Sulfolobus Spindle-shaped Virus Research Articles

Introduction: the secret lives of microbial mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

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

A challenge in virology is quantifying relative virulence (V R) 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 V R using canonical parameters, like maximum specific growth rate (μ max), 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 V R. This metric (I SC) accurately aligns biological phenomena with quantified metrics to determine V R. 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.

Host-Dependent Differences in Replication Strategy of the Sulfolobus Spindle-Shaped Virus Strain SSV9 (a.k.a., SSVK1): Infection Profiles in Hosts of the Family Sulfolobaceae.

The Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying thermophilic virus biology, including archaeal host-virus interactions and biogeography. Several factors make the SSV system amenable to studying archaeal genetic mechanisms (e.g., CRISPRs) as well as virus-host interactions in high temperature acidic environments. Previously, we reported that SSVs exhibited differential infectivity on allopatric vs. sympatric hosts. We also noticed a wide host range for virus strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” double-stranded DNA viruses that infect species of the genus Sulfolobus and release virions via budding rather than host lysis. In this study, we show that SSVs infect hosts representing more than one genus of the family Sulfolobaceae in spot-on-lawn “halo” assays and in liquid culture infection assays. Growth curve analyses support the hypothesis that SSV9 virion release causes cell lysis. While SSV9 appears to lyse allopatric hosts, on a single sympatric host, SSV9 exhibits canonical non-lytic viral release historically reported SSVs. Therefore, the nature of SSV9 lytic-like behavior may be driven by allopatric evolution. The SSV9-infected host growth profile does not appear to be driven by multiplicity of infection (MOI). Greater stability of SSV9 vs. other SSVs (i.e., SSV1) in high temperature, low pH environments may contribute to higher transmission rates. However, neither higher transmission rate nor relative virulence in SSV9 infection seems to alter replication profile in susceptible hosts. Although it is known that CRISPR-Cas systems offer protection against viral infection in prokaryotes, CRISPRS are not reported to be a determinant of virus replication strategy. The mechanisms underlying SSV9 lytic-like behavior remain unknown and are the subject of ongoing investigations. These results suggest that genetic elements, potentially resulting from allopatric evolution, mediate distinct virus-host growth profiles of specific SSV-host strain pairings.

Host-dependent differences in replication and virion release strategy of the Sulfolobus Spindle-shaped Virus strain SSV9 (a.k.a., SSVK1): Lytic replication in hosts of the family Sulfolobaceae

The Sulfolobus Spindle-shaped Virus (SSV) system is a model for studying thermophilic archaeal virus biology. Several factors make the SSV system amenable to studying archaeal genetics and virus-host interactions in extreme environments. It has been shown that populations of Sulfolobus, the natural host, exhibit biogeographic structure. The acidic (pH<4.5) high temperature (65-88°C) habitats have low biodiversity, which diminishes prospects for host switch. SSVs and their hosts are readily cultured in liquid media and on plates. Given the wide geographic separation between various SSV-Sulfolobus habitats, the system is also amenable to studying allopatric versus sympatric virus-host interactions. We previously reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We discovered a strikingly broad host-range for strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species of Sulfolobus and release virus particles via blebbing as a preferred strategy over host lysis (in reported laboratory infections). Here, we show, that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not release virions via blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts, while exhibiting canonical non-lytic virion release (historically reported for SSVs) on a single sympatric host. Lytic versus non-lytic virus release does not appear to be driven by multiplicity of infection. Data suggest that SSV9 is more stable than other SSVs in suspension; however, genetic substrates (e.g., CRISPR profiles) underlying non-lytic versus lytic virion release remain unresolved and are the subject of ongoing investigation.

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.

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.

Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus.

The population diversity and structure of CRISPR-Cas immunity provides key insights into virus–host interactions. Here, we examined two geographically and genetically distinct natural populations of the thermophilic crenarchaeon Sulfolobus islandicus and their interactions with Sulfolobus spindle-shaped viruses (SSVs) and S. islandicus rod-shaped viruses (SIRVs). We found that both virus families can be targeted with high population distributed immunity, whereby most immune strains target a virus using unique unshared CRISPR spacers. In Kamchatka, Russia, we observed high immunity to chronic SSVs that increases over time. In this context, we found that some SSVs had shortened genomes lacking genes that are highly targeted by the S. islandicus population, indicating a potential mechanism of immune evasion. By contrast, in Yellowstone National Park, we found high inter- and intra-strain immune diversity targeting lytic SIRVs and low immunity to chronic SSVs. In this population, we observed evidence of SIRVs evolving immunity through mutations concentrated in the first five bases of protospacers. These results indicate that diversity and structure of antiviral CRISPR-Cas immunity for a single microbial species can differ by both the population and virus type, and suggest that different virus families use different mechanisms to evade CRISPR-Cas immunity.This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

Comparative genetic and genomic analysis of the novel fusellovirus Sulfolobus spindle-shaped virus 10.

Viruses that infect thermophilic Archaea are unique in both their structure and genetic makeup. The lemon-shaped fuselloviruses—which infect members of the order Sulfolobales, growing optimally at 80 °C and pH 3—are some of the most ubiquitous and best studied viruses of the thermoacidophilic Archaea. Nonetheless, much remains to be learned about these viruses. In order to investigate fusellovirus evolution, we have isolated and characterized a novel fusellovirus, Sulfolobus spindle-shaped virus 10 (formerly SSV-L1). Comparative genomic analyses highlight significant similarity with both SSV8 and SSV9, as well as conservation of promoter elements within the Fuselloviridae. SSV10 encodes five ORFs with no homology within or outside of the Fuselloviridae, as well as a putatively functional Cas4-like ORF, which may play a role in evading CRISPR-mediated host defenses. Moreover, we demonstrate the ability of SSV10 to withstand mutation in a fashion consistent with mutagenesis in SSV1.

Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change.

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, Sulfolobus Spindle-shaped Virus 1 (SSV1), is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene VP3, the VP1 gene does not tolerate deletion or transposon insertion. To determine the role of the VP1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with VP1 genes from other SSVs. By analyzing these mutants, we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice

Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties.

Sulfolobus Spindle-Shaped Virus 1 Growth Kinetics

Geothermal and hypersaline environments are rich in viral particles, among which spindle-shaped morphologies predominate. Currently, viruses with spindle- or lemon-shaped virions are unique to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, encompasses spindle-shaped viruses with very short tails, which infect Sulfolobus solfataricus and close relatives. 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. However, our knowledge of fuselloviral life cycles and the relationships between these viruses and their hosts is still limited. As a result, to establish one-step growth curves to study virus replication kinetics and to investigate the mechanism of host binding, the adsorption kinetics of wild-type SSV1 to its Sulfolobus host needed 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

ABSTRACTSimilar 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

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. 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.

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

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. 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.

Virus-induced dormancy in the archaeon Sulfolobus islandicus.

ABSTRACTWe investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood.

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.

Molecular biology of fuselloviruses and their satellites

Fuselloviruses, also known as Sulfolobus Spindle-shaped viruses (SSVs), are "lemon"- or "spindle"-shaped double-stranded DNA viruses. Among them, SSV1, SSV2 and the satellite viruses pSSVx and pSSVi have been investigated at the structural, genetic, transcriptomic, proteomic and biochemical levels, thus becoming models for dissecting DNA replication/gene expression in Archaea. Important progress has been made including elucidation of temporal genome expression during virus infection and induction of replication, SSV1 lysogeny maintenance as well as differentially expression of pSSVx replicase. Future researches focusing on these model systems would yield insightful knowledge of life cycle and DNA replication of fuselloviruses.

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.

Structural studies of E73 from a hyperthermophilic archaeal virus identify the "RH3" domain, an elaborated ribbon-helix-helix motif involved in DNA recognition.

Hyperthermophilic archaeal viruses, including Sulfolobus spindle-shaped viruses (SSVs) such as SSV-1 and SSV-Ragged Hills, exhibit remarkable morphology and genetic diversity. However, they remain poorly understood, in part because their genomes exhibit limited or unrecognizable sequence similarity to genes with known function. Here we report structural and functional studies of E73, a 73-residue homodimeric protein encoded within the SSV-Ragged Hills genome. Despite lacking significant sequence similarity, the nuclear magnetic resonance (NMR) structure reveals clear similarity to ribbon-helix-helix (RHH) domains present in numerous proteins involved in transcriptional regulation. In vitro double-stranded DNA (dsDNA) binding experiments confirm the ability of E73 to bind dsDNA in a nonspecific manner with micromolar affinity, and characterization of the K11E variant confirms the location of the predicted DNA binding surface. E73 is distinct, however, from known RHH domains. The RHH motif is elaborated upon by the insertion of a third helix that is tightly integrated into the structural domain, giving rise to the "RH3" fold. Within the homodimer, this helix results in the formation of a conserved, symmetric cleft distal to the DNA binding surface, where it may mediate protein-protein interactions or contribute to the high thermal stability of E73. Analysis of backbone amide dynamics by NMR provides evidence of a rigid core, fast picosecond to nanosecond time scale NH bond vector motions for residues located within the antiparallel β-sheet region of the proposed DNA-binding surface, and slower microsecond to millisecond time scale motions for residues in the α1-α2 loop. The roles of E73 and its SSV homologues in the viral life cycle are discussed.