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.

Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

UNLABELLED: Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. IMPORTANCE: STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into the virion during infection. The experiments described here highlight the elements of this enzyme that are essential for proper function and also provide supporting evidence that B204 is present in the mature STIV virion.

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.

Archaeal Viruses of the Sulfolobales: Isolation, Infection, and CRISPR Spacer Acquisition.

Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environmental virus mixture isolated from Yellowstone National Park (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012). Experimental studies of isolated genetic elements from this mixture revealed that SMV1 (S ulfolobus Monocauda Virus 1), a tailed spindle-shaped virus, can induce spacer acquisition in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs and the techniques used both to infect laboratory strains with these virus mixtures and to obtain purified virus particles. Secondly, we present the experimental conditions required for activating SMV1-induced spacer acquisition in two different Sulfolobus species.

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.

First experimental evidence for the presence of a CRISPR toxin in sulfolobus.

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.

SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2.

Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze-thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.

Novel insights into gene regulation of the rudivirus SIRV2 infecting Sulfolobus cells.

Microarray analysis of infection by a lytic Sulfolobus rudivirus, SIRV2, revealed both the temporal expression of viral genes and the differential regulation of host genes. A highly susceptible strain derived from Sulfolobus solfataricus P2 with a large genomic deletion spanning CRISPR clusters A to D was infected with SIRV2, and subjected to a microarray analysis. Transcripts from a few viral genes were detected at 15 min post-infection and all except one were expressed within 2 h. The earliest expressed genes were located mainly at the termini of the linear viral genome while later expressed genes were concentrated in the central region. Timing of the expression correlated with the known or predicted functions of the viral gene products and, thus, should facilitate functional characterization of many hypothetical viral genes. Evaluation of the microarray data with quantitative reverse-transcription PCR analyses of a few selected viral genes revealed a good correlation between the two methods. Expression of about 3,000 host genes was examined. Seventy-two were downregulated>2-fold that were mainly associated with stress response and vesicle formation, as well as chromosome structure maintenance, which appears to contribute to host chromosome degradation and cellular collapse. A further 76 host genes were upregulated>2-fold and they were dominated by genes associated with metabolism and membrane transport, including phosphate transport and DNA precursor synthesis. The altered transcriptional patterns suggest that the virus reprograms the host cellular machinery to facilitate its own DNA replication and to inhibit cellular processes required for defense against viruses.

Transcriptomic analysis of the SSV2 infection of Sulfolobus solfataricus with and without the integrative plasmid pSSVi.

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

T(lys), a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes.

While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (T(lys)) was identified. Transcriptional analysis revealed that T(lys) is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and T(ind)) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and T(ind) transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the T(ind) promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1.

Insights into a viral lytic pathway from an archaeal virus-host system.

Archaeal host cells infected by Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. This viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. The STIV protein C92 and the SIRV2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of STIV and SIRV2, respectively. Since SIRV2 and STIV have fundamentally different morphotypes and genome sequences, it is surprising that they share this lysis system. In this study, we have constructed a collection of C92/P98 chimeric proteins and tested their abilities, both in the context of virus replication and alone, to form pyramid lysis structures in S. solfataricus. The results of this study illustrate that these proteins are functionally homologous when expressed as individual chimeric proteins but not when expressed in the context of complete STIV infection.

Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.

Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Fossil record of an archaeal HK97-like provirus.

One of the outstanding questions in biology today is the origin of viruses. We have discovered a protein in the hyperthermophile Sulfolobus solfataricus while following proteome regulation during viral infection that led to the discovery of a fossil provirus. Characterization of the wild type and recombinant protein revealed that it assembled into virus-like particles with a diameter of ~32nm. Sequence and structural analyses showed that the likely proviral capsid protein, Sso2749, is homologous to a protein from Pyrococcus furiosus that forms virus-like particles using the HK-97 major capsid protein fold. The SsP2-provirus appears mosaic and contains proteins with similarity to, among others, eukaryotic herpesviruses and tailed dsDNA bacteriophage families, reinforcing the hypothesis of a common ancestral gene pool across all three domains of life. This is the first description of the HK-97 fold in a crenarchaeal virus and the first direct genomic connection of linocin-like protein cages to a virus.

Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

In vivo virus structures: simultaneous classification, resolution enhancement, and noise reduction in whole-cell electron tomography.

Sulfolobus Turreted Icosahedral Virus (STIV) experiences an extra-cellular environment of near boiling acid (80°C, pH 3) and particles purified under these conditions were previously analyzed by cryo electron microscopy and image reconstruction. Here we describe cryo-tomograms of Solfolobus cells infected with STIV and the maximum likelihood algorithm employed to compute reconstructions of virions within the cell. Virions in four different tomograms were independently reconstructed with an average of 91 particles per tomogram and their structures compared with each other and with the higher resolution single-particle reconstruction from purified virions. The algorithm described here automatically classified and oriented two different particle types within each cell and generated reconstructions of full and empty particles. Because the particles are randomly oriented within the cell, the reconstructions do not suffer from the missing wedge of data absent from the reciprocal-space tomogram. The fact that the particles have icosahedral symmetry is used to dramatically improve the signal to noise ratio in the reconstructions. The reconstructions have approximately 60Å resolution (based on Fourier Shell Correlation analysis among reconstructions computed by the algorithm described here from four different tomograms).

Use of cellular CRISPR (clusters of regularly interspaced short palindromic repeats) spacer-based microarrays for detection of viruses in environmental samples.

It is currently difficult to detect unknown viruses in any given environment. The recent discovery of CRISPR (clusters of regularly interspaced short palindromic repeats) loci within bacterial and archaeal cellular genomes may provide an alternative approach to detect new viruses. It has been shown that the spacer sequences between the direct repeat units of the CRISPR loci are often derived from viruses and likely function as guide sequences to protect the cell from viral infection. The spacer sequences within the CRISPR loci may therefore serve as a record of the viruses that have replicated within the cell. We have cataloged the CRISPR spacer sequences from cellular metagenomic data from high-temperature (>80°C), acidic (pH < 4) hot spring environments located in Yellowstone National Park (YNP). We designed a microarray platform utilizing these CRISPR spacer sequences as potential probes to detect viruses present in YNP hot spring environments. We show that this microarray approach can detect viral sequences directly from virus-enriched environmental samples, detecting new viruses which have not been previously characterized. We further demonstrated that this microarray approach can be used to examine temporal changes in viral populations within the environment. Our results demonstrate that CRISPR spacer sequence-based microarrays will be useful tools for detecting and monitoring viruses from diverse environmental samples.

Particle assembly and ultrastructural features associated with replication of the lytic archaeal virus sulfolobus turreted icosahedral virus.

Little is known about the replication cycle of archaeal viruses. We have investigated the ultrastructural changes of Sulfolobus solfataricus P2 associated with infection by Sulfolobus turreted icosahedral virus (STIV). A time course of a near synchronous STIV infection was analyzed using both scanning and transmission electron microscopy. Assembly of STIV particles, including particles lacking DNA, was observed within cells, and fully assembled STIV particles were visible by 30 h postinfection (hpi). STIV was determined to be a lytic virus, causing cell disruption beginning at 30 hpi. Prior to cell lysis, virus infection resulted in the formation of pyramid-like projections from the cell surface. These projections, which have not been documented in any other host-virus system, appeared to be caused by the protrusion of the cell membrane beyond the bordering S-layer. These structures are thought to be sites at which progeny virus particles are released from infected cells. Based on these observations of lysis, a plaque assay was developed for STIV. From these studies we propose an overall assembly model for STIV.

Transcriptome analysis of infection of the archaeon Sulfolobus solfataricus with Sulfolobus turreted icosahedral virus.

Microarray analysis of infection by Sulfolobus turreted icosahedral virus (STIV) revealed insights into the timing and extent of virus transcription, as well as differential regulation of host genes. Using a microarray containing genes from both the host and the virus, the infection cycle of STIV was studied. Following infection of Sulfolobus solfataricus strain 2-2-12 with STIV, transcription of virus genes was first detected at 8 h postinfection (p.i.), with a peak at 24 h p.i. Lysis of cells was first detected at 32 h p.i. There was little temporal control of the transcription of virus genes, although the three open reading frames on the noncoding strand were transcribed later in the infection process. During the infection, 177 host genes were determined to be differentially expressed, with 124 genes up-regulated and 53 genes down-regulated. The up-regulated genes were dominated by genes associated with DNA replication and repair and those of unknown function, while the down-regulated genes, mostly detected at 32 h p.i., were associated with energy production and metabolism. Examination of infected cells by transmission electron microscopy revealed alterations in cell ultrastructure consistent with the microarray analysis. The observed patterns of transcription suggest that up-regulated genes are likely used by the virus to reprogram the cell for virus replication, while the down-regulated genes reflect the imminent lysis of the cells.

The SSV1 viral integrase is not essential.

Viral integration is a widely conserved characteristic in viruses in all domains of life; however, its necessity is not well understood in many cases. Integration using tyrosine recombinases is one of the most widespread and best characterized mechanisms of integration. We completely removed the tyrosine recombinase integrase from the hyperthermophilic and acidophilic archaeal virus SSV1 using a novel LIPCR technique and found that the virus still replicated and spread in its host Sulfolobus solfataricus without integration. The mutant virus maintained a persistent infection but the integrase-lacking virus was less competitive than the wild-type virus when co-cultured. Based on these results, we discuss the necessity of integration and the possible advantages of this type of replication strategy.