RESUMO
Sulfolobus spindle-shaped virus 1 is the only UV-inducible member of the virus family Fuselloviridae. Originally isolated from Saccharolobus shibatae B12, it can also infect Saccharolobus solfataricus. Like the CI repressor of the bacteriophage λ, the SSV1-encoded F55 transcription repressor acts as a key regulator for the maintenance of the SSV1 carrier state. In particular, F55 binds to tandem repeat sequences located within the promoters of the early and UV-inducible transcripts. Upon exposure to UV light, a temporally coordinated pattern of gene expression is triggered. In the case of the better characterized bacteriophage λ, the switch from lysogenic to lytic development is regulated by a crosstalk between the virus encoded CI repressor and the host RecA, which regulates also the SOS response. For SSV1, instead, the regulatory mechanisms governing the switch from the carrier to the induced state have not been completely unravelled. In this study we have applied an integrated biochemical approach based on a variant of the EMSA assay coupled to mass spectrometry analyses to identify the proteins associated with F55 when bound to its specific DNA promoter sequences. Among the putative F55 interactors, we identified RadA and showed that the archaeal molecular components F55 and RadA are functional homologs of bacteriophage λ (factor CI) and Escherichia coli (RecA) system.
Assuntos
Proteínas Arqueais/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fuselloviridae/genética , Fuselloviridae/metabolismo , Fuselloviridae/patogenicidade , Regiões Promotoras Genéticas , Ligação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo , Sulfolobus/efeitos da radiação , Sulfolobus/virologia , Fatores de Transcrição/genética , Raios Ultravioleta , Proteínas Virais/genéticaRESUMO
Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology.IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses.
Assuntos
Proteínas do Capsídeo/genética , Fuselloviridae/genética , Sulfolobus/virologia , Montagem de Vírus , Fuselloviridae/metabolismo , Genoma Viral , Fontes Termais , Mutação , Sulfolobus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/genéticaRESUMO
UNLABELLED: Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE: Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fuselloviridae/metabolismo , Lipídeos de Membrana/metabolismo , Sulfolobus solfataricus/virologia , Sequência de Aminoácidos , Fuselloviridae/genética , Genoma Viral/genética , Glicosilação , Haloarcula/virologia , Interações Hospedeiro-Patógeno , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Proteínas Virais/metabolismo , Montagem de Vírus/genéticaRESUMO
Fuselloviruses, also known as Sulfolobus Spindle-shaped viruses (SSVs), are "lemon"- or "spindle"-shaped double-stranded DNA viruses. Among them, SSV1, SSV2 and the satellite viruses pSSVx and pSSVi have been investigated at the structural, genetic, transcriptomic, proteomic and biochemical levels, thus becoming models for dissecting DNA replication/gene expression in Archaea. Important progress has been made including elucidation of temporal genome expression during virus infection and induction of replication, SSV1 lysogeny maintenance as well as differentially expression of pSSVx replicase. Future researches focusing on these model systems would yield insightful knowledge of life cycle and DNA replication of fuselloviruses.
Assuntos
Archaea/virologia , Fuselloviridae/metabolismo , Regulação Viral da Expressão Gênica , Sequência de Aminoácidos , Archaea/genética , Archaea/metabolismo , Fuselloviridae/genética , Fuselloviridae/patogenicidade , Fuselloviridae/ultraestrutura , Dados de Sequência Molecular , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein that does not show significant sequence homology with any protein with known three-dimensional structure. EMSA (electrophoretic mobility-shift assay) experiments, DNA footprinting and CD analyses indicate that recombinant C68, purified from Escherichia coli, binds to two different operator sites that are located upstream of its own promoter. The three-dimensional structure, solved by a single-wavelength anomalous diffraction experiment on a selenomethionine derivative, shows that the protein assumes a swapped-hairpin fold, which is a distinctive fold associated with a family of prokaryotic transcription factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features.
Assuntos
Fuselloviridae/metabolismo , Plasmídeos/metabolismo , Sulfolobus/virologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Pegada de DNA , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fuselloviridae/genética , Dados de Sequência Molecular , Regiões Operadoras Genéticas , Plasmídeos/genética , Regiões Promotoras Genéticas , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus Satélites/genética , Vírus Satélites/metabolismo , Selenometionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfolobus/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH