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1.
J Gen Virol ; 102(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33151141

RESUMO

A challenge in virology is quantifying relative virulence (V R) between two (or more) viruses that exhibit different replication dynamics in a given susceptible host. Host growth curve analysis is often used to mathematically characterize virus-host interactions and to quantify the magnitude of detriment to host due to viral infection. Quantifying V R using canonical parameters, like maximum specific growth rate (µ max), can fail to provide reliable information regarding virulence. Although area-under-the-curve (AUC) calculations are more robust, they are sensitive to limit selection. Using empirical data from Sulfolobus Spindle-shaped Virus (SSV) infections, we introduce a novel, simple metric that has proven to be more robust than existing methods for assessing V R. This metric (I SC) accurately aligns biological phenomena with quantified metrics to determine V R. It also addresses a gap in virology by permitting comparisons between different non-lytic virus infections or non-lytic versus lytic virus infections on a given host in single-virus/single-host infections.


Assuntos
Virologia/métodos , Vírus/patogenicidade , Archaea/crescimento & desenvolvimento , Archaea/virologia , Área Sob a Curva , Fuselloviridae/crescimento & desenvolvimento , Fuselloviridae/patogenicidade , Interações Hospedeiro-Patógeno , Modelos Biológicos , Virulência , Replicação Viral , Vírus/crescimento & desenvolvimento
2.
Biochim Biophys Acta Gene Regul Mech ; 1863(5): 194493, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32014611

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/genética
3.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31748395

RESUMO

Fuselloviruses are among the most widespread and best-characterized archaeal viruses. They exhibit remarkable diversity, as the list of members of this family is rapidly growing. However, it has yet to be shown how a fuselloviral genome may undergo variation at the levels of both single nucleotides and sequence stretches. Here, we report the isolation and characterization of four novel spindle-shaped viruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22), from a hot spring in the Philippines. SSV19 is a member of the genus Alphafusellovirus, whereas SSV20-22 belong to the genus Betafusellovirus The genomes of SSV20-SSV22 are identical except for the presence of two large variable regions, as well as numerous sites of single-nucleotide polymorphisms (SNPs) unevenly distributed throughout the genomes and enriched in certain regions, including the gene encoding the putative end filament protein VP4. We show that coinfection of the host with SSV20 and SSV22 led to the formation of an SSV21-like virus, presumably through homologous recombination. In addition, large numbers of SNPs were identified in DNA sequences retrieved by PCR amplification targeting the SSV20-22 vp4 gene from the original enrichment culture, indicating the enormous diversity of SSV20-22-like viruses in the environment. The high variability of VP4 is consistent with its potential role in host recognition and binding by the virus.IMPORTANCE How a virus survives in the arms race with its host is an intriguing question. In this study, we isolated and characterized four novel fuselloviruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22). Interestingly, SSV20-22 differ primarily in two genomic regions and are apparently convertible through homologous recombination during coinfection. Moreover, sites of single-nucleotide polymorphism (SNP) were identified throughout the genomes of SSV20-22 and, notably, enriched in certain regions, including the gene encoding the putative end filament protein VP4, which is believed to be involved in host recognition and binding by the virus.


Assuntos
Fuselloviridae/genética , Sulfolobus/genética , Sequência de Aminoácidos , Vírus de Archaea/genética , Evolução Biológica , Vírus de DNA/genética , DNA Viral/genética , Fuselloviridae/isolamento & purificação , Variação Genética/genética , Genoma Viral , Genômica/métodos , Fontes Termais/virologia , Filipinas , Sulfolobus/isolamento & purificação , Sulfolobus/virologia , Proteínas Virais/metabolismo
4.
Mol Microbiol ; 113(4): 718-727, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31774609

RESUMO

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain.


Assuntos
Resistência à Doença/genética , Proteínas de Fímbrias/metabolismo , Fuselloviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , Rudiviridae/fisiologia , Sulfolobus , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/metabolismo , Sulfolobus/genética , Sulfolobus/virologia , Ligação Viral
5.
Nat Commun ; 10(1): 4797, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641111

RESUMO

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.


Assuntos
Proteínas Arqueais/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sulfolobus solfataricus/citologia , Sulfolobus solfataricus/virologia , Proteínas Arqueais/genética , Parede Celular/genética , Parede Celular/metabolismo , Cromossomos de Archaea , Fuselloviridae , Técnicas de Silenciamento de Genes , Inativação Gênica , Teste de Complementação Genética , Glicosilação , Interações Hospedeiro-Patógeno/genética , Sulfolobus solfataricus/genética
6.
Arch Virol ; 162(10): 3239-3242, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28681144

RESUMO

The complete nucleotide sequence of a novel positive single-stranded (+ss) RNA virus, tentatively named watermelon virus A (WVA), was determined using a combination of three methods: RNA sequencing, small RNA sequencing, and Sanger sequencing. The full genome of WVA is comprised of 8,372 nucleotides (nt), excluding the poly (A) tail, and contains four open reading frames (ORFs). The largest ORF, ORF1 encodes a putative replication-associated polyprotein (RP) with three conserved domains. ORF2 and ORF4 encode a movement protein (MP) and coat protein (CP), respectively. The putative product encoded by ORF3, of an estimated molecular mass of 25 kDa, has no significant similarity with other proteins. Identity and phylogenetic analysis indicate that WVA is a new virus, closely related to members of the family Betaflexiviridae. However, the final taxonomic allocation of WVA within the family is yet to be determined.


Assuntos
Citrullus/virologia , Fuselloviridae , Genoma Viral , Sequência de Bases , RNA/genética
7.
J Virol ; 91(10)2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28148789

RESUMO

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/genética
8.
mBio ; 7(5)2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27624130

RESUMO

UNLABELLED: Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission. IMPORTANCE: The replication of enveloped viruses has been extensively studied in eukaryotes but has remained unexplored for enveloped viruses infecting Archaea Here, we provide a sequential view on the assembly and egress of SSV1, a prototypic archaeal virus. The observed process is highly similar to the budding of eukaryotic enveloped viruses, including human immunodeficiency virus, influenza virus, and Ebola virus. The present study is the first to characterize such a phenomenon in archeal cells, showing that membrane budding is not an exclusive feature of eukaryotic viruses. Our results provide significant insights into the biogenesis and architecture of unique, spindle-shaped virions that infect archaea. Furthermore, our findings open doors for future inquiries into (i) the evolution of the virus budding process, (ii) mechanistic details of virus-mediated membrane scission in Archaea, and (iii) elucidation of virus- and host-encoded molecular players responsible for archaeal membrane and surface remodeling.


Assuntos
Archaea/virologia , Fuselloviridae/fisiologia , Liberação de Vírus , Archaea/ultraestrutura , Tomografia com Microscopia Eletrônica , Fuselloviridae/ultraestrutura
9.
J Virol ; 89(22): 11681-91, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26355093

RESUMO

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/genética
10.
J Virol ; 89(21): 10934-44, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26292330

RESUMO

UNLABELLED: SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (Int(SSV2)). Int(SSV2) alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of Int(SSV2) (residues 81 to 328), which corresponded to the putative product of the 3'-end sequence of the Int(SSV2) gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length Int(SSV2) except for the slightly reduced recombination activity. Our results suggest that, unlike λ integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion. IMPORTANCE: Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.


Assuntos
DNA Nucleotidiltransferases/metabolismo , Fuselloviridae/enzimologia , Integrases/fisiologia , Filogenia , Sulfolobales/virologia , Integração Viral/genética , Sequência de Bases , Cromatografia em Gel , Análise por Conglomerados , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas In Vitro , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Ligação Proteica , Análise de Sequência de DNA , Especificidade por Substrato
11.
J Virol ; 89(12): 6453-61, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25878101

RESUMO

UNLABELLED: Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so far the only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as a molecular switch for the transcriptional regulation of the early viral genes. IMPORTANCE: Functional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.


Assuntos
Fuselloviridae/fisiologia , Regulação Viral da Expressão Gênica , Lisogenia/efeitos da radiação , Sulfolobus/virologia , Fatores de Transcrição/metabolismo , Replicação Viral , Regiões Promotoras Genéticas , Ligação Proteica , Sulfolobus/efeitos da radiação , Raios Ultravioleta , Proteínas Virais/metabolismo
12.
Biochimie ; 118: 322-32, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25891845

RESUMO

Fuselloviruses SSV1 and SSV2 are model systems to investigate virus-host relationships in stably infected cells thanks to their temperate nature. Although they are very similar in morphology, genome organization and gene synteny, their replication is induced by different stimuli, i.e.: by UV-light exposure (for SSV1) and by the growth progression of the host (for SSV2). In this study, we have analysed global gene expression in SSV1- and SSV2-lysogens of Sulfolobus solfataricus P2 in the absence of any stimuli. Additionally, the interplay among SSV1, SSV2 and the host has been investigated in a double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the host-viral interaction in the frame of the CRISPR-response.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Fuselloviridae , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica em Archaea/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transcriptoma , Proteínas Virais/biossíntese , Proteínas Virais/genética
13.
Extremophiles ; 19(2): 539-46, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25479832

RESUMO

The Fuselloviridae prototype member Sulfolobus spindle-shaped virus 1 is a model of UV-inducible viruses infecting Crenarchaeota. Previous works on SSV1 UV induction were bases on empirically determined parameters that have not yet been standardized. Thus, in many peer reviewed literature, it is not clear how the fluence and irradiance have been determined. Here, we describe a protocol for the UV induction of SSV1 replication, which is based on the combination of the following instrumentally monitored parameters: (1) the fluence; (2) the irradiance; (3) the exposure time, and (4) the exposure distance. With the aim of finding a good balance between the viral replication induction and the host cells viability, UV-irradiated cultures were monitored for their ability to recover in the aftermath of the UV exposure. This UV irradiation procedure has been set up using the well-characterized Sulfolobus solfataricus P2 strain as model system to study host-virus interaction.


Assuntos
Fuselloviridae/efeitos da radiação , Sulfolobus/virologia , Raios Ultravioleta , Virologia/métodos , Ativação Viral , Fuselloviridae/fisiologia
14.
Virology ; 474: 105-9, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25463608

RESUMO

The structure and assembly of many icosahedral and helical viruses are well-characterized. However, the molecular basis for the unique spindle-shaped morphology of many viruses that infect Archaea remains unknown. To understand the architecture and assembly of these viruses, the spindle-shaped virus SSV1 was examined using cryo-EM, providing the first 3D-structure of a spindle-shaped virus as well as insight into SSV1 biology, assembly and evolution. Furthermore, a geometric framework underlying the distinct spindle-shaped structure is proposed.


Assuntos
Fuselloviridae/ultraestrutura , Archaea/virologia , Simulação por Computador , Microscopia Crioeletrônica , Evolução Molecular , Fuselloviridae/genética , Fuselloviridae/fisiologia , Imageamento Tridimensional , Modelos Moleculares , Vírion/ultraestrutura , Montagem de Vírus
15.
Nucleic Acids Res ; 42(9): 5993-6011, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24682827

RESUMO

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.


Assuntos
Fuselloviridae/química , Proteínas Virais/química , Fatores de Transcrição Winged-Helix/química , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Sulfolobus/virologia
16.
Extremophiles ; 18(3): 473-89, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24562787

RESUMO

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 Genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
J Virol ; 88(4): 2354-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24335300

RESUMO

Viruses with spindle-shaped virions are abundant in diverse environments. Over the years, such viruses have been isolated from a wide range of archaeal hosts. Evolutionary relationships between them remained enigmatic, however. Here, using structural proteins as markers, we define familial ties among these "dark horses" of the virosphere and segregate all spindle-shaped viruses into two distinct evolutionary lineages, corresponding to Bicaudaviridae and Fuselloviridae. Our results illuminate the utility of structure-based virus classification and bring additional order to the virosphere.


Assuntos
Archaea/virologia , Vírus de Archaea/genética , Vírus de Archaea/ultraestrutura , Vírus de DNA/genética , Vírus de DNA/ultraestrutura , Modelos Moleculares , Filogenia , Vírus de Archaea/classificação , Sequência de Bases , Mapeamento Cromossômico , Vírus de DNA/classificação , Evolução Molecular , Fuselloviridae/classificação , Fuselloviridae/genética , Marcadores Genéticos/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas Estruturais Virais/genética
18.
Virology ; 441(2): 126-34, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23579037

RESUMO

The fusellovirus SSV2 and the integrative plasmid pSSVi, which constitute a unique helper-satellite virus system, replicate in Sulfolobus solfataricus P2. In this study, we investigated the interplay among SSV2, pSSVi and their host by transcriptomic analysis. Following infection of S. solfataricus P2, SSV2 activated its promoters in a temporal and distributive fashion, starting from the transcription of ORF305. Expression of several host genes encoding DNA replication and transcription proteins was up-regulated, suggesting that SSV2 depended heavily on the host replication machinery for its replication. SSV2 gene expression appeared to follow a similar pattern in S. solfataricus P2 harboring pSSVi to that in S. solfataricus P2 lacking the plasmid. Several early genes of the virus were transcribed earlier and more efficiently in the presence of pSSVi than in its absence. These results provide valuable clues to the understanding of the three-way interactions among SSV2, pSSVi and the host.


Assuntos
Fuselloviridae/crescimento & desenvolvimento , Plasmídeos , Sulfolobus solfataricus/virologia , Transcriptoma , Fuselloviridae/genética , Interações Microbianas , Vírus Satélites/genética , Vírus Satélites/crescimento & desenvolvimento , Sulfolobus solfataricus/genética , Transcrição Genética
19.
Mol Microbiol ; 80(2): 481-91, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21385233

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that - unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus- DNA is targeted in this archaeon.


Assuntos
Fuselloviridae/crescimento & desenvolvimento , Sulfolobus solfataricus/genética , DNA Arqueal/genética , DNA Viral/genética , Fuselloviridae/genética , Fuselloviridae/imunologia , Plasmídeos , Recombinação Genética , Transfecção
20.
Biochem J ; 435(1): 157-66, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21208189

RESUMO

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 & purificação
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