Sulfolobus islandicus Employs Orc1-2-Mediated DNA Damage Response in Defense against Infection by SSV2.

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

Genomics, Transcriptomics, and Proteomics of SSV1 and Related Fusellovirus: A Minireview.

Saccharolobus spindle-shaped virus 1 (SSV1) was one of the first viruses identified in the archaeal kingdom. Originally isolated from a Japanese species of Saccharolobus back in 1984, it has been extensively used as a model system for genomic, transcriptomic, and proteomic studies, as well as to unveil the molecular mechanisms governing the host-virus interaction. The purpose of this mini review is to supply a compendium of four decades of research on the SSV1 virus.

Structural insights into a spindle-shaped archaeal virus with a sevenfold symmetrical tail.

Archaeal viruses with a spindle-shaped virion are abundant and widespread in extremely diverse environments. However, efforts to obtain the high-resolution structure of a spindle-shaped virus have been unsuccessful. Here, we present the structure of SSV19, a spindle-shaped virus infecting the hyperthermophilic archaeon Sulfolobus sp. E11-6. Our near-atomic structure reveals an unusual sevenfold symmetrical virus tail consisting of the tailspike, nozzle, and adaptor proteins. The spindle-shaped capsid shell is formed by seven left-handed helical strands, constructed of the hydrophobic major capsid protein, emanating from the highly glycosylated tail assembly. Sliding between adjacent strands is responsible for the variation of a virion in size. Ultrathin sections of the SSV19-infected cells show that SSV19 virions adsorb to the host cell membrane through the tail after penetrating the S-layer. The tailspike harbors a putative endo-mannanase domain, which shares structural similarity to a Bacteroides thetaiotaomicro endo-mannanase. Molecules of glycerol dibiphytanyl glycerol tetraether lipid were observed in hydrophobic clefts between the tail and the capsid shell. The nozzle protein resembles the stem and clip domains of the portals of herpesviruses and bacteriophages, implying an evolutionary relationship among the archaeal, bacterial, and eukaryotic viruses.

Construction of a Fusellovirus with a Minimal Set of Genes.

A large number of fuselloviruses have been found in acidic hot springs around the globe. They share a set of highly conserved genes (core genes) and possess a varying number of less-conserved genes (non-core genes). However, the functions of most of these genes are unknown. Recent studies show that as many as half of these genes tolerate mutation. In this study, we conducted a genetic analysis on Saccharolobus spindle-shaped virus 22 (SSV22), an alphafusellovirus with fewer open reading frames (ORFs) than most of the isolated fuselloviruses. Both deletion and frame-shift mutations were introduced into nearly all of the 26 ORFs of the viral genome. A total of 17 ORFs were indispensable, and two additional ORFs were required for the optimal infectivity of the virus. Deletion of either VP2 or VP3, the two structural proteins, did not affect the morphology or infectivity of the virus. An infectious SSV22 derivative carrying a minimal genome of 20 ORFs was obtained. The SSV22 capsid was capable of accommodating a genome as large as ∼18 kb, or ∼7 kb larger than that of the wild-type virus. The viral capsid varied in both the length and width, but not in shape, with the size of the genome. Our results will facilitate the analysis of crucial protein-protein interactions between SSV22 and the host during viral infection and help explore the use of SSV22 as a vector for DNA delivery in potential applications.

New insights into the diversity and evolution of the archaeal mobilome from three complete genomes of Saccharolobus shibatae.

Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. It serves as a natural host to the extensively studied spindle-shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus-host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus-to-plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.

Quantifying relative virulence: when µmax fails and AUC alone just is not enough.

A challenge in virology is quantifying relative virulence (VR) 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 VR using canonical parameters, like maximum specific growth rate (µmax), 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 VR. This metric (ISC) accurately aligns biological phenomena with quantified metrics to determine VR. 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.

The interaction between the F55 virus-encoded transcription regulator and the RadA host recombinase reveals a common strategy in Archaea and Bacteria to sense the UV-induced damage to the host DNA.

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.

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus.

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.

Novel Sulfolobus Fuselloviruses with Extensive Genomic Variations.

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.

CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection.

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.

The complete nucleotide sequence and genome organization of a novel betaflexivirus infecting Citrullus lanatus.

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.

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.

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.

Eukaryotic-Like Virus Budding in Archaea.

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.

Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids.

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.

Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions.

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.

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1.

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.

Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system.

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.

A standardized protocol for the UV induction of Sulfolobus spindle-shaped virus 1.

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.

Structural insights into the architecture of the hyperthermophilic Fusellovirus SSV1.

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.

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.

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.