Insights into the biology of archaeal viruses by high-throughput approaches

Get Complete Project Material File(s) Now! »

Insights into the biology of hyperthermophilic archaeal viruses.

Fusiform and filamentous VLPs are highly abundant and widely distributed in archaeadominated habitats. The two groups of viruses are represented by fusellovirus SSV1 and rudivirus SIRV2 which have been among the first archaeal viruses to be isolated from geothermal environments where they can infect Sulfolobus cells. SSV1 has a rather broad host range (Schleper et al., 1992), whereas SIRV2 can only infect a limited number of strains of S. islandicus (Bize et al., 2009). The two viruses serve as model systems for the study of hyperthermophilic archaeal viruses.
SSV1. The genome of SSV1 was shown to be present in S. shibatae B12 in two forms: as a linear form within the host chromosome and as free, circular episomal copies in the cytoplasm (Yeats et al., 1982). UV irradiation is a strong stimulus to enhance the production of lemonshaped particles encasing the circular form of the viral genome (Martin et al., 1984). SSV1 was initially called SAV-1 due to misclassification of its natural host as a strain of S. acidocaldarius. It is a temperate virus and infection results in a lysogenic cycle leading to growth recovery of cultures even after stimulation (Schleper et al., 1992). The capacity to integrate into the cellular genome at a specific site within a tRNA-Arginine gene (Reiter et al., 1989) has been used to establish one of the first genetic systems in Archaea (Schleper et al., 1992). As a result, the viral tyrosine recombinase has been extensively studied (Muskhelishvili et al., 1993; Serre et al., 2002; Letzelter et al., 2004; Zhan et al., 2012). Development of genetic tools has also allowed systematic analysis of the functions of viral open reading frames (ORFs) and effects of their deletions on virus fitness (Stedman et al., 1999; Iverson and Stedman, 2012). In particular, the integrase gene has been shown to be non-essential for infection (Clore and Stedman, 2007). Interestingly, unlike the situation found in bacteriophages, upon viral genome integration, the integrase gene is partitioned in two fragments (Reiter et al., 1989). Several isolates are now known to be similar to SSV1 in morphology, genomic content, replication strategy, etc. (Stedman et al., 2003; Wiedenheft et al., 2004; Redder et al., 2009); nevertheless, SSV1 remains to be a model to understand the biology of spindle-shaped viruses. Using genome-wide microarray, it was shown that there is a tight regulation of gene expression timing, reminiscent of bacteriophages and eukaryotic viruses. The transcription starts with a small UV-specific transcript and continues with early and late transcripts towards the end of the viral cycle (Frols et al., 2007a; Fusco et al., 2013).
Interestingly, there was no marked difference detected in the transcriptome of the host S. solfataricus, in line with the postulated egress of SSV1 by budding through the cytoplasmic membrane without lysis of the host (Martin et al., 1984). Most of the particles released are uniform in size (60×100 nm) although up to 1% of viral population can be larger, the maximum length being about 300 nm (Reiter et al., 1988). Recently, the structure of SSV1 was examined by cryo-electron microscopy (cryo-EM) and 3D image reconstruction. A model of SSV1 structure has been proposed despite the fact that resolution was severely limited by particle size, lack of global symmetry, structural heterogeneity, and a small number of particles considered (Stedman et al., 2015). In particular, the presence of an actual lipid membrane encasing the virion body could not be verified and remained controversial up to now. Thus, one of the main objectives of my PhD was to perform a comprehensive biochemical characterization of SSV1 virions which is described in the Chapter 4. Briefly, we showed that SSV1 is a lipid-containing virus composed of glycrosylated proteins and hostderived lipids encasing the nucleoprotein filament (Quemin et al., 2015). These findings provide insights into the architecture of unique archaeal viruses and are used as a foundation for ongoing studies targeting the interactions of SSV1 with its host Sulfolobus (Quemin et al., in preparation). We recently obtained significant insights into the assembly and release strategy utilized by SSV1 virions which are presented in the Chapter 5.

Virus-host interactions in Archaea: state-of-the-art.

Members of the third domain of life, the archaea, were initially regarded as exotic microorganisms capable of growing in conditions which are hostile to humans. Among other intriguing features, they are now known to host unique viruses classified into exclusive viral families. Several studies have permitted the isolation of highly diverse viruses characterized by atypical virion shapes and mysterious genomic contents. The research undertaken in the past thirty years has improved our appreciation of the virosphere associated with archaea.
However, the study of archaeal viruses imposes serious constraints and the collection of virushost systems found in laboratories is far from representing the situation observed in natural environments. The isolation and characterization are indeed limited due to the need of culturing cells under extreme conditions of temperature, pH, salinity, pressure, etc. which are complicated to set up in laboratory. Another restriction comes from the viruses themselves which tend to be produced in low titers rendering analysis by classical techniques often challenging.
Using high-throughput approaches, one can neglect some of these factors and overcome the major difficulties linked to the research on archaeal viruses. In the Chapter 1, the editorial outlines the recent insights that have been obtained on the infection cycle of hyperthermoacidophilic virus-host models, namely SSV1, SIRV2, and STIV (Quemin et al., 2014). We put a particular emphasis on data covering structural genomics, whole-genome microarrays or RNA-sequencing, as well as large-scale proteomic analysis of infected cells. In fact, comparative genomics defined the structure and/or function of more than 10% of the ORFs identified in viral genomes. Additional insights came from screens for interactions or wholetranscriptome analyses in the case of SIRV2. Viral and host gene expression through the course of infection varies and a tight timing of transcription has been described for SSV1 with early, middle and late genes. Considering the proteome, STIV infection was shown to induce significant differences in protein levels and, more importantly, in post-translational modification profiles. Together these studies highlight the rapid development of highthroughput methods in the field of archaeal viruses and help to define interesting targets that should be the focus of intensive research in the near future. Moreover, recent studies trying to decipher the sequential events of the viral life cycle have led to major breakthroughs in the field. The review proposed in the Chapter 2 has been written during the framework of my PhD. It summarizes the available information concerning the virus-host interplay in Archaea with a focus on hyperthermo-acidophilic virus-host systems (Quemin and Quax, 2015). We discuss the possibility that appendages, which are observed to decorate virion termini in various families and can even form complex structures, are required during the entry process of these viruses. In the same line, novel strategies employed for egress have been recently described and are reported in great detail. The molecular mechanisms of virus-host interactions in archaea are also compared to the ways bacterial and eukaryotic viruses interact with their respective hosts. Together with the harsh environmental conditions, the characteristics of archaeal cell surface, i.e. cytoplasmic membrane and S-layer, might render the delivery of viral nucleic acids and the release of viral progeny quite difficult. Therefore, the host specificities in terms of ecology and biology could have compelled viruses to adapt and employ uncommon strategies that we are just starting to discover and understand.

Strategies for Viral Escape from the Host Cell

The last and essential step of the viral infection cycle is escape of viral particles from the host cell. So far, the egress mechanism has been analyzed for only a small subset of archaeal viruses (Torsvik and Dundas, 1974; Bize et al., 2009; Brumfield et al., 2009; Snyder et al., 2013a). Some viruses are completely lytic, while others are apparently stably produced without causing evident cell lysis (Bettstetter et al., 2003). In addition, there are temperate archaeal viruses with a lysogenic life cycle for which induction of virion production in some cases leads to cell disruption (Janekovic et al., 1983; Schleper et al., 1992; Prangishvili et al., 2006b).
The release mechanisms utilized by archaeal viruses can be divided in two categories: those for which the cell membrane is disrupted and those where the membrane integrity remains intact. The strategy for egress is linked with the assembly mechanism of new virions. Some archaeal viruses are known to mature inside the cell cytoplasm and provoke lysis, such as STIV1 (Sulfolobus turreted icosahedral virus) and SIRV2 (Bize et al., 2009; Brumfield et al., 2009; Fu et al., 2010). However, most non-lytic viruses undergo final maturation concomitantly with passage through the cell membrane (Roine and Bamford, 2012) or even in the extracellular environment, as observed for ATV (Haring et al., 2005c).

Architecture of spindle-shaped virions: the case-study of SSV1.

SSV1 has been one of the first archaeal viruses to be isolated and is the prototypical member of the Fuselloviridae family (Martin et al., 1984; Pina et al., 2011). Like most fuselloviruses, SSV1 virions are lemon-shaped and possess short filamentous appendages at one end. In order to improve our understanding on the architecture of spindle-shaped archaeal viruses, we carried out a comprehensive biochemical characterization of SSV1 virions (Quemin et al., 2015) (Chapter 4). As a prerequisite, we established large-scale virus production and purification methods which were not available before. Indeed, recent structural analysis by cryo-EM and 3D reconstruction could not conclude on the organization of SSV1 virions being limited in the resolution by the number of particles considered and heterogeneity within sample (Stedman et al., 2015). In addition, the presence of lipids and/or viral envelope could not be addressed and has remained controversial up to now (Martin et al., 1984; Reiter et al., 1987).
In agreement with previous reports, the virions were found to contain four virus-encoded structural proteins: VP1, VP2, VP3 and VP4 – formerly known as C792 (Reiter et al., 1987; Redder et al., 2009). The MCP VP1 maturates through proteolytic cleavage of a precursor molecule and together with VP3 and VP4 undergo post-translational glycosylation. Notably, the viral DNA-binding protein VP2 is not essential for virus infectivity and for most of the fuselloviruses, no homologous ORF has been identified in the viral genome (Redder et al., 2009; Iverson and Stedman, 2012). For the first time, our findings suggest that another cellular DNA-binding protein included in the viral particles, Sso7d, can replace VP2. Sso7d is the most abundant chromatin remodeling protein in the host and a member of the 7-kDa protein superfamily (Koster et al., 2015). Hence, it is likely that the viral and cellular DNAbinding proteins play a similar function in condensing the circular dsDNA genome in SSV1 virions. Furthermore, we could unambiguously resolve the controversy concerning the viral architecture and specifically the presence of lipids in the viral particles. We identified GDGT lipids in highly purified SSV1 virions using mass spectrometry techniques. These lipids are specific to archaea and structurally distinct from their bacterial and eukaryotic counterparts.
They display an ether linkage between the glycerol moiety and the hydrocarbon chains forming a covalently-bound monolayer (De Rosa et al., 1986).

Table of contents :

RESUME
ABSTRACT
Key words
INTRODUCTION
The third domain of life.
Highly diversified archaea.
Unique archaeal virosphere.
Sulfolobus, a model for hyperthermophilic archaea.
Cell surface characteristics.
Cell surface appendages.
Insights into the biology of hyperthermophilic archaeal viruses.
SSV1.
SIRV2.
Virus-host interactions in Archaea: state-of-the-art.
CHAPTER 1
Insights into the biology of archaeal viruses by high-throughput approaches
CHAPTER 2
Virus-host interactions in Archaea – the best is yet to come.
CHAPTER 3
Unique spindle-shaped viruses in Archaea.
CHAPTER 4
One update on the architecture of SSV1 virions.
CHAPTER 5
The egress of SSV1 or how to bud from an archaeon.
CHAPTER 6
Unravelling the early stages of SIRV2 infection.
DISCUSSION
Successful spindle-shaped archaeal viruses.
Architecture of spindle-shaped virions: the case-study of SSV1.
SSV1 as a model for lipid-containing viruses infecting archaea
SIRV2 as a model for non-enveloped viruses infecting archaea.
Concluding remarks and future perspectives.
REFERENCES
ACKNOWLEDGMENTS
MEMBERS OF THE JURY

GET THE COMPLETE PROJECT