RESUMEN
Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.
Asunto(s)
Archaea/virología , Virus de Archaea/aislamiento & purificación , Bacteriófagos/aislamiento & purificación , Manantiales de Aguas Termales/virología , Rudiviridae/genética , Rudiviridae/aislamiento & purificación , Archaea/genética , Archaea/aislamiento & purificación , Virus de Archaea/clasificación , Virus de Archaea/genética , Virus de Archaea/fisiología , Bacteriófagos/clasificación , Bacteriófagos/genética , Bacteriófagos/fisiología , Genoma Viral , Manantiales de Aguas Termales/química , Japón , Lipothrixviridae/clasificación , Lipothrixviridae/genética , Lipothrixviridae/aislamiento & purificación , Lipothrixviridae/fisiología , Metagenoma , Filogenia , Rudiviridae/clasificación , Replicación ViralRESUMEN
Many proteins of viruses infecting hyperthermophilic Crenarchaeota have no detectable homologs in current databases, hampering our understanding of viral evolution. We used sensitive database search methods and structural modeling to show that a nucleocapsid protein (TP1) of Thermoproteus tenax virus 1 (TTV1) is a derivative of the Cas4 nuclease, a component of the CRISPR-Cas adaptive immunity system that is encoded also by several archaeal viruses. In TTV1, the Cas4 gene was split into two, with the N-terminal portion becoming TP1, and lost some of the catalytic amino acid residues, apparently resulting in the inactivation of the nuclease. To our knowledge, this is the first described case of exaptation of an enzyme for a virus capsid protein function.
Asunto(s)
Proteínas Arqueales/genética , Endonucleasas/genética , Evolución Molecular , Lipothrixviridae/enzimología , Lipothrixviridae/genética , Proteínas de la Nucleocápside/genética , Proteínas Arqueales/metabolismo , Sistemas CRISPR-Cas , Endonucleasas/metabolismo , Proteínas de la Nucleocápside/metabolismo , Thermoproteus/genética , Thermoproteus/virologíaRESUMEN
The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central ß-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29, AvtR can also act as an activator of another gene, gp30. Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.
Asunto(s)
Acidianus/virología , Proteínas de Unión al ADN/química , Lipothrixviridae/química , Proteínas Virales/química , Acidianus/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Análisis Mutacional de ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Lipothrixviridae/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Virales/genéticaRESUMEN
Linear viruses with double-stranded DNA genomes are classified into two families, Lipothrixviridae and Rudiviridae. The members of these two families, all of which infect hyperhermophilic members of the domain Archaea, differ significantly in the complexity of their virions as well as in their mechanisms of genome replication. However, recent structural and genomic studies have revealed a robust evolutionary link between members of the two families. To acknowledge this relationship we propose to unify the two families into the new taxonomic order "Ligamenvirales".
Asunto(s)
Archaea/virología , Lipothrixviridae/clasificación , Rudiviridae/clasificación , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Análisis por Conglomerados , ADN/genética , ADN Viral/genética , Orden Génico , Genoma Viral , Lipothrixviridae/genética , Lipothrixviridae/crecimiento & desarrollo , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Rudiviridae/genética , Rudiviridae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Virión/ultraestructura , Replicación ViralRESUMEN
Acidianus filamentous virus 1 (AFV1) (Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (>85 degrees C and pH <3) of Yellowstone National Park, WY. The AFV1 20.8-kb, linear, double-stranded DNA genome encodes 40 putative open reading frames whose sequences generally show little similarity to other genes in the sequence databases. Because three-dimensional structures are more conserved than sequences and hence are more effective at revealing function, we set out to determine protein structures from putative AFV1 open reading frames (ORF). The crystal structure of ORF157 reveals an alpha+beta protein with a novel fold that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear double-stranded DNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined.
Asunto(s)
Acidianus/virología , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Lipothrixviridae/química , Lipothrixviridae/enzimología , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cristalografía por Rayos X , ADN/metabolismo , Análisis Mutacional de ADN , Desoxirribonucleasas/genética , Manantiales de Aguas Termales/microbiología , Lipothrixviridae/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Proteínas Virales/genéticaRESUMEN
Acidianus filamentous virus 1 (AFV1), a member of the Lipothrixviridae family, infects the hyperthermophilic, acidophilic crenarchaeaon Acidianus hospitalis. The virion, covered with a lipidic outer shell, is 9,100-A long and contains a 20.8-kb linear dsDNA genome. We have identified the two major coat proteins of the virion (MCPs; 132 and 140 amino acids). They bind DNA and form filaments when incubated with linear dsDNA. A C-terminal domain is identified in their crystal structure with a four-helix-bundle fold. In the topological model of the virion filament core, the genomic dsDNA superhelix wraps around the AFV1-132 basic protein, and the AFV1-140 basic N terminus binds genomic DNA, while its lipophilic C-terminal domain is imbedded in the lipidic outer shell. The four-helix bundle fold of the MCPs from AFV1 is identical to that of the coat protein (CP) of Sulfolobus islandicus rod-shaped virus (SIRV), a member of the Rudiviridae family. Despite low sequence identity between these proteins, their high degree of structural similarity suggests that they could have derived from a common ancestor and could thus define an yet undescribed viral lineage.
Asunto(s)
Proteínas de la Cápside/química , Lipothrixviridae/química , Pliegue de Proteína , Acidianus/virología , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Genoma Viral , Lipothrixviridae/genética , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Sulfolobus/químicaRESUMEN
Screening for viruses in samples taken from acidic hot springs of Kamchatka (Russia) revealed a collection of morphotypes, including linear, spherical and complex fusiform shapes, which show partial similarity to those found in acidic geothermal environments in other geographical locations. One of the viruses, Acidianus filamentous virus 9, AFV9, was isolated and its structure and genome were studied in detail.
Asunto(s)
Acidianus/virología , Manantiales de Aguas Termales/virología , Lipothrixviridae/aislamiento & purificación , Acidianus/clasificación , Acidianus/genética , Ácidos , Genoma Viral , Interacciones Huésped-Patógeno , Lipothrixviridae/genética , Lipothrixviridae/ultraestructura , Datos de Secuencia Molecular , Filogenia , Federación de Rusia , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/clasificación , Virión/genética , Virión/aislamiento & purificación , Virión/ultraestructuraRESUMEN
Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters.
Asunto(s)
Acidianus/virología , Genoma Viral/genética , Lipothrixviridae/clasificación , Lipothrixviridae/ultraestructura , Secuencia Conservada/genética , Microscopía por Crioelectrón , Orden Génico , Lipothrixviridae/genética , Lipothrixviridae/aislamiento & purificación , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Operón , Análisis de Secuencia de ADN , Secuencias Repetidas Terminales/genética , Proteínas no Estructurales Virales/genética , Proteínas Estructurales Virales/análisis , Virión/química , Virión/ultraestructuraRESUMEN
The objective of this work was to identify novel viral 'evasion' genes without homology in the database through functional assays. Using this approach, the 'unassigned', conserved murine gammaherpesvirus ORF20 gene was shown to localize in the nucleus and to induce cell-cycle arrest followed by apoptosis in both mouse and human cells. Such growth-arrested cells did not express phospho-histone H3, demonstrating that the virus protein caused arrest at the G2 stage of the cell cycle. To characterize the mechanism further, Western blots of ORF20-recombinant lentivirus-infected cells were developed with antibodies to cyclin B1, Cdc2 and phospho-Tyr-15-Cdc2. This analysis revealed a relative increase in cyclin B and phospho-Tyr-15-Cdc2, from 24 to 72 h after infection with recombinant lentivirus. The demonstration that Cdc2 is in its inactive phosphorylated form and the clearly increased levels of cyclin B indicated that the virus gene blocks the progression of cells into mitosis by acting at the level of the Cdc2-cyclin B complex. To confirm this result, the Cdc2-cyclin B complex in ORF20-expressing cells was shown to be essentially without kinase activity. As the ORF20 gene is conserved in all herpesvirus, it may be presumed to have evolved to fulfil an important, as yet undefined, biological role in host-cell modification.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclina B/antagonistas & inhibidores , Lipothrixviridae/genética , Sistemas de Lectura Abierta/genética , Animales , Apoptosis , Ciclo Celular , Línea Celular , Fase G2 , Humanos , Etiquetado Corte-Fin in Situ , Cinética , RatonesRESUMEN
The crenarchaeote Sulfolobus spp. is a host for a remarkably large spectrum of viruses and plasmids. The genetic elements characterized so far indicate a large degree of novelty in terms of morphology (viruses) and in terms of genome content (plasmids and viruses). The viruses and conjugative plasmids encode a great number of conserved proteins of unknown function due to the lack of sequence similarity to functionally characterized proteins. These apparently essential proteins remain to be studied and should help to understand the physiology and genetics of the respective genetic elements as well as the host. Sulfolobus is one of the best-studied archaeons and could develop into an important model organism of the crenarchaea and the archaea.
Asunto(s)
Virus de Archaea/genética , Plásmidos/genética , Sulfolobus/genética , Sulfolobus/virología , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Virus de Archaea/clasificación , Virus de Archaea/aislamiento & purificación , Secuencia de Bases , Conjugación Genética , ADN de Archaea/genética , ADN de Archaea/aislamiento & purificación , Fuselloviridae/genética , Fuselloviridae/aislamiento & purificación , Genoma Viral , Guttaviridae/genética , Guttaviridae/aislamiento & purificación , Lipothrixviridae/genética , Lipothrixviridae/aislamiento & purificación , Datos de Secuencia Molecular , Plásmidos/aislamiento & purificación , Rudiviridae/genética , Rudiviridae/aislamiento & purificación , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Sulfolobus/enzimologíaRESUMEN
A novel filamentous virus, AFV2, from the hyperthermophilic archaeal genus Acidianus shows structural similarity to lipothrixviruses but differs from them in its unusual terminal and core structures. The double-stranded DNA genome contains 31,787 bp and carries eight open reading frames homologous to those of other lipothrixviruses, a single tRNA(Lys) gene containing a 12-bp archaeal intron, and a 1,008-bp repeat-rich region near the center of the genome.
Asunto(s)
Genoma Viral , Lipothrixviridae/clasificación , Lipothrixviridae/genética , Manantiales de Aguas Termales/virología , Lipothrixviridae/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN de Transferencia de Lisina/genética , Proteínas Virales/genética , Virión/genéticaRESUMEN
The remarkable diversity of the morphologies of viruses found in terrestrial hydrothermal environments with temperatures >80 degrees C is unprecedented for aquatic ecosystems. The best-studied viruses from these habitats have been assigned to novel viral families: Fuselloviridae, Lipothrixviridae and Rudiviridae. They all have double-stranded DNA genomes and infect hyperthermophilic crenarchaea of the orders Sulfolobales and Thermoproteales. Representatives of the different viral families share a few homologous ORFs (open reading frames). However, about 90% of all ORFs in the seven sequenced genomes show no significant matches to sequences in public databases. This suggests that these hyperthermophilic viruses have exceptional biochemical solutions for biological functions. Specific features of genome organization, as well as strategies for DNA replication, suggest that phylogenetic relationships exist between crenarchaeal rudiviruses and the large eukaryal DNA viruses: poxviruses, the African swine fever virus and Chlorella viruses. Sequence patterns at the ends of the linear genome of the lipothrixvirus AFV1 are reminiscent of the telomeric ends of linear eukaryal chromosomes and suggest that a primitive telomeric mechanism operates in this virus.
Asunto(s)
Virus de Archaea/genética , Genoma Viral , Virus de la Fiebre Porcina Africana/genética , Secuencia de Bases , Virus ADN/genética , Planeta Tierra , Genoma , Lipothrixviridae/genética , Microscopía Electrónica , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Sulfolobus/genética , Telómero/ultraestructuraRESUMEN
We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23-130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes.
Asunto(s)
Acidianus/virología , Calor , Lipothrixviridae/clasificación , Lipothrixviridae/aislamiento & purificación , Acidianus/crecimiento & desarrollo , Secuencia de Bases , Genoma Viral , Lipothrixviridae/genética , Lipothrixviridae/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/ultraestructuraRESUMEN
The double-stranded DNA genomes of the viruses SIRV1 and SIRV2, which infect the extremely thermophilic archaeon Sulfolobus and belong to the family Rudiviridae, were sequenced. They are linear, covalently closed at the ends, and 32,312 and 35,502 bp long, respectively, with an A+T content of 75%. The genomes of SIRV1 and SIRV2 carry inverted terminal repeats of 2029 and 1628 bp, respectively, which contain multiple direct repeats. SIRV1 and SIRV2 genomes contain 45 and 54 ORFs, respectively, of which 44 are homologous to one another. Their predicted functions include a DNA polymerase, a Holliday junction resolvase, and a dUTPase. The genomes consist of blocks with well-conserved sequences separated by nonconserved sequences. Recombination, gene duplication, horizontal gene transfer, and substitution of viral genes by homologous host genes have contributed to their evolution. The finding of head-to-head and tail-to-tail linked replicative intermediates suggests that the linear genomes replicate by the same mechanism as the similarly organized linear genomes of the eukaryal poxviruses, African swine fever virus and Chlorella viruses. SIRV1 and SIRV2 both contain motifs that resemble the binding sites for Holliday junction resolvases of eukaryal viruses and may use common mechanisms for resolution of replicative intermediates. The results suggest a common origin of the replication machineries of the archaeal rudiviruses and the above-mentioned eukaryal viruses. About 1/3 of the ORFs of each rudivirus have homologs in the Sulfolobus virus SIFV of the family Lipothrixviridae, indicating that the two viral families form a superfamily. The finding of inverted repeats of at least 0.8 kb at the termini of the linear genome of SIFV supports this inference.