Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs.

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named 'Suismacovirus', comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses' genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

Temperate Bacteriophages-The Powerful Indirect Modulators of Eukaryotic Cells and Immune Functions.

Bacteriophages are natural biological entities that limit the growth and amplification of bacteria. They are important stimulators of evolutionary variability in bacteria, and currently are considered a weapon against antibiotic resistance of bacteria. Nevertheless, apart from their antibacterial activity, phages may act as modulators of mammalian immune responses. In this paper, we focus on temperate phages able to execute the lysogenic development, which may shape animal or human immune response by influencing various processes, including phagocytosis of bacterial invaders and immune modulation of mammalian host cells.

ViralRecall-A Flexible Command-Line Tool for the Detection of Giant Virus Signatures in 'Omic Data.

Giant viruses are widespread in the biosphere and play important roles in biogeochemical cycling and host genome evolution. Also known as nucleo-cytoplasmic large DNA viruses (NCLDVs), these eukaryotic viruses harbor the largest and most complex viral genomes known. Studies have shown that NCLDVs are frequently abundant in metagenomic datasets, and that sequences derived from these viruses can also be found endogenized in diverse eukaryotic genomes. The accurate detection of sequences derived from NCLDVs is therefore of great importance, but this task is challenging owing to both the high level of sequence divergence between NCLDV families and the extraordinarily high diversity of genes encoded in their genomes, including some encoding for metabolic or translation-related functions that are typically found only in cellular lineages. Here, we present ViralRecall, a bioinformatic tool for the identification of NCLDV signatures in 'omic data. This tool leverages a library of giant virus orthologous groups (GVOGs) to identify sequences that bear signatures of NCLDVs. We demonstrate that this tool can effectively identify NCLDV sequences with high sensitivity and specificity. Moreover, we show that it can be useful both for removing contaminating sequences in metagenome-assembled viral genomes as well as the identification of eukaryotic genomic loci that derived from NCLDV. ViralRecall is written in Python 3.5 and is freely available on GitHub: https://github.com/faylward/viralrecall.

Fundamental Difficulties Prevent the Reconstruction of the Deep Phylogeny of Viruses.

The extension of virology beyond its traditional medical, veterinary, or agricultural applications, now called environmental virology, has shown that viruses are both the most numerous and diverse biological entities on Earth. In particular, virus isolations from unicellular eukaryotic hosts (heterotrophic and photosynthetic protozoans) revealed numerous viral types previously unexpected in terms of virion structure, gene content, or mode of replication. Complemented by large-scale metagenomic analyses, these discoveries have rekindled interest in the enigma of the origin of viruses, for which a description encompassing all their diversity remains not available. Several laboratories have repeatedly tackled the deep reconstruction of the evolutionary history of viruses, using various methods of molecular phylogeny applied to the few shared "core" genes detected in certain virus groups (e.g., the Nucleocytoviricota). Beyond the practical difficulties of establishing reliable homology relationships from extremely divergent sequences, I present here conceptual arguments highlighting several fundamental limitations plaguing the reconstruction of the deep evolutionary history of viruses, and even more the identification of their unique or multiple origin(s). These arguments also underline the risk of establishing premature high level viral taxonomic classifications. Those limitations are direct consequences of the random mechanisms governing the reductive/retrogressive evolution of all obligate intracellular parasites.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

Structural Determinants within the Adenovirus Early Region 1A Protein Spacer Region Necessary for Tumorigenesis.

It has long been established that group A human adenoviruses (HAdV-A12, -A18, and -A31) can cause tumors in newborn rodents, with tumorigenicity related to the presence of a unique spacer region located between conserved regions 2 and 3 within the HAdV-A12 early region 1A (E1A) protein. Group B adenoviruses are weakly oncogenic, whereas most of the remaining human adenoviruses are nononcogenic. In an attempt to understand better the relationship between the structure of the AdE1A spacer region and oncogenicity of HAdVs, the structures of synthetic peptides identical or very similar to the adenovirus 12 E1A spacer region were determined and found to be α-helical using nuclear magnetic resonance (NMR) spectroscopy. This contrasts significantly with some previous suggestions that this region is unstructured. Using available predictive algorithms, the structures of spacer regions from other E1As were also examined, and the extent of the predicted α-helix was found to correlate reasonably well with the tumorigenicity of the respective virus. We suggest that this may represent an as-yet-unknown binding site for a partner protein required for tumorigenesis.IMPORTANCE This research analyzed small peptides equivalent to a region within the human adenovirus early region 1A protein that confers, in part, tumor-inducing properties to various degrees on several viral strains in rats and mice. The oncogenic spacer region is α-helical, which contrasts with previous suggestions that this region is unstructured. The helix is characterized by a stretch of amino acids rich in alanine residues that are organized into a hydrophobic, or "water-hating," surface that is considered to form a major site of interaction with cellular protein targets that mediate tumor formation. The extent of α-helix in E1A from other adenovirus species can be correlated to a limited degree to the tumorigenicity of that virus. Some serotypes show significant differences in predicted structural propensity, suggesting that the amino acid type and physicochemical properties are also of importance.

Epigenetic and epitranscriptomic regulation of viral replication.

Eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and RNAs. Whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone H3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in DNA virus-infected cells. However, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections. By contrast, the various covalent modifications added to RNAs, termed epitranscriptomic modifications, can positively regulate mRNA translation and/or stability, and both DNA and RNA viruses have evolved to utilize epitranscriptomic modifications as a means to maximize viral gene expression. As a consequence, both chromatin and RNA modifications could serve as novel targets for the development of antivirals. In this Review, we discuss how host epigenetic and epitranscriptomic processes regulate viral gene expression at the levels of chromatin and RNA function, respectively, and explore how viruses modify, avoid or utilize these processes in order to regulate viral gene expression.

Giant virus diversity and host interactions through global metagenomics.

Our current knowledge about nucleocytoplasmic large DNA viruses (NCLDVs) is largely derived from viral isolates that are co-cultivated with protists and algae. Here we reconstructed 2,074 NCLDV genomes from sampling sites across the globe by building on the rapidly increasing amount of publicly available metagenome data. This led to an 11-fold increase in phylogenetic diversity and a parallel 10-fold expansion in functional diversity. Analysis of 58,023 major capsid proteins from large and giant viruses using metagenomic data revealed the global distribution patterns and cosmopolitan nature of these viruses. The discovered viral genomes encoded a wide range of proteins with putative roles in photosynthesis and diverse substrate transport processes, indicating that host reprogramming is probably a common strategy in the NCLDVs. Furthermore, inferences of horizontal gene transfer connected viral lineages to diverse eukaryotic hosts. We anticipate that the global diversity of NCLDVs that we describe here will establish giant viruses-which are associated with most major eukaryotic lineages-as important players in ecosystems across Earth's biomes.

VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells.

Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.

Pressure-driven release of viral genome into a host nucleus is a mechanism leading to herpes infection.

Many viruses previously have been shown to have pressurized genomes inside their viral protein shell, termed the capsid. This pressure results from the tight confinement of negatively charged viral nucleic acids inside the capsid. However, the relevance of capsid pressure to viral infection has not been demonstrated. In this work, we show that the internal DNA pressure of tens of atmospheres inside a herpesvirus capsid powers ejection of the viral genome into a host cell nucleus. To our knowledge, this provides the first demonstration of a pressure-dependent mechanism of viral genome penetration into a host nucleus, leading to infection of eukaryotic cells.

Assessing the viral content of uncultured picoeukaryotes in the global-ocean by single cell genomics.

Viruses are the most abundant biological entities on Earth and have fundamental ecological roles in controlling microbial communities. Yet, although their diversity is being increasingly explored, little is known about the extent of viral interactions with their protist hosts as most studies are limited to a few cultivated species. Here, we exploit the potential of single-cell genomics to unveil viral associations in 65 individual cells of 11 essentially uncultured stramenopiles lineages sampled during the Tara Oceans expedition. We identified viral signals in 57% of the cells, covering nearly every lineage and with narrow host specificity signal. Only seven out of the 64 detected viruses displayed homologies to known viral sequences. A search for our viral sequences in global ocean metagenomes showed that they were preferentially found at the DCM and within the 0.2-3 µm size fraction. Some of the viral signals were widely distributed, while others geographically constrained. Among the viral signals we detected an endogenous mavirus virophage potentially integrated within the nuclear genome of two distant uncultured stramenopiles. Virophages have been previously reported as a cell's defence mechanism against other viruses, and may therefore play an important ecological role in regulating protist populations. Our results point to single-cell genomics as a powerful tool to investigate viral associations in uncultured protists, suggesting a wide distribution of these relationships, and providing new insights into the global viral diversity.

Virus Control of Cell Metabolism for Replication and Evasion of Host Immune Responses.

Over the last decade, there has been significant advances in the understanding of the cross-talk between metabolism and immune responses. It is now evident that immune cell effector function strongly depends on the metabolic pathway in which cells are engaged in at a particular point in time, the activation conditions, and the cell microenvironment. It is also clear that some metabolic intermediates have signaling as well as effector properties and, hence, topics such as immunometabolism, metabolic reprograming, and metabolic symbiosis (among others) have emerged. Viruses completely rely on their host's cell energy and molecular machinery to enter, multiply, and exit for a new round of infection. This review explores how viruses mimic, exploit or interfere with host cell metabolic pathways and how, in doing so, they may evade immune responses. It offers a brief outline of key metabolic pathways, mitochondrial function and metabolism-related signaling pathways, followed by examples of the mechanisms by which several viral proteins regulate host cell metabolic activity.

Extracellular RNA in viral-host interactions: Thinking outside the cell.

Small RNAs and their associated RNA interference (RNAi) pathways underpin diverse mechanisms of gene regulation and genome defense across all three kingdoms of life and are integral to virus-host interactions. In plants, fungi and many animals, an ancestral RNAi pathway exists as a host defense mechanism whereby viral double-stranded RNA is processed to small RNAs that enable recognition and degradation of the virus. While this antiviral RNAi pathway is not generally thought to be present in mammals, other RNAi mechanisms can influence infection through both viral- and host-derived small RNAs. Furthermore, a burgeoning body of data suggests that small RNAs in mammals can function in a non-cell autonomous manner to play various roles in cell-to-cell communication and disease through their transport in extracellular vesicles. While vesicular small RNAs have not been proposed as an antiviral defense pathway per se, there is increasing evidence that the export of host- or viral-derived RNAs from infected cells can influence various aspects of the infection process. This review discusses the current knowledge of extracellular RNA functions in viral infection and the technical challenges surrounding this field of research. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.

Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range.

While single-stranded DNA (ssDNA) was once thought to be a relatively rare genomic architecture for viruses, modern metagenomics sequencing has revealed circular ssDNA viruses in most environments and in association with diverse hosts. In particular, circular ssDNA viruses encoding a homologous replication-associated protein (Rep) have been identified in the majority of eukaryotic supergroups, generating interest in the ecological effects and evolutionary history of circular Rep-encoding ssDNA viruses (CRESS DNA) viruses. This review surveys the explosion of sequence diversity and expansion of eukaryotic CRESS DNA taxonomic groups over the last decade, highlights similarities between the well-studied geminiviruses and circoviruses with newly identified groups known only through their genome sequences, discusses the ecology and evolution of eukaryotic CRESS DNA viruses, and speculates on future research horizons.

Evolution of the Large Nucleocytoplasmic DNA Viruses of Eukaryotes and Convergent Origins of Viral Gigantism.

The Nucleocytoplasmic Large DNA Viruses (NCLDV) of eukaryotes (proposed order "Megavirales") comprise an expansive group of eukaryotic viruses that consists of the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, Pithoviridae, and Mimiviridae, as well as Pandoraviruses, Molliviruses, and Faustoviruses that so far remain unaccounted by the official virus taxonomy. All these viruses have double-stranded DNA genomes that range in size from about 100 kilobases (kb) to more than 2.5 megabases. The viruses with genomes larger than 500kb are informally considered "giant," and the largest giant viruses surpass numerous bacteria and archaea in both particle and genome size. The discovery of giant viruses has been highly unexpected and has changed the perception of viral size and complexity, and even, arguably, the entire concept of a virus. Given that giant viruses encode multiple proteins that are universal among cellular life forms and are components of the translation system, the quintessential cellular molecular machinery, attempts have been made to incorporate these viruses in the evolutionary tree of cellular life. Moreover, evolutionary scenarios of the origin of giant viruses from a fourth, supposedly extinct domain of cellular life have been proposed. However, despite all the differences in the genome size and gene repertoire, the NCLDV can be confidently defined as monophyletic group, on the strength of the presence of about 40 genes that can be traced back to their last common ancestor. Using several most strongly conserved genes from this ancestral set, a well-resolved phylogenetic tree of the NCLDV was built and employed as the scaffold to reconstruct the history of gene gain and loss throughout the course of the evolution of this group of viruses. This reconstruction reveals extremely dynamic evolution that involved extensive gene gain and loss in many groups of viruses and indicates that giant viruses emerged independently in several clades of the NCLDV. Thus, these giants of the virus world evolved repeatedly from smaller and simpler viruses, rather than from a fourth domain of cellular life, and captured numerous genes, including those for translation system components, from eukaryotes, along with some bacterial genes. Even deeper evolutionary reconstructions reveal apparent links between the NCLDV and smaller viruses of eukaryotes, such as adenoviruses, and ultimately, derive all these viruses from tailless bacteriophages.

Emerging Proviral Roles of Caspases during Lytic Replication of Gammaherpesviruses.

Due to their roles in the regulation of programmed cell death and inflammation, the cellular caspase proteases are considered antiviral factors. However, recent studies have revealed examples of proviral functions for caspases. Here, we review a growing body of literature on the role of caspases in promoting the replication of human gammaherpesviruses. We propose that gammaherpesviruses have evolved ways to redirect these enzymes and to use their activation to support viral replication and immune evasion.

Prion-like Domains in Eukaryotic Viruses.

Prions are proteins that can self-propagate, leading to the misfolding of proteins. In addition to the previously demonstrated pathogenic roles of prions during the development of different mammalian diseases, including neurodegenerative diseases, they have recently been shown to represent an important functional component in many prokaryotic and eukaryotic organisms and bacteriophages, confirming the previously unexplored important regulatory and functional roles. However, an in-depth analysis of these domains in eukaryotic viruses has not been performed. Here, we examined the presence of prion-like proteins in eukaryotic viruses that play a primary role in different ecosystems and that are associated with emerging diseases in humans. We identified relevant functional associations in different viral processes and regularities in their presence at different taxonomic levels. Using the prion-like amino-acid composition computational algorithm, we detected 2679 unique putative prion-like domains within 2,742,160 publicly available viral protein sequences. Our findings indicate that viral prion-like proteins can be found in different viruses of insects, plants, mammals, and humans. The analysis performed here demonstrated common patterns in the distribution of prion-like domains across viral orders and families, and revealed probable functional associations with different steps of viral replication and interaction with host cells. These data allow the identification of the viral prion-like proteins as potential novel regulators of viral infections.