Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Identification of diverse RNA viruses in Obscuromonas flagellates (Euglenozoa: Trypanosomatidae: Blastocrithidiinae).

Trypanosomatids (Euglenozoa) are a diverse group of unicellular flagellates predominately infecting insects (monoxenous species) or circulating between insects and vertebrates or plants (dixenous species). Monoxenous trypanosomatids harbor a wide range of RNA viruses belonging to the families Narnaviridae, Totiviridae, Qinviridae, Leishbuviridae, and a putative group of tombus-like viruses. Here, we focus on the subfamily Blastocrithidiinae, a previously unexplored divergent group of monoxenous trypanosomatids comprising two related genera: Obscuromonas and Blastocrithidia. Members of the genus Blastocrithidia employ a unique genetic code, in which all three stop codons are repurposed to encode amino acids, with TAA also used to terminate translation. Obscuromonas isolates studied here bear viruses of three families: Narnaviridae, Qinviridae, and Mitoviridae. The latter viral group is documented in trypanosomatid flagellates for the first time. While other known mitoviruses replicate in the mitochondria, those of trypanosomatids appear to reside in the cytoplasm. Although no RNA viruses were detected in Blastocrithidia spp., we identified an endogenous viral element in the genome of B. triatomae indicating its past encounter(s) with tombus-like viruses.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We employed protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a supefamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains, and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

All organisms, from animals to bacteria, are subject to genetic parasites, such as viruses and transposons. Genetic parasites are pieces of nucleic acids (DNA or RNA) that can use a cell's machinery to copy themselves at the expense of their hosts. This often leads to the host's demise, so organisms evolved many types of defense mechanisms. One of the most ancient and common forms of defense against viruses and transposons is the targeted restriction of nucleic acids, that is, deployment of host enzymes that can destroy or restrict nucleic acids containing specific sequence motifs or modifications. In bacteria, many of the restriction enzymes targeting parasitic genetic elements are formed by fusions of proteins from the so-called McrBC systems with a protein domain called EVE. EVE and other functionally similar domains are a part of proteins that recognize and bind modified bases in nucleic acids. Enzymes can use the ability of these specificity domains to bind modified bases to detect non-host nucleic acids. Bell et al. conducted a comprehensive computational search for McrBC systems and discovered a large and highly diverse branch of this family with unusual characteristic structural and functional domains. These features include regions that form long alpha-helices (coils) that coil with other alpha-helices (known as coiled-coils), as well as several distinct enzymatic domains that break down nucleic acids (known as nucleases). They call these systems CoCoNuTs (coiled-coiled nuclease tandems). All CoCoNuTs contain domains, including EVE-like ones, which are predicted to interact with components of the RNA-based systems responsible for producing proteins in the cell (translation), suggesting that the CoCoNuTs have an important impact on protein abundance and RNA metabolism. Bell et al.'s findings will be of interest to scientists working on prokaryotic immunity and virulence. Furthermore, similarities between CoCoNuTs and components of eukaryotic RNA-degrading systems suggest evolutionary connections between this diverse family of bacterial predicted RNA restriction systems and RNA regulatory pathways of eukaryotes. Further deciphering the mechanisms of CoCoNuTs could shed light on how certain pathways of RNA metabolism and regulation evolved, and how they may contribute to advances in biotechnology.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Balance between asymmetric allocation and repair of somatic damage in unicellular life forms as an ancient form of r/K selection.

Over the course of multiple divisions, cells accumulate diverse nongenetic, somatic damage including misfolded and aggregated proteins and cell wall defects. If the rate of damage accumulation exceeds the rate of dilution through cell growth, a dedicated mitigation strategy is required to prevent eventual population collapse. Strategies for somatic damage control can be divided into two categories, asymmetric allocation and repair, which are not, in principle, mutually exclusive. We explore a mathematical model to identify the optimal strategy, maximizing the total cell number, over a wide range of environmental and physiological conditions. The optimal strategy is primarily determined by extrinsic, damage-independent mortality and the physiological model for damage accumulation that can be either independent (linear) or increasing (exponential) with respect to the prior accumulated damage. Under the linear regime, the optimal strategy is either exclusively repair or asymmetric allocation, whereas under the exponential regime, the optimal strategy is a combination of asymmetry and repair. Repair is preferred when extrinsic mortality is low, whereas at high extrinsic mortality, asymmetric damage allocation becomes the strategy of choice. We hypothesize that at an early stage of life evolution, optimization over repair and asymmetric allocation of somatic damage gave rise to r and K selection strategists.

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

Defence systems and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations.

Widespread photosynthesis reaction centre barrel proteins are necessary for haloarchaeal cell division.

Cell division is fundamental to all cellular life. Most archaea depend on either the prokaryotic tubulin homologue FtsZ or the endosomal sorting complex required for transport for division but neither system has been robustly characterized. Here, we show that three of the four photosynthesis reaction centre barrel domain proteins of Haloferax volcanii (renamed cell division proteins B1/2/3 (CdpB1/2/3)) play important roles in cell division. CdpB1 interacts directly with the FtsZ membrane anchor SepF and is essential for cell division, whereas deletion of cdpB2 and cdpB3 causes a major and a minor division defect, respectively. Orthologues of CdpB proteins are also involved in cell division in other haloarchaea, indicating a conserved function of these proteins. Phylogenetic analysis shows that photosynthetic reaction centre barrel proteins are widely distributed among archaea and appear to be central to cell division in most if not all archaea.

Mriyaviruses: Small Relatives of Giant Viruses.

The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCP) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name "Mriyaviricetes" (after Ukrainian Mriya, dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family "Gamadviridae". The previously characterized members of these families, Yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism.

Megataxonomy and global ecology of the virosphere.

Nearly all organisms are hosts to multiple viruses that collectively appear to be the most abundant biological entities in the biosphere. With recent advances in metagenomics and metatranscriptomics, the known diversity of viruses substantially expanded. Comparative analysis of these viruses using advanced computational methods culminated in the reconstruction of the evolution of major groups of viruses and enabled the construction of a virus megataxonomy, which has been formally adopted by the International Committee on Taxonomy of Viruses. This comprehensive taxonomy consists of six virus realms, which are aspired to be monophyletic and assembled based on the conservation of hallmark proteins involved in capsid structure formation or genome replication. The viruses in different major taxa substantially differ in host range and accordingly in ecological niches. In this review article, we outline the latest developments in virus megataxonomy and the recent discoveries that will likely lead to reassessment of some major taxa, in particular, split of three of the current six realms into two or more independent realms. We then discuss the correspondence between virus taxonomy and the distribution of viruses among hosts and ecological niches, as well as the abundance of viruses versus cells in different habitats. The distribution of viruses across environments appears to be primarily determined by the host ranges, i.e. the virome is shaped by the composition of the biome in a given habitat, which itself is affected by abiotic factors.

Diversity, origin, and evolution of the ESCRT systems.

Endosomal sorting complexes required for transport (ESCRT) play key roles in protein sorting between membrane-bounded compartments of eukaryotic cells. Homologs of many ESCRT components are identifiable in various groups of archaea, especially in Asgardarchaeota, the archaeal phylum that is currently considered to include the closest relatives of eukaryotes, but not in bacteria. We performed a comprehensive search for ESCRT protein homologs in archaea and reconstructed ESCRT evolution using the phylogenetic tree of Vps4 ATPase (ESCRT IV) as a scaffold and using sensitive protein sequence analysis and comparison of structural models to identify previously unknown ESCRT proteins. Several distinct groups of ESCRT systems in archaea outside of Asgard were identified, including proteins structurally similar to ESCRT-I and ESCRT-II, and several other domains involved in protein sorting in eukaryotes, suggesting an early origin of these components. Additionally, distant homologs of CdvA proteins were identified in Thermoproteales which are likely components of the uncharacterized cell division system in these archaea. We propose an evolutionary scenario for the origin of eukaryotic and Asgard ESCRT complexes from ancestral building blocks, namely, the Vps4 ATPase, ESCRT-III components, wH (winged helix-turn-helix fold) and possibly also coiled-coil, and Vps28-like domains. The last archaeal common ancestor likely encompassed a complex ESCRT system that was involved in protein sorting. Subsequent evolution involved either simplification, as in the TACK superphylum, where ESCRT was co-opted for cell division, or complexification as in Asgardarchaeota. In Asgardarchaeota, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was already established.IMPORTANCEAll eukaryotic cells possess complex intracellular membrane organization. Endosomal sorting complexes required for transport (ESCRT) play a central role in membrane remodeling which is essential for cellular functionality in eukaryotes. Recently, it has been shown that Asgard archaea, the archaeal phylum that includes the closest known relatives of eukaryotes, encode homologs of many components of the ESCRT systems. We employed protein sequence and structure comparisons to reconstruct the evolution of ESCRT systems in archaea and identified several previously unknown homologs of ESCRT subunits, some of which can be predicted to participate in cell division. The results of this reconstruction indicate that the last archaeal common ancestor already encoded a complex ESCRT system that was involved in protein sorting. In Asgard archaea, ESCRT systems evolved toward greater complexity, and in particular, the connection between ESCRT and the ubiquitin system that was previously considered a eukaryotic signature was established.

Diversity of Bathyarchaeia viruses in metagenomes and virus-encoded CRISPR system components.

Bathyarchaeia represent a class of archaea common and abundant in sedimentary ecosystems. Here we report 56 metagenome-assembled genomes of Bathyarchaeia viruses identified in metagenomes from different environments. Gene sharing network and phylogenomic analyses led to the proposal of four virus families, including viruses of the realms Duplodnaviria and Adnaviria, and archaea-specific spindle-shaped viruses. Genomic analyses uncovered diverse CRISPR elements in these viruses. Viruses of the proposed family "Fuxiviridae" harbor an atypical Type IV-B CRISPR-Cas system and a Cas4 protein that might interfere with host immunity. Viruses of the family "Chiyouviridae" encode a Cas2-like endonuclease and two mini-CRISPR arrays, one with a repeat identical to that in the host CRISPR array, potentially allowing the virus to recruit the host CRISPR adaptation machinery to acquire spacers that could contribute to competition with other mobile genetic elements or to inhibit host defenses. These findings present an outline of the Bathyarchaeia virome and offer a glimpse into their counter-defense mechanisms.

Defense systems and horizontal gene transfer in bacteria.

Horizontal gene transfer (HGT) is a fundamental process in the evolution of prokaryotes, making major contributions to diversification and adaptation. Typically, HGT is facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages that generally impose fitness costs on their hosts. However, a substantial fraction of bacterial genes is involved in defense mechanisms that limit the propagation of MGEs, raising the possibility that they can actively restrict HGT. Here we examine whether defense systems curb HGT by exploring the connections between HGT rate and the presence of 73 defense systems in 12 bacterial species. We found that only 6 defense systems, 3 of which are different CRISPR-Cas subtypes, are associated with the reduced gene gain rate on the scale of species evolution. The hosts of such defense systems tend to have a smaller pangenome size and harbor fewer phage-related genes compared to genomes lacking these systems, suggesting that these defense mechanisms inhibit HGT by limiting the integration of prophages. We hypothesize that restriction of HGT by defense systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and fitness effect of HGT in bacterial populations.

Microbial diversity and ecological complexity emerging from environmental variation and horizontal gene transfer in a simple mathematical model.

Microbiomes are generally characterized by high diversity of coexisting microbial species and strains that remains stable within a broad range of conditions. However, under fixed conditions, microbial ecology conforms with the exclusion principle under which two populations competing for the same resource within the same niche cannot coexist because the less fit population inevitably goes extinct. To explore the conditions for stabilization of microbial diversity, we developed a simple mathematical model consisting of two competing populations that could exchange a single gene allele via horizontal gene transfer (HGT). We found that, although in a fixed environment, with unbiased HGT, the system obeyed the exclusion principle, in an oscillating environment, within large regions of the phase space bounded by the rates of reproduction and HGT, the two populations coexist. Moreover, depending on the parameter combination, all three major types of symbiosis obtained, namely, pure competition, host-parasite relationship and mutualism. In each of these regimes, certain parameter combinations provided for synergy, that is, a greater total abundance of both populations compared to the abundance of the winning population in the fixed environments. These findings show that basic phenomena that are universal in microbial communities, environmental variation and HGT, provide for stabilization of microbial diversity and ecological complexity.

Bacterial defense systems exhibit synergistic anti-phage activity.

Bacterial defense against phage predation involves diverse defense systems acting individually and concurrently, yet their interactions remain poorly understood. We investigated >100 defense systems in 42,925 bacterial genomes and identified numerous instances of their non-random co-occurrence and negative association. For several pairs of defense systems significantly co-occurring in Escherichia coli strains, we demonstrate synergistic anti-phage activity. Notably, Zorya II synergizes with Druantia III and ietAS defense systems, while tmn exhibits synergy with co-occurring systems Gabija, Septu I, and PrrC. For Gabija, tmn co-opts the sensory switch ATPase domain, enhancing anti-phage activity. Some defense system pairs that are negatively associated in E. coli show synergy and significantly co-occur in other taxa, demonstrating that bacterial immune repertoires are largely shaped by selection for resistance against host-specific phages rather than negative epistasis. Collectively, these findings demonstrate compatibility and synergy between defense systems, allowing bacteria to adopt flexible strategies for phage defense.

ICTV Virus Taxonomy Profile: Kolmioviridae 2024.

Kolmioviridae is a family for negative-sense RNA viruses with circular, viroid-like genomes of about 1.5-1.7 kb that are maintained in mammals, amphibians, birds, fish, insects and reptiles. Deltaviruses, for instance, can cause severe hepatitis in humans. Kolmiovirids encode delta antigen (DAg) and replicate using host-cell DNA-directed RNA polymerase II and ribozymes encoded in their genome and antigenome. They require evolutionary unrelated helper viruses to provide envelopes and incorporate helper virus proteins for infectious particle formation. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Kolmioviridae, which is available at ictv.global/report/kolmioviridae.

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.