A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus–host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus–host interactions, we studied the life cycle of the enveloped, ∼2-μm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus–host interactions in Archaea.

On topology and knotty entanglement in protein folding

We investigate aspects of topology in protein folding. For this we numerically simulate the temperature driven folding and unfolding of the slipknotted archaeal virus protein AFV3-109. Due to knottiness the (un)folding is a topological process, it engages the entire backbone in a collective fashion. Accordingly we introduce a topological approach to model the process. Our simulations reveal that the (un)folding of AFV3-109 slipknot proceeds through a folding intermediate that has the topology of a trefoil knot. We observe that the final slipknot causes a slight swelling of the folded AFV3-109 structure. We disclose the relative stability of the strands and helices during both the folding and unfolding processes. We confirm results from previous studies that pointed out that it can be very demanding to simulate the formation of knotty self-entanglement, and we explain how the problems are circumvented: The slipknotted AFV3-109 protein is a very slow folder with a topologically demanding pathway, which needs to be properly accounted for in a simulation description. When we either increase the relative stiffness of bending, or when we decrease the speed of ambient cooling, the rate of slipknot formation rapidly increases.

ORF4 of the Temperate Archaeal Virus SNJ1 Governs the Lysis-Lysogeny Switch and Superinfection Immunity

ABSTRACT Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes. IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

Discovery and Characterization of Thermoproteus Spherical Piliferous Virus 1: a Spherical Archaeal Virus Decorated with Unusual Filaments

ABSTRACT We describe the discovery of an archaeal virus, one that infects archaea, tentatively named Thermoproteus spherical piliferous virus 1 (TSPV1), which was purified from a Thermoproteales host isolated from a hot spring in Yellowstone National Park (USA). TSPV1 packages an 18.65-kb linear double-stranded DNA (dsDNA) genome with 31 open reading frames (ORFs), whose predicted gene products show little homology to proteins with known functions. A comparison of virus particle morphologies and gene content demonstrates that TSPV1 is a new member of the Globuloviridae family of archaeal viruses. However, unlike other Globuloviridae members, TSPV1 has numerous highly unusual filaments decorating its surface, which can extend hundreds of micrometers from the virion. To our knowledge, similar filaments have not been observed in any other archaeal virus. The filaments are remarkably stable, remaining intact across a broad range of temperature and pH values, and they are resistant to chemical denaturation and proteolysis. A major component of the filaments is a glycosylated 35-kDa TSPV1 protein (TSPV1 GP24). The filament protein lacks detectable homology to structurally or functionally characterized proteins. We propose, given the low host cell densities of hot spring environments, that the TSPV1 filaments serve to increase the probability of virus attachment and entry into host cells. IMPORTANCE High-temperature environments have proven to be an important source for the discovery of new archaeal viruses with unusual particle morphologies and gene content. Our isolation of Thermoproteus spherical piliferous virus 1 (TSPV1), with numerous filaments extending from the virion surface, expands our understanding of viral diversity and provides new insight into viral replication in high-temperature environments.

Virus-borne mini-CRISPR arrays are involved in interviral conflicts

CRISPR-Cas immunity is at the forefront of antivirus defense in bacteria and archaea and specifically targets viruses carrying protospacers matching the spacers catalogued in the CRISPR arrays. Here, we perform deep sequencing of the CRISPRome—all spacers contained in a microbiome—associated with hyperthermophilic archaea of the order Sulfolobales recovered directly from an environmental sample and from enrichment cultures established in the laboratory. The 25 million CRISPR spacers sequenced from a single sampling site dwarf the diversity of spacers from all available Sulfolobales isolates and display complex temporal dynamics. Comparison of closely related virus strains shows that CRISPR targeting drives virus genome evolution. Furthermore, we show that some archaeal viruses carry mini-CRISPR arrays with 1–2 spacers and preceded by leader sequences but devoid of cas genes. Closely related viruses present in the same population carry spacers against each other. Targeting by these virus-borne spacers represents a distinct mechanism of heterotypic superinfection exclusion and appears to promote archaeal virus speciation.