gives information on the localization and interaction of virion components. In the present study, controlled dissociation was used to address the virion architecture of HRPV-1. A comparative lipid analysis of HRPV-1 and its host was also carried out. Our results show that the unique virion type is composed of a flexible membrane decorated with the glycosylated spikes of VP4 and internal membrane protein VP3. The circular ssDNA genome resides inside the viral membrane vesicle without detected association to any nucleoproteins.     

Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.     

New,Closely Related Haloarchaeal Viral Elements with Different Nucleic Acid Types

During the search for haloarchaeal viruses, we isolated and characterized a new pleomorphic lipid-containing virus, Haloarcula hispanica pleomorphic virus 1 (HHPV-1), that infects the halophilic archaeon Haloarcula hispanica. The virus contains a circular double-stranded DNA genome of 8,082 bp in size. The organization of the genome shows remarkable synteny and amino acid sequence similarity to the genome and predicted proteins of the halovirus HRPV-1, a pleomorphic single-stranded DNA virus that infects a halophilic archaeon Halorubrum sp. Analysis of the two halovirus sequences, as well as the entire nucleotide sequence of the 10.8-kb pHK2-plasmid and a 12.6-kb chromosomal region in Haloferax volcanii, allows us to suggest a new group of closely related viruses with genomes of either single-stranded or double-stranded DNA. Currently, closely related viruses are considered to have the same genome type. Our observation clearly contradicts this categorization and indicates that we should reconsider the way we classify viruses. Our results also provide a new example of related viruses where the viral structural proteins have not diverged as much as the proteins associated with genome replication. This result further strengthens the proposal for higher-order classification to be based on virion architecture rather than on genome type or replication mechanism.Metagenomic studies have increased the amount of information on the nucleotide sequence space in our environment. It has also increased our awareness of the viral abundance and diversity not recognized before (16, 24, 26). Along with this new information, we have learned to acknowledge the significance of viruses in the evolution and behavior of other organisms (55). To reveal the dynamics and molecular interactions in the interplay between a particular virus and its host, however, isolation of single viruses and their hosts is needed. Even though a number of viruses pathogenic to humans, domestic animals, and plants, as well as some bacteriophages, have been studied in great detail, much of the diversity of the archaeal viruses has remained unknown. By the year 2007 only 44 archaeal viruses had been described (2). That embraces less than 1% of all reported viruses. Although the diversity among these few isolated archaeal viruses is considerable, a head-and-tail morphology is prevalent among isolated viruses infecting euryarchaeal cells. In contrast, viruses of Crenarchaeota are diverse and often unusual with no viruses having a head-tail morphology (53).Archaeal haloviruses infect euryarchaeal hosts living in environments up to saturated salt. This makes them an interesting group of viruses that reside in a very restricted habitat. In samples taken from high salt environments, the Dead Sea and Spanish solar salterns, viral morphotypes most often observed were spindle-shaped, head-and-tail or tailless icosahedral particles (25, 31, 47). Isolated haloviruses, however, do not seem to reflect the proportions of different morphotypes found in the nature as nearly all of the isolates possess a head-and-tail morphology (2). Molecular level studies on only two spindle-shaped (10, 11) and one tailless icosahedral particle have been carried out (37, 51). Virus-like particles of other morphologies have also been observed in high-salt environments (47), but only one additional morphotype has been described in detail (50). This recently isolated lipid containing halovirus, HRPV-1, is the first archaeal virus containing a single-stranded DNA (ssDNA) genome (50). It infects Halorubrum sp. and has a pleomorphic appearance with glycosylated spike structures protruding from its external membrane (49, 50).The evolution of prokaryotic viral genome sequences is very fast (18), and the assessment of viral relationships using homology of the genome sequences applies only to closely related viruses (17, 19). Current higher-order classification of viruses is based on the host organism, the nature of the genome (RNA/DNA, single stranded versus double stranded) and the virion morphology. Recently, a higher-order clustering of virus families has been proposed based on common principles of virion architectures as well as on the fold of the major capsid protein (1, 6, 12, 13, 42). Consequently, major capsid proteins most probably belong to the vertically inherited viral “self” (4), whereas proteins involved in replication of the viral genome can be swapped by horizontal exchange (21, 63). The proposal is based on observations that structurally related viruses have been found to infect organisms that reside in all three domains of life.We have isolated a new pleomorphic virus infecting Haloarcula hispanica (Har. hispanica pleomorphic virus 1 [HHPV-1]). Here, we determine the molecular constituents of HHPV-1 and its genetic relatedness to other archaeal viruses and putative proviruses. Sequence homology and gene order (synteny) shows distinct genomic regions shared between four genetic elements separating replication, virus assembly, and integration functions. Surprisingly, in spite of the close relatedness of HRPV-1 and HHPV-1, the genome types of these two viruses differ (ssDNA and dsDNA, respectively).     

Identification and characterization of SNJ2, the first temperate pleolipovirus integrating into the genome of the SNJ1‐lysogenic archaeal strain

Proviral regions have been identified in the genomes of many haloarchaea, but only a few archaeal halophilic temperate viruses have been studied. Here, we report a new virus, SNJ2, originating from archaeal strain Natrinema sp. J7‐1. We demonstrate that this temperate virus coexists with SNJ1 virus and is dependent on SNJ1 for efficient production. Here, we show that SNJ1 is an icosahedral membrane‐containing virus, whereas SNJ2 is a pleomorphic one. Instead of producing progeny virions and forming plaques, SNJ2 integrates into the host tRNA^Met gene. The virion contains a discontinuous, circular, double‐stranded DNA genome of 16 992 bp, in which both nicks and single‐stranded regions are present preceded by a ‘GCCCA’ motif. Among 25 putative SNJ2 open reading frames (ORFs), five of them form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Furthermore, SNJ2‐like proviruses containing the conserved gene cluster were identified in the chromosomes of archaea belonging to 10 different genera. Comparison of SNJ2 and these proviruses suggests that they employ a similar integration strategy into a tRNA gene.     

Transfection of haloarchaea by the DNAs of spindle and round haloviruses and the use of transposon mutagenesis to identify non‐essential regions

Spindle‐shaped halovirus His2 and spherical halovirus SH1 represent ecologically dominant virus morphotypes in high‐salt environments. Both have linear dsDNA genomes with inverted terminal repeat sequences and terminal proteins, and probably replicate using protein priming. As a first step towards conventional genetic analyses on these viruses, we show that purified viral DNAs can transfect host cells. Intact terminal proteins were essential for this process. Despite the narrow host ranges of these viruses, at least under laboratory conditions, their DNAs were able to transfect a wide range of haloarchaeal species, demonstrating that the cytoplasms of diverse haloarchaea possess all the factors necessary for viral DNA synthesis and virion assembly. Transposon mutagenesis of viral DNAs was then used in conjunction with transfection to produce recombinant viruses, and to then map the insertion sites to identify non‐essential genes. The inserts in 34 His2 mutants were mapped precisely, and most clustered in a few, specific regions, particularly in the inverted terminal repeats and near the ends of ORFs. The results are consistent with the small genome size and densely packed, often overlapping ORFs that are transcribed as long operons. This study is the first demonstration of transfection and transposon mutagenesis in protein‐primed archaeal viruses.     

Related haloarchaeal pleomorphic viruses contain different genome types

Archaeal viruses have been the subject of recent interest due to the diversity discovered in their virion architectures. Recently, a new group of haloarchaeal pleomorphic viruses has been discovered. It is distinctive in terms of the virion morphology and different genome types (ssDNA/dsDNA) harboured by rather closely related representatives. To date there are seven isolated viruses belonging to this group. Most of these share a cluster of five conserved genes, two of which encode major structural proteins. Putative proviruses and proviral remnants containing homologues of the conserved gene cluster were also identified suggesting a long-standing relationship of these viruses with their hosts. Comparative genomic analysis revealed three different ways of the genome organization, which possibly reflect different replication strategies employed by these viruses. The dsDNA genomes of two of these viruses were shown to contain single-strand interruptions. Further studies on one of the genomes suggested that the interruptions are located along the genome in a sequence-specific manner and exhibit polarity in distribution.     

The Structure of the NTPase That Powers DNA Packaging into Sulfolobus Turreted Icosahedral Virus 2

Biochemical reactions powered by ATP hydrolysis are fundamental for the movement of molecules and cellular structures. One such reaction is the encapsidation of the double-stranded DNA (dsDNA) genome of an icosahedrally symmetric virus into a preformed procapsid with the help of a genome-translocating NTPase. Such NTPases have been characterized in detail from both RNA and tailed DNA viruses. We present four crystal structures and the biochemical activity of a thermophilic NTPase, B204, from the nontailed, membrane-containing, hyperthermoacidophilic archaeal dsDNA virus Sulfolobus turreted icosahedral virus 2. These are the first structures of a genome-packaging NTPase from a nontailed, dsDNA virus with an archaeal host. The four structures highlight the catalytic cycle of B204, pinpointing the molecular movement between substrate-bound (open) and empty (closed) active sites. The protein is shown to bind both single-stranded and double-stranded nucleic acids and to have an optimum activity at 80°C and pH 4.5. The overall fold of B204 places it in the FtsK-HerA superfamily of P-loop ATPases, whose cellular and viral members have been suggested to share a DNA-translocating mechanism.     

Virion architecture unifies globally distributed pleolipoviruses infecting halophilic archaea

Our understanding of the third domain of life, Archaea, has greatly increased since its establishment some 20 years ago. The increasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomorphic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unselectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are embedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.     

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.     

Viral diversity in hot springs of Pozzuoli, Italy, and characterization of a unique archaeal virus, Acidianus bottle-shaped virus, from a new family, the Ampullaviridae

Virus-like particles with five different morphotypes were observed in an enriched environmental sample from a hot, acidic spring (87 to 93 degrees C, pH 1.5) in Pozzuoli, Italy. The morphotypes included rigid rods, flexible filaments, and novel, exceptional forms. Particles of each type were isolated, and they were shown to represent viable virions of five novel viruses which infect members of the hyperthermophilic archaeal genus Acidianus. One of these, named the Acidianus bottle-shaped virus, ABV, exhibits a previously unreported morphotype. The bottle-shaped virion carries an envelope which encases a funnel-shaped core. The pointed end of the virion is likely to be involved in adsorption and channeling of viral DNA into host cells. The broad end exhibits 20 (+/- 2) thin filaments which appear to be inserted into a disk, or ring, and are interconnected at their bases. These filaments are apparently not involved in adsorption. ABV virions contain six proteins in the size range 15 to 80 kDa and a 23.9-kb linear, double-stranded DNA genome. Virus replication does not cause lysis of host cells. On the basis of its unique morphotype and structure, we propose to assign ABV to a new viral family, the Ampullaviridae.     

Novel haloarchaeal viruses from Lake Retba infecting Haloferax and Halorubrum species

The diversity of archaeal viruses is severely undersampled compared with that of viruses infecting bacteria and eukaryotes, limiting our understanding on their evolution and environmental impacts. Here, we describe the isolation and characterization of four new viruses infecting halophilic archaea from the saline Lake Retba, located close to Dakar on the coast of Senegal. Three of the viruses, HRPV10, HRPV11 and HRPV12, have enveloped pleomorphic virions and should belong to the family Pleolipoviridae, whereas the forth virus, HFTV1, has an icosahedral capsid and a long non-contractile tail, typical of bacterial and archaeal members of the order Caudovirales. Comparative genomic and phylogenomic analyses place HRPV10, HRPV11 and HRPV12 into the genus Betapleolipovirus, whereas HFTV1 appears to be most closely related to the unclassified Halorubrum virus HRTV-4. Differently from HRTV-4, HFTV1 encodes host-derived minichromosome maintenance helicase and PCNA homologues, which are likely to orchestrate its genome replication. HFTV1, the first archaeal virus isolated on a Haloferax strain, could also infect Halorubrum sp., albeit with an eightfold lower efficiency, whereas pleolipoviruses nearly exclusively infected autochthonous Halorubrum strains. Mapping of the metagenomic sequences from this environment to the genomes of isolated haloarchaeal viruses showed that these known viruses are underrepresented in the available viromes.     

From small hosts come big viruses: the complete genome of a second Ostreococcus tauri virus, OtV-1

Ostreococcus tauri virus (OtV-1) is a large double-stranded DNA virus and a prospective member of the family Phycodnaviridae , genus Prasinovirus . OtV-1 infects the unicellular marine green alga O. tauri , the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNA-encoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N -myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history.     

Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1

By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix, and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

A snapshot of viral evolution from genome analysis of the tectiviridae family

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.     

Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus

The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.     

Signatures of selection in natural populations adapted to chronic pollution

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.     

Diversity,taxonomy, and evolution of archaeal viruses of the class Caudoviricetes

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus–host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Comparative genomics and host range analysis reveals the remarkable diversity and evolution of tailed archaeal viruses of the order Caudoviricetes, which together with their bacterial relatives arguably represent the most abundant and widespread virus group on our planet.     

Comparative Analysis of the Mosaic Genomes of Tailed Archaeal Viruses and Proviruses Suggests Common Themes for Virion Architecture and Assembly with Tailed Viruses of Bacteria

Tailed double-stranded DNA viruses (order Caudovirales) represent the dominant morphotype among viruses infecting bacteria. Analysis and comparison of complete genome sequences of tailed bacterial viruses provided insights into their origin and evolution. Structural and genomic studies have unexpectedly revealed that tailed bacterial viruses are evolutionarily related to eukaryotic herpesviruses. Organisms from the third domain of life, Archaea, are also infected by viruses that, in their overall morphology, resemble tailed viruses of bacteria. However, high-resolution structural information is currently unavailable for any of these viruses, and only a few complete genomes have been sequenced so far. Here we identified nine proviruses that are clearly related to tailed bacterial viruses and integrated into chromosomes of species belonging to four different taxonomic orders of the Archaea. This more than doubled the number of genome sequences available for comparative studies. Our analyses indicate that highly mosaic tailed archaeal virus genomes evolve by homologous and illegitimate recombination with genomes of other viruses, by diversification, and by acquisition of cellular genes. Comparative genomics of these viruses and related proviruses revealed a set of conserved genes encoding putative proteins similar to virion assembly and maturation, as well as genome packaging proteins of tailed bacterial viruses and herpesviruses. Furthermore, fold prediction and structural modeling experiments suggest that the major capsid proteins of tailed archaeal viruses adopt the same topology as the corresponding proteins of tailed bacterial viruses and eukaryotic herpesviruses. Data presented in this study strongly support the hypothesis that tailed viruses infecting archaea share a common ancestry with tailed bacterial viruses and herpesviruses.