The Experts below are selected from a list of 156 Experts worldwide ranked by ideXlab platform
Kenneth M Stedman - One of the best experts on this subject based on the ideXlab platform.
-
extreme mutation tolerance nearly half of the archaeal fusellovirus sulfolobus spindle shaped virus 1 genes are not required for virus function including the minor capsid protein gene vp3
Journal of Virology, 2017Co-Authors: Eric A Iverson, David A Goodman, Madeline E Gorchels, Kenneth M StedmanAbstract: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.
-
differential virus host ranges of the Fuselloviridae of hyperthermophilic archaea implications for evolution in extreme environments
Frontiers in Microbiology, 2012Co-Authors: Ruben Michael Ceballos, Caleb D Marceau, Joshua Ovila Marceau, Steven Morris, Adam J Clore, Kenneth M StedmanAbstract:An emerging model for investigating virus-host interactions in hyperthermophilic Archaea is the Fusellovirus-Sulfolobus system. The host, Sulfolobus, is a hyperthermophilic acidophile endemic to sulfuric volcanic-driven hot springs worldwide. The Fuselloviruses, also known as Sulfolobus Spindle-shaped Viruses (SSVs), are “lemon” or “spindle” shaped double-stranded DNA viruses that are also found worldwide. Although a few studies have addressed the host-range for the type virus, SSV1, using common Sulfolobus strains, a comprehensive host-range study for SSV-Sulfolobus systems has not been performed. Herein, we examine six bona fide SSV strains (SSV1, SSV2, SSV3, SSVL1, SSVK1, SSVRH) and their respective infection characteristics on multiple hosts from the family Sulfolobaceae. A “halo” assay was used to determine virus infectivity and host susceptibility. Different SSV strains have different host-ranges with SSV1 exhibiting the narrowest host-range and SSVRH exhibiting the broadest host range. There is no correlation between geographic separation of viruses and their hosts and their relative infectivity and susceptibility. In contrast to previous reports, SSVs can infect hosts beyond the genus Sulfolobus. Furthermore, the Fusellovirus-Sulfolobus system appears to exhibit host-advantage. This work provides a foundation for understanding Fusellovirus biology and virus-host co-evolution in extreme ecosystems, a rapidly emerging field of study.
-
comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses
Journal of Virology, 2004Co-Authors: Blake Wiedenheft, Kenneth M Stedman, Francisco F Roberto, Deborah A Willits, Annekathrin Gleske, Luisa Zoeller, Jamie C Snyder, Trevor Douglas, Mark J YoungAbstract:The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of approximately 15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.
-
relationships between fuselloviruses infecting the extremely thermophilic archaeon sulfolobus ssv1 and ssv2
Research in Microbiology, 2003Co-Authors: Kenneth M Stedman, Hans Peter Arnold, Ingelore Holz, Hien Phan, Roger A Garrett, Wolfram ZilligAbstract:The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.
Mart Krupovic - One of the best experts on this subject based on the ideXlab platform.
-
Eukaryotic-Like Virus Budding in Archaea
mBio, 2016Co-Authors: Emmanuelle R. J. Quemin, David Prangishvili, Patrick Forterre, Petr Chlanda, Mart Sachse, Mart KrupovicAbstract: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 eu-karyotic 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 bacteria and archaea. Here, we provide a sequential view on the assembly and egress of SSV1, a pro-totypic 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 archaeal 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.
-
Eukaryotic-Like Virus Budding in Archaea
American Society for Microbiology, 2016Co-Authors: Emmanuelle R. J. Quemin, David Prangishvili, Patrick Forterre, Petr Chlanda, Martin Sachse, Mart KrupovicAbstract: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
-
Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein and host-derived lipids
Journal of Virology, 2015Co-Authors: Emmanuelle R. J. Quemin, David Prangishvili, Maija K. Pietilä, Hanna M. Oksanen, P. Forterre, W. Irene C. Rijpstra, Stefan Schouten, Dennis H. Bamford, Mart KrupovicAbstract: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. Thelarger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogeneticallydistant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one ofthe first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactionsin Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conductedan extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structuralproteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergoposttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition tothe 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, resolvinga long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolatedfrom the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmicmembrane, likely during progeny egress.
Claire Geslin - One of the best experts on this subject based on the ideXlab platform.
-
Research Article Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
2016Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, Le M. Romancer, H. Van TilbeurghAbstract:Copyright © 2013 N. Leulliot et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiformmembers of the family Fuselloviridae or the genus Salterprovirus.The 18 kb dsDNAgenome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome.We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å.Theprotein forms dimers both in solution and in the crystal.The fold of PAV1-137 is a four
-
Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
Archaea, 2013Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, M. Le Romancer, H. Van TilbeurghAbstract:Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 A. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.
-
PAV1, the First Virus-Like Particle Isolated from a Hyperthermophilic Euryarchaeote, “Pyrococcus abyssi”
Journal of Bacteriology, 2003Co-Authors: Claire Geslin, Gaël Erauso, M. Le Romancer, M. Gaillard, G. Perrot, Daniel PrieurAbstract:We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "Pyrococcus abyssi" previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the Fuselloviridae, isolated from Sulfolobus shibatae. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of Thermococcales isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.
-
recherche et caracterisation de particules virales chez les thermococcales archaea hyperthermophiles description du premier virus de pyrococcus abyssi pav1
2002Co-Authors: Claire GeslinAbstract:Ces dernieres annees, un certain nombre de nouveaux virus aux caracteristiques tout a fait originales ont ete decrits chez les Archaea hyperthermophiles. D'une facon surprenante, tous ont ete obtenus a partir du phylum des Crenarchaeota, principalement parmi les representants de l'ordre des Sulfolobales, tandis qu'aucun virus n'a ete decrit pour les membres hyperthermophiles du phylum des Euryarchaeota. Dans cette these nous rapportons la caracterisation de la premiere particule de type viral ou " VU " pour "Virus-like particle ", isolee d'une Euryarchaeota hyperthermophile, Pyrococcus abyssi souche GE23. Cette particule, nommee PAV1, a la forme d'un citron (120 nm x 80 nm) et ressemble a SSV1 de Sulfolobus sp. , le virus modele de la famille Fuselloviridae composee exclusivement de virus archaeens. Nous avons montre que PAV1 se maintient dans son hote dans un etat porteur. Les particules de PAV1 contiennent un ADN double-brin circulaire de 18 kb qui est aussi present a un nombre de copies eleve sous forme libre dans le cytoplasme de l'hote. Ce genome a ete clone et entierement sequence. 24 cadres ouverts de lecture ont ete identifies. La recherche de motifs structuraux et de domaines conserves a notamment revele une similitude significative entre l'ORF 678 de PAV1 et un domaine sialidase que l'on retrouve chez les enzymes lytiques virales. Par ailleurs, l'ORF 183 montre une certaine homologie avec une proteine, identifiee dans plusieurs plasmides d'Archaea hyperthermophiles, qui pourrait etre un regulateur transcriptionnel. La derniere partie de cette etude a ete consacree a une approche de la diversite et de la distribution geographique de virus associes a des micro-organismes hydrothermaux hyperthermophiles et neutrophiles. Nous avons realise une recherche systematique sur des echantillons collectes de differents sites hydrothermaux situes au niveau de la dorsale du Pacifique Oriental (9ʿN et 13ʿN), de la dorsale medio Atlantique (36ʿN et 37ʿN) et de la dorsale centrale Indienne (25ʿS). 94 cultures d'enrichissement ont ete obtenues a 85ʿC a partir de 107 echantillons bruts. Des VLPs ont ete detectees par microscopie electronique dans 16 cultures d'enrichissement differentes. Parmi les differents morphotypes decouverts, la forme "citron" prevaut, mais des bâtonnets rigides, des filaments et des formes uniques pleomorphes ont egalement ete observes, ce qui laisse presager une importante diversite virale chez les Euryarchaeota hyperthermophiles.
Patrizia Contursi - One of the best experts on this subject based on the ideXlab platform.
-
unravelling the role of the f55 regulator in the transition from lysogeny to uv induction of sulfolobus spindle shaped virus 1
Journal of Virology, 2015Co-Authors: Salvatore Fusco, Qunxin She, Simonetta Bartolucci, Gabriella Fiorentino, Patrizia ContursiAbstract:ABSTRACT 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.
-
STRUCTURAL AND FUNCTIONAL STUDIES OF STF76 FROM THE SULFOLOBUS ISLANDICUS PLASMID–VIRUS PSSVX: A NOVEL PECULIAR MEMBER OF THE WINGED HELIX–TURN–HELIX TRANSCRIPTION FACTOR FAMILY
Università degli studi di Padova, 2014Co-Authors: Luciano Pirone, Patrizia Contursi, Biancamaria Farina, Salvatore Fusco, Luigi Russo, Simonetta Bartolucci, Roberto Fattorusso, Emilia PedoneAbstract:Seven families of double-stranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and therefore represent model systems for detailed studies of archaeal virus biology. Two distinct genetic elements, SSV2 and pSSVx, belong to Fuselloviridae and coexist in the same Sulfolobus islandicus REY15/4 host, thus representing one of the few known two-virus systems in Archaea (1). pSSVx is a satellite virus that generates virus particles with the help of SSV2-associated packaging mechanisms. The transcriptional pattern of pSSVx undergoes a temporal variation of gene expression during its own life cycle, thus providing a good model for studying regulation of gene expression in Archaea (2). This genetic element encodes four TFs possibly implicated in the regulation of gene expression, i.e. ORF-c68, ORF51, ORF91 and ORF76. Among these, ORF76, here named Stf76 (Sulfolobus transcription factor 76 aminoacid protein), has homologs in almost all conjugative and cryptic plasmids from Sulfolobus (3), thus suggesting a relevant role for this protein in replication and/or maintenance of the plasmid. In this study, we have performed a detailed structural and functional characterization of Stf76. The corresponding gene has been cloned, expressed in Escherichia coli and the recombinant protein purified to homogeneity. To elucidate its interaction with the identified DNA operator sequence, analyses regarding its DNA binding capabilities by means of EMSA, circular dichroism, spectrofluorimetric and isothermal titration calorimetry experiments have been performed. Moreover, a structural study has been undertaken by Nuclear Magnetic Resonance spectroscopy leading to:(i) the solution structure of Stf76 based on CS-Rosetta approach, (ii) the characterization of the Stf76-DNA interaction by chemical shift perturbation analysis, (iii) a structural model describing the interaction of a single Stf76 monomer with its DNA operator. Altogether these results contribute to elucidate the regulatory mechanism underpinning the role of this protein (4). REFERENCES: 1. ARNOLD, H.P., ET AL.(1999) THE GENETIC ELEMENT PSSVX OF THE EXTREMELY THERMOPHILIC CRENARCHAEON SULFOLOBUS IS A HYBRID BETWEEN A PLASMID AND A VIRUS. MOL MICROBIOL, 34, 217-226. 2. CONTURSI, P., ET.AL. (2010) TRANSCRIPTION TERMINATION IN THE PLASMID/VIRUS HYBRID PSSVX FROM SULFOLOBUS ISLANDICUS. EXTREMOPHILES, 14, 453-463. 3. LIPPS, G. (2006) PLASMIDS AND VIRUSES OF THE THERMOACIDOPHILIC CRENARCHAEOTE SULFOLOBUS. EXTREMOPHILES, 10, 17-28. 4. CONTURSI P., FARINA B., PIRONE L. ET AL. NUCLEIC ACIDS RES. ACCEPTED 28 FEB. 2014
-
Structure, function and unexplored properties of fuselloviruses-encoded transcription factors
'Oxford University Press (OUP)', 2014Co-Authors: Salvatore Fusco, Qunxin She, Simonetta Bartolucci, Emilia Pedone, Patrizia ContursiAbstract:Objective: Archaeal viruses are predicted to encode for a wide variety of transcriptional regulators. The majority of these gene products do not have detectable homologues in the databases other than in related hyperthermophilic viral genomes, thus leading to the necessity of performing structural and functional analyses to unravel their role. Archaeal transcription regulators resemble the bacterial ones at structural level but operate in an eukaryal-like transcriptional context (1). Therefore, it is expected that in this “hybrid” transcriptional apparatus, peculiar and not yet characterized mechanisms for the regulation of expression are employed and novel folds and/or functions might be discovered. Fuselloviridae is one of the best-characterized families of crenarchaeal viruses, whose members have been used as model systems to understand the mechanisms underpinning the transcription process at structural and functional level. Methods: Putative transcription factors encoded by pSSVx and SSV1 have been studied with the purpose of determining their structure and/or dissecting their regulative mechanisms. In this regard, a combination of approaches, ranging from DNA-protein interaction assays to structure determination techniques, has been used. Results: The discovery and characterization of the SSV1-encoded regulator F55 (2) helped to shed light on the molecular mechanisms governing the maintenance of the SSV1 lysogeny. This protein has been proved to bind, in vitro and in vivo, to the viral genome at the promoters of the UV-inducible (Tind), of the early transcripts (T5 and T6) as well as of its own transcript (Tlys). Indeed, band-shift and chromatin immunoprecipitation assays confirmed the interaction of F55 with these regulative regions and its dissociation soon after UV irradiation of SSV1 lysogenic cells. The structure of two proteins encoded by the hybrid plasmid/virus pSSVx has been solved by nuclear magnetic resonance spectroscopy and X-ray diffraction, revealing that ORFc-68 bears a looped-hinged-helix motif (3) whereas Stf76 assumes a winged helix-turn-helix fold (4). These DNA-binding proteins interact specifically with target sequences located within promoters of their gene, thus regulating their own expression. Despite the resemblance of the overall fold to that of bacterial transcription regulators, both ORFc68 and Stf76 display peculiar structural features. Interestingly, a 28aa-long stretch of the Stf76 amino acid sequence (named Pep37V-64R) that encompasses the DNA-binding domain was in silico predicted to be a potential cationic antimicrobial peptide (CAMP) and tested for antimicrobial and antifungal activities towards several strains. It was revealed that both Gram+ and Gram- strains were sensitive to the action of Pep37V-64R, possibly as a consequence of membrane disaggregation and/or of the retained ability of Pep37V-64R to interact with nucleic acids. Further biochemical and structural studies on this peptide are underway. Conclusion: Altogether these analyses expand the current knowledge about the structures adopted by archaeal transcription factors and their mechanisms of action. Moreover, it has been revealed that the domain of Achaea might represent a potential reservoir of bioactive peptides. References: 1. Contursi et al. (2013) Extremophiles, 17(6):881-895 2. Fusco et al., (2013) J Virol, 87(10):5926-5936 3. Contursi et al., (2011) Biochem J, 435(1):157-166 4. Contursi et al., (2014) Nucleic Acids Res, [Epub ahead of print] doi: 10.1093/nar/gku21
-
p55, a newly identified Ribbon-Helix-Helix transcription factor encoded by the fusellovirus SSV1: elucidation of an archaeal host-virus relationship
2012Co-Authors: Salvatore Fusco, Qunxin She, Simonetta Bartolucci, Patrizia ContursiAbstract:Sulfolobus spindle shaped virus 1 (SSV1) is the type member of the archaeal Fuselloviridae virus family. Viruses belonging to this family share similar features such as genome size, structure and organization as well as viron morphology. The replication of SSV1 has shown to be strongly enhanced by UV-light irradiation. Moreover, the physical map of eleven transcripts (T1-9, Tx and Tind) is available and them transcription patterns have been investigated before and after UV-irradiation of lysogenic cells. Nevertheless, the molecular bases of the viral life cycle induction are still poorly known and unclear. Recently, microarray analyses led to the identification of a twelfth viral transcript, referred as Tp55, which showed high level of expression under condition of no-UV irradiation (carrier stage). This transcript and in turn the encoded protein, named p55, have never been showed to be expressed after UV-light irradiation. In silico analyses of p55 protein sequence have suggested that it could adopt a Ribbon-Helix-Helix fold (RHH), a structural motif typically found in negative transcription regulators. Such kind of regulators has been identified in Bacteria, Archaea and Bacteriophages, but no RHH motifs have been characterized in eukaryotic proteins. Several RHH transcription factors are important for host-pathogen interactions, thus suggesting a convergent evolution of the regulation machineries underlying several unrelated viral life cycles. Unlike the better-characterized Helix-Turn-Helix motif, the DNA-binding-specificity determinants of the RHH motif are located within its amino-terminal -strand. The functional unit of the RHH domain is a twofold symmetric dimer that is capable to bind multiple sites within operons that are arranged as inverted or tandem repeats. Several DNA tandem repeats, that act as targets for p55, have shown to be located in a region of the SSV1 genome critical for the UV-induction. The binding specificity of p55 to these sequences has been tested by band shift assays
H. Van Tilbeurgh - One of the best experts on this subject based on the ideXlab platform.
-
Research Article Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
2016Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, Le M. Romancer, H. Van TilbeurghAbstract:Copyright © 2013 N. Leulliot et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiformmembers of the family Fuselloviridae or the genus Salterprovirus.The 18 kb dsDNAgenome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome.We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å.Theprotein forms dimers both in solution and in the crystal.The fold of PAV1-137 is a four
-
Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi
Archaea, 2013Co-Authors: Nicolas Leulliot, Sophie Quevillon-cheruel, Marc Graille, Claire Geslin, Didier Flament, M. Le Romancer, H. Van TilbeurghAbstract:Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 A. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.
-
ORF157 from the Archaeal Virus Acidianus Filamentous Virus 1 Defines a New Class of Nuclease
Journal of virology, 2010Co-Authors: Adeline Goulet, David Prangishvili, Nicolas Leulliot, H. Van Tilbeurgh, Peter Redder, Mery Pina, Laura Vera, Julie Lichière, Miguel Ortiz-lombardía, Valérie CampanacciAbstract:The properties of double-stranded DNA (dsDNA) viruses that infect Crenarchaea living in acidic hot springs (pH 1.5 to 3 and 75 to 95°C) are radically different from those of the viruses that infect Bacteria and Eukarya. Not only are the shapes of these viruses distinct from those of all other known viruses, but ∼80% of their open reading frames (ORFs) do not share any sequence similarity with ORFs of other viruses or of cellular life forms apart from other archaeal viruses (44). Seven novel viral families have been created to categorize their unique characteristics: the spindle-shaped Fuselloviridae, the filamentous Rudiviridae and Lipothrixviridae, the bottle-shaped Ampullaviridae, the droplet-shaped Guttaviridae, the spherical Globuloviridae, the two-tailed Bicaudaviridae, and the unclassified Sulfolobus turreted icosahedral virus (STIV) (43). The study of archaeal viruses is still in its infancy compared to that of eukaryal and bacterial viruses, and little is known regarding crenarchaeal virus life cycles, virus-host relationships, genetics, or biochemistry. Further studies of these viruses are expected to provide genetic, biochemical, and evolutionary insight into their crenarchaeal hosts and the requirements for life in the harsh environments. Transcription cycles of the fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) (16) and the rudiviruses Sulfolobus islandicus rod-shaped virus 1 and 2 (SIRV1 and SIRV2) (24) have been analyzed. Also, recent results concerning the lytic viruses STIV and SIRV2 shed new light on their replication cycle and interaction with their hosts (7, 9, 38). Because structures generally are much better conserved than sequences, structural studies have aimed at uncovering functional and evolutionary relationships that are not apparent from the primary sequence. To date, 11 protein structures from crenarchaeal viruses have been reported (18, 19, 22, 23, 25-30, 33). Most of these proteins share structural similarity with proteins of known function: three winged-helix proteins are likely involved in transcriptional regulation (two from the fusellovirus SSV1 [27, 33] and one from STIV [28]); one glycosyltransferase from STIV displays the GT-A fold (30); one adaptor protein from SSV1 is similar to the repressor of primer (ROP) of Escherichia coli (26); and the major coat protein of STIV has revealed the first evolutionary relationship spanning the three domains of life (25). The structures of the highly conserved ORFs among these viruses, ORF109 of lipothrixvirus AFV3 (22) and ORFB116 from STIV (29), suggest that they are DNA-binding proteins that function in transcriptional regulation. Filamentous viruses, the most abundant morphotype in these extreme environments, form the new viral order Ligamenvirales, which is divided into the Rudiviridae and Lipothrixviridae families (43). Lipothrixviruses (Acidianus filamentous virus 1 to 9, Thermoproteus tenax virus 1 to 3, and Sulfolobus islandicus filamentous virus) (8, 43), which were the first enveloped filamentous viruses with linear dsDNA genomes discovered, infect acidophilic and hyperthermophilic Crenarchaea. They are classified into α, β, γ, and δ genera based on their genomic properties and on the diversity of their terminal appendages, which are involved in host cell recognition. AFV1 is a γ-lipothrixvirus isolated from an acidic hot spring in Yellowstone National Park, where the temperature is above 85°C and the pH below 3 (6). The linear, double-stranded 20.8-kb DNA genome of AFV1 encodes 40 putative ORFs, 32% of which are homologous to viral ORFs from the lipothrixvirus SIFV and the rudiviruses SIRV1 and SIRV2. The predicted products generally are too dissimilar to the sequences in the public databases to allow functional assignment; only two glycosyltransferases, two CopG-like proteins, and one transcription regulator have been detected (6). It is highly unlikely that all of the encoded proteins consist entirely of unique protein folds serving novel functions. Therefore, to get insight into the biology of the Lipothrixviridae, we performed crystallographic studies of the AFV1 proteome. Although the solved structures of AFV1-102 (23) and the homologs AFV1-99 (19) and SIFV-014 (18) have not revealed any structural homologs, they have suggested that the proteins are involved in protein-protein interaction and are minor structural components, respectively. Here, we report the crystal structure of AFV1-157 and its biochemical characterization. AFV1-157 is a 157-residue protein with one homolog in the fusellovirus Sulfolobus spindle-shaped virus Ragged Hills (SSVRH). It has a novel α+β fold that remotely resembles the nucleotidyltransferase topology. We demonstrated that (i) AFV1-157 exhibits in vitro nuclease activity that degrades linear dsDNA, and (ii) the E86 residue is essential for the nuclease activity.
