Structural atlas of a human gut crassvirus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments.

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids.

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit ß-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

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.

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.

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: Plectroviridae.

Members of the family Plectroviridae produce particles that are non-enveloped rigid rods (70-280×10-16 nm). The supercoiled, circular, single-stranded DNA genome of about 4.5-8.3 kb, encodes 4-13 proteins. Viruses of this family infect cell wall-less bacteria, adsorbing to the bacterial surface, replicating their DNA by a rolling-circle mechanism or transposition, and releasing progeny from cells by extrusion, without killing the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Plectroviridae which is available at ictv.global/report/plectroviridae.

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.

ICTV Virus Taxonomy Profile: Retroviridae 2021.

Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80-100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

ICTV Virus Taxonomy Profile: Finnlakeviridae.

Finnlakeviridae is a family of icosahedral, internal membrane-containing bacterial viruses with circular, single-stranded DNA genomes. The family includes the genus, Finnlakevirus, with the species, Flavobacterium virus FLiP. Flavobacterium phage FLiP was isolated with its Gram-negative host bacterium from a boreal freshwater habitat in Central Finland in 2010. It is the first described single-stranded DNA virus with an internal membrane and shares minimal sequence similarity with other known viruses. The virion organization (pseudo T=21 dextro) and major capsid protein fold (double-ß-barrel) resemble those of Pseudoalteromonas phage PM2 (family Corticoviridae), which has a double-stranded DNA genome. A similar major capsid protein fold is also found in other double-stranded DNA viruses in the kingdom Bamfordvirae. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Finnlakeviridae, which is available at ictv.global/report/finnlakeviridae.

ICTV Virus Taxonomy Profile: Tristromaviridae.

Tristromaviridae is a family of viruses with linear, double-stranded DNA genomes of 16-18 kbp. The flexible, filamentous virions (400±20 nm×30±3 nm) consist of an envelope and an inner core constructed from two structural units: a rod-shaped helical nucleocapsid and a nucleocapsid-encompassing matrix protein layer. Tristromaviruses are lytic and infect hyperthermophilic archaea of the order Thermoproteales. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Tristromaviridae, which is available at www.ictv.global/report/tristromaviridae.

In-depth analysis of the replication cycle of Orpheovirus.

BACKGROUND: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS: We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

ICTV Virus Taxonomy Profile: Globuloviridae.

The family Globuloviridae comprises enveloped viruses with linear, double-stranded DNA genomes of about 21-28 kbp. The virions are spherical with a diameter of 70-100 nm. No information is available about genome replication. Globuloviruses infect hyperthermophilic archaea belonging to the genera Pyrobaculum and Thermoproteus, which thrive in extreme geothermal environments. Infection does not cause lysis of host cells and is noncytocidal. The viral genome does not integrate into the host chromosome. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Globuloviridae, which is available at www.ictv.global/report/globuloviridae.

A Novel Type of Polyhedral Viruses Infecting Hyperthermophilic Archaea.

Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated.IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, "Portogloboviridae."

Cedratvirus lausannensis - digging into Pithoviridae diversity.

Amoeba-infecting viruses have raised scientists' interest due to their novel particle morphologies, their large genome size and their genomic content challenging previously established dogma. We report here the discovery and the characterization of Cedratvirus lausannensis, a novel member of the Megavirales, with a 0.75-1 µm long amphora-shaped particle closed by two striped plugs. Among numerous host cell types tested, the virus replicates only in Acanthamoeba castellanii leading to host cell lysis within 24 h. C. lausannensis was resistant to ethanol, hydrogen peroxide and heating treatments. Like 30 000-year-old Pithovirus sibericum, C. lausannensis enters by phagocytosis, releases its genetic content by fusion of the internal membrane with the inclusion membrane and replicates in intracytoplasmic viral factories. The genome encodes 643 proteins that confirmed the grouping of C. lausannensis with Cedratvirus A11 as phylogenetically distant members of the family Pithoviridae. The 575,161 bp AT-rich genome is essentially devoid of the numerous repeats harbored by Pithovirus, suggesting that these non-coding repetitions might be due to a selfish element rather than particular characteristics of the Pithoviridae family. The discovery of C. lausannensis confirms the contemporary worldwide distribution of Pithoviridae members and the characterization of its genome paves the way to better understand their evolution.

Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology.

The largest known DNA viruses infect Acanthamoeba and belong to two markedly different families. The Megaviridae exhibit pseudo-icosahedral virions up to 0.7 µm in diameter and adenine-thymine (AT)-rich genomes of up to 1.25 Mb encoding a thousand proteins. Like their Mimivirus prototype discovered 10 y ago, they entirely replicate within cytoplasmic virion factories. In contrast, the recently discovered Pandoraviruses exhibit larger amphora-shaped virions 1 µm in length and guanine-cytosine-rich genomes up to 2.8 Mb long encoding up to 2,500 proteins. Their replication involves the host nucleus. Whereas the Megaviridae share some general features with the previously described icosahedral large DNA viruses, the Pandoraviruses appear unrelated to them. Here we report the discovery of a third type of giant virus combining an even larger pandoravirus-like particle 1.5 µm in length with a surprisingly smaller 600 kb AT-rich genome, a gene content more similar to Iridoviruses and Marseillevirus, and a fully cytoplasmic replication reminiscent of the Megaviridae. This suggests that pandoravirus-like particles may be associated with a variety of virus families more diverse than previously envisioned. This giant virus, named Pithovirus sibericum, was isolated from a >30,000-y-old radiocarbon-dated sample when we initiated a survey of the virome of Siberian permafrost. The revival of such an ancestral amoeba-infecting virus used as a safe indicator of the possible presence of pathogenic DNA viruses, suggests that the thawing of permafrost either from global warming or industrial exploitation of circumpolar regions might not be exempt from future threats to human or animal health.

Unification of the globally distributed spindle-shaped viruses of the Archaea.

Viruses with spindle-shaped virions are abundant in diverse environments. Over the years, such viruses have been isolated from a wide range of archaeal hosts. Evolutionary relationships between them remained enigmatic, however. Here, using structural proteins as markers, we define familial ties among these "dark horses" of the virosphere and segregate all spindle-shaped viruses into two distinct evolutionary lineages, corresponding to Bicaudaviridae and Fuselloviridae. Our results illuminate the utility of structure-based virus classification and bring additional order to the virosphere.

Discovery of two novel viruses expands the diversity of single-stranded DNA and single-stranded RNA viruses infecting a cosmopolitan marine diatom.

Recent studies have suggested that diatom viruses are an important factor affecting diatom population dynamics, which in turn are important in considering marine primary productivity. The marine planktonic diatom Chaetoceros tenuissimus Meunier is a cosmopolitan species and often causes blooms off the western coast of Japan. To date, two viruses, C. tenuissimus DNA virus (CtenDNAV) type I and CtenRNAV type I, have been identified that potentially affect C. tenuissimus population dynamics in the natural environment. In this study, we successfully isolated and characterized two additional novel viruses (CtenDNAV type II and CtenRNAV type II). This paper reports the basic characteristics of these new viruses isolated from surface water or sediment from the Hiroshima Bay, Japan. The physiological and morphological characteristics of the two new viruses were similar to those of the previously isolated viruses. However, the amino acid sequences of the structural proteins of CtenDNAV type II and CtenRNAV type II were clearly distinct from those of both type I viruses, with identity scores of 38.3% and 27.6%, respectively. Our results suggest that at least four genetically distinct viruses sharing the same diatom host are present in western Japan and affect the population dynamics of C. tenuissimus. Moreover, the result that CtenRNAV type II lysed multiple diatom species indicates that RNA viruses may affect various diatom populations in the natural environment.

DNA viruses: the really big ones (giruses).

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences than on extensive details about individual viruses.