An archaeal virus-encoded anti-CRISPR protein inhibits type III-B immunity by inhibiting Cas RNP complex turnover.

CRISPR-Cas systems are widespread in prokaryotes and provide adaptive immune against viral infection. Viruses encode a type of proteins called anti-CRISPR to evade the immunity. Here, we identify an archaeal virus-encoded anti-CRISPR protein, AcrIIIB2, that inhibits Type III-B immunity. We find that AcrIIIB2 inhibits Type III-B CRISPR-Cas immunity in vivo regardless of viral early or middle-/late-expressed genes to be targeted. We also demonstrate that AcrIIIB2 interacts with Cmr4α subunit, forming a complex with target RNA and Cmr-α ribonucleoprotein complex (RNP). Furtherly, we discover that AcrIIIB2 inhibits the RNase activity, ssDNase activity and cOA synthesis activity of Cmr-α RNP in vitro under a higher target RNA-to-Cmr-α RNP ratio and has no effect on Cmr-α activities at the target RNA-to-Cmr-α RNP ratio of 1. Our results suggest that once the target RNA is cleaved by Cmr-α RNP, AcrIIIB2 probably inhibits the disassociation of cleaved target RNA, therefore blocking the access of other target RNA substrates. Together, our findings highlight the multiple functions of a novel anti-CRISPR protein on inhibition of the most complicated CRISPR-Cas system targeting the genes involved in the whole life cycle of viruses.

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host.

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.

Lytic archaeal viruses infect abundant primary producers in Earth's crust.

The continental subsurface houses a major portion of life's abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

ICTV Virus Taxonomy Profile: Thaspiviridae 2021.

Members of the family Thaspiviridae have linear dsDNA genomes of 27 to 29 kbp and are the first viruses known to infect mesophilic ammonia-oxidizing archaea of the phylum Thaumarchaeota. The spindle-shaped virions of Nitrosopumilus spindle-shaped virus 1 possess short tails at one pole and measure 64±3 nm in diameter and 112±6 nm in length. This morphology is similar to that of members of the families Fuselloviridae and Halspiviridae. Virus replication is not lytic but leads to growth inhibition of the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Thaspiviridae, which is available at ictv.global/report/thaspiviridae.

ICTV Virus Taxonomy Profile: Portogloboviridae.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae.

Prokaryotic virus host predictor: a Gaussian model for host prediction of prokaryotic viruses in metagenomics.

BACKGROUND: Viruses are ubiquitous biological entities, estimated to be the largest reservoirs of unexplored genetic diversity on Earth. Full functional characterization and annotation of newly discovered viruses requires tools to enable taxonomic assignment, the range of hosts, and biological properties of the virus. Here we focus on prokaryotic viruses, which include phages and archaeal viruses, and for which identifying the viral host is an essential step in characterizing the virus, as the virus relies on the host for survival. Currently, the method for determining the viral host is either to culture the virus, which is low-throughput, time-consuming, and expensive, or to computationally predict the viral hosts, which needs improvements at both accuracy and usability. Here we develop a Gaussian model to predict hosts for prokaryotic viruses with better performances than previous computational methods. RESULTS: We present here Prokaryotic virus Host Predictor (PHP), a software tool using a Gaussian model, to predict hosts for prokaryotic viruses using the differences of k-mer frequencies between viral and host genomic sequences as features. PHP gave a host prediction accuracy of 34% (genus level) on the VirHostMatcher benchmark dataset and a host prediction accuracy of 35% (genus level) on a new dataset containing 671 viruses and 60,105 prokaryotic genomes. The prediction accuracy exceeded that of two alignment-free methods (VirHostMatcher and WIsH, 28-34%, genus level). PHP also outperformed these two alignment-free methods much (24-38% vs 18-20%, genus level) when predicting hosts for prokaryotic viruses which cannot be predicted by the BLAST-based or the CRISPR-spacer-based methods alone. Requiring a minimal score for making predictions (thresholding) and taking the consensus of the top 30 predictions further improved the host prediction accuracy of PHP. CONCLUSIONS: The Prokaryotic virus Host Predictor software tool provides an intuitive and user-friendly API for the Gaussian model described herein. This work will facilitate the rapid identification of hosts for newly identified prokaryotic viruses in metagenomic studies.

Viruses in Extreme Environments, Current Overview, and Biotechnological Potential.

Virus research has advanced significantly since the discovery of the tobacco mosaic virus (TMV), the characterization of its infection mechanisms and the factors that determine their pathogenicity. However, most viral research has focused on pathogenic viruses to humans, animals and plants, which represent only a small fraction in the virosphere. As a result, the role of most viral genes, and the mechanisms of coevolution between mutualistic viruses, their host and their environment, beyond pathogenicity, remain poorly understood. This review focuses on general aspects of viruses that interact with extremophile organisms, characteristics and examples of mechanisms of adaptation. Finally, this review provides an overview on how knowledge of extremophile viruses sheds light on the application of new tools of relevant use in modern molecular biology, discussing their value in a biotechnological context.

Viral Hijack of Filamentous Surface Structures in Archaea and Bacteria.

The bacterial and archaeal cell surface is decorated with filamentous surface structures that are used for different functions, such as motility, DNA exchange and biofilm formation. Viruses hijack these structures and use them to ride to the cell surface for successful entry. In this review, we describe currently known mechanisms for viral attachment, translocation, and entry via filamentous surface structures. We describe the different mechanisms used to exploit various surface structures bacterial and archaeal viruses. This overview highlights the importance of filamentous structures at the cell surface for entry of prokaryotic viruses.

ICTV Virus Taxonomy Profile: Ovaliviridae.

Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae.

Anti-CRISPR Proteins in Archaea.

Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas immune systems. To date, Acrs inhibiting types I, II, III, V, and VI CRISPR-Cas systems have been characterized. While most known Acrs are derived from bacterial phages and prophages, very few have been characterized in the domain Archaea, despite the nearly ubiquitous presence of CRISPR-Cas in archaeal cells. Here we summarize the discovery and characterization of the archaeal Acrs with the representatives encoded by a model archaeal virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2). AcrID1 inhibits subtype I-D CRISPR-Cas immunity through direct interaction with the large subunit Cas10d of the effector complex, and AcrIIIB1 inhibits subtype III-B CRISPR-Cas immunity through a mechanism interfering with middle/late gene targeting. Future development of efficient screening methods will be key to uncovering the diversity of archaeal Acrs.

Structure and assembly of archaeal viruses.

Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among bacteriophages or viruses of eukaryotes. However, recent environmental studies have shown that archaeal viruses are widespread also in moderate ecosystems, where they play an important ecological role by influencing the turnover of microbial communities, with a global impact on the carbon and nitrogen cycles. In this review, we summarize recent advances in understanding the molecular details of virion organization and assembly of archaeal viruses. We start by briefly introducing the 20 officially recognized families of archaeal viruses and then outline the similarities and differences of archaeal virus assembly with the morphogenesis pathways used by bacterial and eukaryotic viruses, and discuss the evolutionary implications of these observations. Generally, the assembly of the icosahedral archaeal viruses closely follows the mechanisms employed by evolutionarily related bacterial and eukaryotic viruses with the HK97 fold and double jelly-roll major capsid proteins, emphasizing the overall conservation of these pathways over billions of years of evolution. By contrast, archaea-specific viruses employ unique virion assembly mechanisms. We also highlight some of the molecular adaptations underlying the stability of archaeal viruses in extreme environments. Despite considerable progress during the past few years, the archaeal virosphere continues to represent one of the least studied parts of the global virome, with many molecular features awaiting to be discovered and characterized.

An Uncultivated Virus Infecting a Nanoarchaeal Parasite in the Hot Springs of Yellowstone National Park.

The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses had been described that infect the Nanoarchaeota. Here, we identify a virus infecting Nanoarchaeota by the use of a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6-kb circular DNA genome coding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single-celled organism that is itself an ectobiont of another single-celled organism.IMPORTANCE Here, we present evidence of the first virus found to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. On the basis of annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family, further demonstrating the remarkable genetic diversity of archaeal viruses.

A packing for A-form DNA in an icosahedral virus.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

Survey of high-resolution archaeal virus structures.

Archaeal viruses exhibit diverse morphologies whose structures are just beginning to be explored at high-resolution. In this review, we update recent findings on archaeal structural proteins and virion architectures and place them in the biological context in which these viruses replicate. We conclude that many of the unusual structural features and dynamics of archaeal viruses aid their replication and survival in the chemically harsh environments, in which they replicate. Furthermore, we should expect to find more novel features from examining the high-resolution structures of additional archaeal viruses.

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.

Structural basis for assembly of vertical single ß-barrel viruses.

The vertical double ß-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 Å resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single ß-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double ß-barrel MCPs.

Tolerance of Sulfolobus SMV1 virus to the immunity of I-A and III-B CRISPR-Cas systems in Sulfolobus islandicus.

Sulfolobus islandicus Rey15A encodes one Type I-A and two Type III-B systems, all of which are active in mediating nucleic acids interference. However, the effectiveness of each CRISPR system against virus infection was not tested in this archaeon. Here we constructed S. islandicus strains that constitutively express the antiviral immunity from either I-A, or III-B, or I-A plus III-B systems against SMV1 and tested the response of each host to SMV1 infection. We found that, although both CRISPR immunities showed a strong inhibition to viral DNA replication at an early stage of incubation, the host I-A CRISPR immunity gradually lost the control on virus proliferation, allowing accumulation of cellular viral DNA and release of a large number of viral particles. In contrast, the III-B CRISPR immunity showed a tight control on both viral DNA replication and virus particle formation. Furthermore, the SMV1 tolerance to the I-A CRISPR immunity did not result from the occurrence of escape mutations, suggesting the virus probably encodes an anti-CRISPR protein (Acr) to compromise the host I-A CRISPR immunity. Together, this suggests that the interplay between viral Acrs and CRISPR-Cas systems in thermophilic archaea could have shaped the stable virus-host relationship that is observed for many archaeal viruses.

New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures.

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.

Characterization of the lytic archaeal virus Drs3 infecting Methanobacterium formicicum.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.