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1.
Nat Commun ; 11(1): 5993, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239638

RESUMO

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.


Assuntos
Proteínas Arqueais/antagonistas & inibidores , Proteínas Associadas a CRISPR/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Sulfolobus/genética , Proteínas Virais/metabolismo , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas/genética , Clivagem do DNA , Interações Hospedeiro-Patógeno/genética , Domínios Proteicos/genética , Proteínas Repressoras/genética , Rudiviridae/genética , Rudiviridae/metabolismo , Rudiviridae/patogenicidade , Sulfolobus/virologia , Proteínas Virais/genética , Proteínas Virais/ultraestrutura
2.
Trends Microbiol ; 28(11): 913-921, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32499102

RESUMO

Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas immune systems. To date, Acrs inhibiting types I, II, III, V, and VI CRISPR-Cas systems have been characterized. While most known Acrs are derived from bacterial phages and prophages, very few have been characterized in the domain Archaea, despite the nearly ubiquitous presence of CRISPR-Cas in archaeal cells. Here we summarize the discovery and characterization of the archaeal Acrs with the representatives encoded by a model archaeal virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2). AcrID1 inhibits subtype I-D CRISPR-Cas immunity through direct interaction with the large subunit Cas10d of the effector complex, and AcrIIIB1 inhibits subtype III-B CRISPR-Cas immunity through a mechanism interfering with middle/late gene targeting. Future development of efficient screening methods will be key to uncovering the diversity of archaeal Acrs.


Assuntos
Archaea/imunologia , Proteínas Arqueais/imunologia , Vírus de Archaea/fisiologia , Sistemas CRISPR-Cas , Rudiviridae/fisiologia , Archaea/genética , Archaea/virologia , Proteínas Arqueais/genética , Vírus de Archaea/genética , Rudiviridae/genética
3.
ISME J ; 14(7): 1821-1833, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32322010

RESUMO

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.


Assuntos
Vírus de Archaea , Rudiviridae , Vírus , Vírus de Archaea/genética , Vírus de DNA/genética , Genoma Viral , Humanos , Itália , Rudiviridae/genética
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.
Cell ; 179(2): 448-458.e11, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31564454

RESUMO

Bacteria and archaea possess a striking diversity of CRISPR-Cas systems divided into six types, posing a significant barrier to viral infection. As part of the virus-host arms race, viruses encode protein inhibitors of type I, II, and V CRISPR-Cas systems, but whether there are natural inhibitors of the other, mechanistically distinct CRISPR-Cas types is unknown. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB1, encoded by the Sulfolobus virus SIRV2. AcrIIIB1 exclusively inhibits CRISPR-Cas subtype III-B immunity mediated by the RNase activity of the accessory protein Csx1. AcrIIIB1 does not appear to bind Csx1 but, rather, interacts with two distinct subtype III-B effector complexes-Cmr-α and Cmr-γ-which, in response to protospacer transcript binding, are known to synthesize cyclic oligoadenylates (cOAs) that activate the Csx1 "collateral" RNase. Taken together, we infer that AcrIIIB1 inhibits type III-B CRISPR-Cas immunity by interfering with a Csx1 RNase-related process.


Assuntos
Proteínas Associadas a CRISPR/fisiologia , Sistemas CRISPR-Cas , Interações Hospedeiro-Patógeno , Rudiviridae/metabolismo , Sulfolobus/virologia , Ribonucleases/metabolismo
6.
Environ Microbiol ; 21(6): 2002-2014, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30451355

RESUMO

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.


Assuntos
Archaea/virologia , Vírus de Archaea/isolamento & purificação , Bacteriófagos/isolamento & purificação , Fontes Termais/virologia , Rudiviridae/genética , Rudiviridae/isolamento & purificação , Archaea/genética , Archaea/isolamento & purificação , Vírus de Archaea/classificação , Vírus de Archaea/genética , Vírus de Archaea/fisiologia , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Genoma Viral , Fontes Termais/química , Japão , Lipothrixviridae/classificação , Lipothrixviridae/genética , Lipothrixviridae/isolamento & purificação , Lipothrixviridae/fisiologia , Metagenoma , Filogenia , Rudiviridae/classificação , Replicação Viral
7.
RNA Biol ; 16(4): 557-565, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30146914

RESUMO

Carrier state viral infection constitutes an equilibrium state in which a limited fraction of a cellular population is infected while the remaining cells are transiently resistant to infection. This type of infection has been characterized for several bacteriophages but not, to date, for archaeal viruses. Here we demonstrate that the rudivirus SIRV3 can produce a host-dependent carrier state infection in the model crenarchaeon Sulfolobus. SIRV3 only infected a fraction of a Sulfolobus islandicus REY15A culture over several days during which host growth was unimpaired and no chromosomal DNA degradation was observed. CRISPR spacer acquisition from SIRV3 DNA was induced by coinfecting with the monocaudavirus SMV1 and it was coincident with increased transcript levels from subtype I-A adaptation and interference cas genes. However, this response did not significantly affect the carrier state infection of SIRV3 and both viruses were maintained in the culture over 12 days during which SIRV3 anti-CRISPR genes were shown to be expressed. Transcriptome and proteome analyses demonstrated that most SIRV3 genes were expressed at varying levels over time whereas SMV1 gene expression was generally low. The study yields insights into the basis for the stable infection of SIRV3 and the resistance to the different host CRISPR-Cas interference mechanisms. It also provides a rationale for the commonly observed coinfection of archaeal cells by different viruses in natural environments.


Assuntos
Sistemas CRISPR-Cas/genética , Imunidade , Rudiviridae/genética , Sulfolobus/genética , Sulfolobus/imunologia , Coinfecção/virologia , DNA Viral/genética , Genoma Viral , Heterozigoto , Interações Hospedeiro-Patógeno/genética , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfolobus/crescimento & desenvolvimento , Proteínas Virais/metabolismo
8.
Viruses ; 10(12)2018 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-30544778

RESUMO

Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Rudiviridae/genética , Sulfolobus/virologia , DNA Viral/genética , Técnicas de Inativação de Genes , Genoma Viral , Mutação , Reação em Cadeia da Polimerase , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Biochem Mol Biol Educ ; 46(5): 547-554, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30369034

RESUMO

Contemporary biology is currently undergoing a revolution, driven by the availability of high-throughput technologies and a wide variety of bioinformatics tools. However, bioinformatics education and practice is still in its infancy in most of the African continent. Consequently, concerted efforts have been made in recent years to incorporate bioinformatics modules into biological sciences curriculum of African Universities. Despite this, one aspect of bioinformatics that is yet to be incorporated is structural bioinformatics. In this article, we report on a structural bioinformatics project carried out by final year project students in a Nigerian university. The target protein was the thermoacidophilic Sulfolobus islandicus rod-shaped virus 1 (SIRV1) Rep protein, which was further characterized using various free, user-friendly and online sequence-based and structure-based bioinformatics tools. This exercise gave students the opportunity to generate new data, interpret the data, and acquire collaborative research skills. In this report, emphasis is placed on analysis of the data generated to further encourage analytical skills. By sharing this experience, it is anticipated that other similar institutions would adopt parallel strategies to expose undergraduate students to structural biology, and increase awareness of freely available bioinformatics tools for tackling pertinent biological questions. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):547-554, 2018.


Assuntos
Biologia Computacional/educação , Rudiviridae/química , Estudantes , Universidades
10.
Nat Microbiol ; 3(4): 461-469, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29507349

RESUMO

Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR-Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3. We show that a 4 kb deletion in the SIRV2 genome dramatically reduces infectivity in Sulfolobus islandicus LAL14/1 that carries functional CRISPR-Cas subtypes I-A, I-D and III-B. Subsequent insertion of a single gene from SIRV3, gp02 (AcrID1), which is conserved in the deleted fragment, successfully restored infectivity. We demonstrate that AcrID1 protein inhibits the CRISPR-Cas subtype I-D system by interacting directly with Cas10d protein, which is required for the interference stage. Sequence and structural analysis of AcrID1 show that it belongs to a conserved family of compact, dimeric αß-sandwich proteins characterized by extreme pH and temperature stability and a tendency to form protein fibres. We identify about 50 homologues of AcrID1 in four archaeal viral families demonstrating the broad distribution of this group of anti-CRISPR proteins.


Assuntos
Proteínas Associadas a CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas/fisiologia , Proteínas Repressoras/metabolismo , Rudiviridae/patogenicidade , Sulfolobus/virologia , Proteínas Associadas a CRISPR/metabolismo , Proteínas Repressoras/genética , Rudiviridae/genética , Sulfolobus/genética
11.
Viruses ; 9(7)2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28718834

RESUMO

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod-shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function. The SIRV2_Gp1 protein is highly expressed during early stages of infection and it is the only protein that is encoded twice on the viral genome. It harbours a helix-turn-helix motif and was therefore hypothesized to bind DNA. The DNA-binding behavior of SIRV2_Gp1 was characterized with electrophoretic mobility shift assays and atomic force microscopy. We provide evidence that the protein interacts with DNA and that it forms large aggregates, thereby causing extreme condensation of the DNA. Furthermore, the N-terminal domain of the protein mediates toxicity to the viral host Sulfolobus. Our findings may lead to biotechnological applications, such as the development of a toxic peptide for the containment of pathogenic bacteria, and add to our understanding of the Rudiviral infection cycle.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Rudiviridae/metabolismo , Sulfolobus/virologia , Proteínas Virais/metabolismo , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Genoma Viral , Conformação de Ácido Nucleico , Domínios Proteicos , Rudiviridae/genética , Proteínas Virais/química , Vírion , Liberação de Vírus
12.
J Virol ; 91(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28424282

RESUMO

Viral factories are compartmentalized centers for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by prototypical archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in the model archaeon Sulfolobus This rod-shaped virus belongs to the viral family Rudiviridae, carrying linear double-stranded DNA (dsDNA) genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that of the four host DNA polymerases (DNA polymerase I [Dpo1] to Dpo4), only Dpo1 participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life.IMPORTANCE The organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication center by a virus infecting members of the Archaea domain.


Assuntos
Rudiviridae/crescimento & desenvolvimento , Sulfolobus/virologia , Montagem de Vírus , Replicação Viral , Proteínas Arqueais/análise , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/análise , Interações Hospedeiro-Parasita , Microscopia , Sulfolobus/química , Proteínas Virais/análise
13.
Nucleic Acids Res ; 44(18): 8799-8809, 2016 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-27407114

RESUMO

Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional 'brush-like' structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.


Assuntos
Replicação do DNA , DNA Viral/genética , Genoma Viral , Sequências Repetidas Invertidas , Células Cultivadas , DNA Viral/química , Hibridização In Situ , Rudiviridae/genética , Replicação Viral
14.
Science ; 348(6237): 914-7, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25999507

RESUMO

Extremophiles, microorganisms thriving in extreme environmental conditions, must have proteins and nucleic acids that are stable at extremes of temperature and pH. The nonenveloped, rod-shaped virus SIRV2 (Sulfolobus islandicus rod-shaped virus 2) infects the hyperthermophilic acidophile Sulfolobus islandicus, which lives at 80°C and pH 3. We have used cryo-electron microscopy to generate a three-dimensional reconstruction of the SIRV2 virion at ~4 angstrom resolution, which revealed a previously unknown form of virion organization. Although almost half of the capsid protein is unstructured in solution, this unstructured region folds in the virion into a single extended α helix that wraps around the DNA. The DNA is entirely in the A-form, which suggests a common mechanism with bacterial spores for protecting DNA in the most adverse environments.


Assuntos
DNA Forma A/metabolismo , Rudiviridae/metabolismo , Sulfolobus/genética , Sulfolobus/virologia , Vírion/ultraestrutura , Sequência de Aminoácidos , Microscopia Crioeletrônica , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Rudiviridae/ultraestrutura , Esporos Bacterianos/genética , Esporos Bacterianos/virologia
15.
J Mol Biol ; 427(12): 2179-91, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25828870

RESUMO

The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5' → 3' ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.


Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/metabolismo , Óperon , Rudiviridae/enzimologia , Proteínas Virais/metabolismo , DNA Helicases/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Rudiviridae/genética , Proteínas Virais/genética
16.
J Mol Biol ; 426(22): 3683-3688, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25277654

RESUMO

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Sulfolobus solfataricus/metabolismo , Apoptose , Western Blotting , Proteínas Associadas a CRISPR/genética , Cromossomos de Archaea/metabolismo , DNA Arqueal/metabolismo , Técnicas de Inativação de Genes , Mutação/genética , Regiões Promotoras Genéticas/genética , Rudiviridae/patogenicidade , Sulfolobus solfataricus/crescimento & desenvolvimento , Sulfolobus solfataricus/virologia
17.
J Virol ; 88(17): 10264-8, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24965447

RESUMO

Sulfolobus mutants resistant to archaeal lytic virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) were isolated, and mutations were identified in two gene clusters, cluster sso3138 to sso3141 and cluster sso2386 and sso2387, encoding cell surface and type IV secretion proteins, respectively. The involvement of the mutations in the resistance was confirmed by genetic complementation. Blocking of virus entry into the mutants was demonstrated by the lack of early gene transcription, strongly supporting the idea of a role of the proteins in SIRV2 entry.


Assuntos
Proteínas Arqueais/metabolismo , Interações Hospedeiro-Parasita , Proteínas de Membrana/metabolismo , Rudiviridae , Sulfolobus/metabolismo , Sulfolobus/virologia , Internalização do Vírus , Teste de Complementação Genética , Família Multigênica , Mutação
18.
J Virol ; 88(12): 7105-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24696494

RESUMO

Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication.


Assuntos
Proteínas Arqueais/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rudiviridae/metabolismo , Sulfolobus/metabolismo , Sulfolobus/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , DNA Arqueal/genética , DNA Arqueal/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Rudiviridae/química , Rudiviridae/genética , Sulfolobus/genética , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
19.
Proc Natl Acad Sci U S A ; 111(10): 3829-34, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24567401

RESUMO

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.


Assuntos
Modelos Moleculares , Complexos Multiproteicos/metabolismo , Conformação Proteica , Rudiviridae/metabolismo , Sulfolobus/virologia , Proteínas Virais/metabolismo , Liberação de Vírus/fisiologia , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli , Complexos Multiproteicos/química , Plasmídeos/genética , Saccharomyces cerevisiae , Proteínas Virais/química
20.
J Virol ; 87(24): 13379-85, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24089554

RESUMO

A decisive step in a virus infection cycle is the recognition of a specific receptor present on the host cell surface, subsequently leading to the delivery of the viral genome into the cell interior. Until now, the early stages of infection have not been thoroughly investigated for any virus infecting hyperthermophilic archaea. Here, we present the first study focusing on the primary interactions between the archaeal rod-shaped virus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) (family Rudiviridae) and its hyperthermoacidophilic host, S. islandicus. We show that SIRV2 adsorption is very rapid, with the majority of virions being irreversibly bound to the host cell within 1 min. We utilized transmission electron microscopy and whole-cell electron cryotomography to demonstrate that SIRV2 virions specifically recognize the tips of pilus-like filaments, which are highly abundant on the host cell surface. Following the initial binding, the viral particles are found attached to the sides of the filaments, suggesting a movement along these appendages toward the cell surface. Finally, we also show that SIRV2 establishes superinfection exclusion, a phenomenon not previously described for archaeal viruses.


Assuntos
Rudiviridae/metabolismo , Sulfolobus/virologia , Vírion/fisiologia , Internalização do Vírus , Fímbrias Bacterianas/virologia , Rudiviridae/ultraestrutura , Vírion/ultraestrutura , Ligação Viral
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