Giant Viruses Research Articles

GIANT VIRUSES - A RELIC OF THE PAST AND CURIOSITY OF THE PRESENT

Discoveries in Microbiology and its subfield Inframicrobiology – which is now known as the independent science of Virology – have always followed the same trend from larger microorganisms to increasingly smaller infectious agents. That held true until recently when scientists confirmed the existence of giant viruses. Although they were initially thought to be comparable to Gram-positive cocci, molecular biology technologies revealed their true viral nature. This new viral type is present in fresh and marine waters, and although commensal in humans, it contributes to human pathologies, especially lower respiratory tract infections – it represents the etiological agent of more than 20% of pneumonia cases without a precise etiology. These large, but paradoxically recently discovered viruses are ancient (as it is demonstrated by their re-awakening from the melting Siberian permafrost) and are leading researchers to question their ecological significance in direct connection with the global warming phenomenon and probable new emergent pathogens. The aim of this paper is to briefly present milestones in virology research and elaborate on the recently discovered senior viruses – giant viruses – along with their main characteristics, implications in human pathology, and ecological significance.

A novel capsid protein network allows the characteristic internal membrane structure of Marseilleviridae giant viruses

Marseilleviridae is a family of giant viruses, showing a characteristic internal membrane with extrusions underneath the icosahedral vertices. However, such large objects, with a maximum diameter of 250 nm are technically difficult to examine at sub-nanometre resolution by cryo-electron microscopy. Here, we tested the utility of 1 MV high-voltage cryo-EM (cryo-HVEM) for single particle structural analysis (SPA) of giant viruses using tokyovirus, a species of Marseilleviridae, and revealed the capsid structure at 7.7 Å resolution. The capsid enclosing the viral DNA consisted primarily of four layers: (1) major capsid proteins (MCPs) and penton proteins, (2) minor capsid proteins (mCPs), (3) scaffold protein components (ScPCs), and (4) internal membrane. The mCPs showed a novel capsid lattice consisting of eight protein components. ScPCs connecting the icosahedral vertices supported the formation of the membrane extrusions, and possibly act like tape measure proteins reported in other giant viruses. The density on top of the MCP trimer was suggested to include glycoproteins. This is the first attempt at cryo-HVEM SPA. We found the primary limitations to be the lack of automated data acquisition and software support for collection and processing and thus achievable resolution. However, the results pave the way for using cryo-HVEM for structural analysis of larger biological specimens.

Giant Viruses as a Source of Novel Enzymes for Biotechnological Application.

The global demand for industrial enzymes has been increasing in recent years, and the search for new sources of these biological products is intense, especially in microorganisms. Most known viruses have limited genetic machinery and, thus, have been overlooked by the enzyme industry for years. However, a peculiar group of viruses breaks this paradigm. Giant viruses of the phylum Nucleocytoviricota infect protists (i.e., algae and amoebae) and have complex genomes, reaching up to 2.7 Mb in length and encoding hundreds of genes. Different giant viruses have robust metabolic machinery, especially those in the Phycodnaviridae and Mimiviridae families. In this review, we present some peculiarities of giant viruses that infect protists and discuss why they should be seen as an outstanding source of new enzymes. We revisited the genomes of representatives of different groups of giant viruses and put together information about their enzymatic machinery, highlighting several genes to be explored in biotechnology involved in carbohydrate metabolism, DNA replication, and RNA processing, among others. Finally, we present additional evidence based on structural biology using chitinase as a model to reinforce the role of giant viruses as a source of novel enzymes for biotechnological application.

Amoebal Tubulin Cleavage Late during Infection Is a Characteristic Feature of Mimivirus but Not of Marseillevirus.

Mimivirus and Marseillevirus infections of Acanthamoeba castellanii, like most other viral infections, induce cytopathic effects (CPE). The details of how they bring about CPE and to what extent and how they modify the host cytoskeletal network are unclear. In this study, we compared the rearrangement of the host cytoskeletal network induced by Mimivirus and Marseillevirus upon infection. We show that while both Mimivirus and Marseillevirus infections of A. castellanii cells cause retraction of acanthopodia and depolymerization of the host actin filament network, the Mimivirus infection also results in characteristic cleavage of the host tubulin, a phenomenon not previously reported with any intracellular pathogens. Furthermore, we show that the amoebal tubulin cleavage during Mimivirus infection is a post-replicative event. Because time-lapse microscopy showed that Mimivirus infection leads to the bursting of cells, releasing the virus, we hypothesize that tubulin cleavage together with actin depolymerization during the later stages of Mimivirus assembly is essential for cell lysis due to apoptotic/necrotic cell death. We also characterize the Mimivirus-encoded gp560, a Zn metalloprotease, however, the purified gp560 protein was unable to cleave the commercially available porcine brain tubulin. While protein synthesis is essential for causing the morphological changes in the case of Mimivirus, the proteins which are packaged in the viral capsid along with the genome are sufficient to induce CPE in the case of Marseillevirus. IMPORTANCE In general, intracellular pathogens target the cytoskeletal network to enable their life cycle inside the host. Pathogen-induced changes in the host cell morphology usually accompany global changes in the cytoskeleton resulting in cytopathic effects. While viruses have been shown to use the host actin cytoskeleton for entry and transport during early infection, the role of microtubules in the viral life cycle is only beginning to emerge. Here, we show that the giant viruses Mimivirus and Marseillevirus both induce depolymerization of the actin filament, Mimivirus also causes a characteristic cleavage of tubulin not previously reported for any intracellular pathogen. Because tubulin cleavage occurs late during infection, we hypothesize that tubulin cleavage aids in cell death and lysis rather than establishing infection. The different strategies used by viruses with similar host niches may help them survive in competition.

Diversity and genomics of giant viruses in the North Pacific Subtropical Gyre.

Large double-stranded DNA viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are ubiquitous members of marine ecosystems that are important agents of mortality for eukaryotic plankton. Although giant viruses are known to be prevalent in marine systems, their activities in oligotrophic ocean waters remain unclear. Oligotrophic gyres constitute the majority of the ocean and assessing viral activities in these regions is therefore critical for understanding overall marine microbial processes. In this study, we generated 11 metagenome-assembled genomes (MAGs) of giant viruses from samples previously collected from Station ALOHA in the North Pacific Subtropical Gyre. Phylogenetic analyses revealed that they belong to the orders Imitervirales (n = 6), Algavirales (n = 4), and Pimascovirales (n = 1). Genome sizes ranged from ~119-574 kbp, and several of the genomes encoded predicted TCA cycle components, cytoskeletal proteins, collagen, rhodopsins, and proteins potentially involved in other cellular processes. Comparison with other marine metagenomes revealed that several have broad distribution across ocean basins and represent abundant viral constituents of pelagic surface waters. Our work sheds light on the diversity of giant viruses present in oligotrophic ocean waters across the globe.

An atlas of the blood virome in healthy individuals

Emerging evidence indicates that gut virome plays a role in human health and disease, however, much less is known about the viral communities in blood. Here we conducted a direct metatranscriptomic sequencing of virus-like-particles in blood from 1200 healthy individuals, without prior amplification to avoid potential amplification bias and with a strictly bioinformatic and manual check for candidate viral reads to reduce false-positive matches. We identified 55 different viruses from 36 viral families, including 24 human DNA, RNA and retroviruses in 70% of the studied pools. The study showed that anelloviruses are widely distributed and dominate the blood virome in healthy individuals. Human herpesviruses and pegivirus-1 are commonly prevalent in asymptomatic humans. We identified the prevalence of RNA viruses often causing acute infection, like HEV, HPIV, RSV and HCoV-HKU1, revealing of a transmissible risk of asymptomatic infection. Several viruses possible related to transfusion safety were identified, including human Merkel cell polyomavirus, papillomavirus, parvovirus B19 and herpesvirus 8 in addition to HBV. In addition, phages in Caudovirales and Microviridae, were commonly found in pools of samples with a very low abundance; a few sequences for invertebrate, plant and giant viruses were found in some of individuals; however, the remaining 31 viruses mostly reflect extensive contamination from commercial reagents and the work environments. In conclusion, this study is the first comprehensive investigation of blood virome in healthy individuals by metatranscriptomic sequencing of VLP in China. Further investigation of potential false positives representing a major challenge for the identification of novel viruses in mNGS, will offer a systemic idea and means to reveal true viral infections of human.

A giant virus genome is densely packaged by stable nucleosomes within virions

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

Near-atomic, non-icosahedrally averaged structure of giant virus Paramecium bursaria chlorella virus 1

Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.

Multiple waves of viral invasions in Symbiodiniaceae algal genomes.

Dinoflagellates from the family Symbiodiniaceae are phototrophic marine protists that engage in symbiosis with diverse hosts. Their large and distinct genomes are characterized by pervasive gene duplication and large-scale retroposition events. However, little is known about the role and scale of horizontal gene transfer (HGT) in the evolution of this algal family. In other dinoflagellates, high levels of HGTs have been observed, linked to major genomic transitions, such as the appearance of a viral-acquired nucleoprotein that originated via HGT from a large DNA algal virus. Previous work showed that Symbiodiniaceae from different hosts are actively infected by viral groups, such as giant DNA viruses and ssRNA viruses, that may play an important role in coral health. Latent viral infections may also occur, whereby viruses could persist in the cytoplasm or integrate into the host genome as a provirus. This hypothesis received experimental support; however, the cellular localization of putative latent viruses and their taxonomic affiliation are still unknown. In addition, despite the finding of viral sequences in some genomes of Symbiodiniaceae, viral origin, taxonomic breadth, and metabolic potential have not been explored. To address these questions, we searched for putative viral-derived proteins in thirteen Symbiodiniaceae genomes. We found fifty-nine candidate viral-derived HGTs that gave rise to twelve phylogenies across ten genomes. We also describe the taxonomic affiliation of these virus-related sequences, their structure, and their genomic context. These results lead us to propose a model to explain the origin and fate of Symbiodiniaceae viral acquisitions.

Endogenous giant viruses contribute to intraspecies genomic variability in the model green alga Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a unicellular eukaryotic alga that has been studied as a model organism for decades. Despite an extensive history as a model system, phylogenetic and genetic characteristics of viruses infecting this alga have remained elusive. We analyzed high-throughput genome sequence data of C. reinhardtii field isolates, and in six we discovered sequences belonging to endogenous giant viruses that reach up to several 100kb in length. In addition, we have also discovered the entire genome of a closely related giant virus that is endogenized within the genome of Chlamydomonas incerta, the closest sequenced relative of C. reinhardtii. Endogenous giant viruses add hundreds of new gene families to the host strains, highlighting their contribution to the pangenome dynamics and interstrain genomic variability of C. reinhardtii. Our findings suggest that the endogenization of giant viruses may have important implications for structuring the population dynamics and ecology of protists in the environment.

Viral origin of eukaryotic type IIA DNA topoisomerases.

Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation, and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologs and have no orthologs in Archaea. Interestingly, eukaryotic Topo IIAs have close homologs in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses. Topo IIAs are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses on a dataset combining viral and cellular Topo IIA homologs. Topo IIAs encoded by Bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both Bacteria and Caudoviricetes from the data set, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long-branch attraction artifact. The topologies of our trees suggest that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

Past and present giant viruses diversity explored through permafrost metagenomics

Giant viruses are abundant in aquatic environments and ecologically important through the metabolic reprogramming of their hosts. Less is known about giant viruses from soil even though two of them, belonging to two different viral families, were reactivated from 30,000-y-old permafrost samples. This suggests an untapped diversity of Nucleocytoviricota in this environment. Through permafrost metagenomics we reveal a unique diversity pattern and a high heterogeneity in the abundance of giant viruses, representing up to 12% of the sum of sequence coverage in one sample. Pithoviridae and Orpheoviridae-like viruses were the most important contributors. A complete 1.6 Mb Pithoviridae-like circular genome was also assembled from a 42,000-y-old sample. The annotation of the permafrost viral sequences revealed a patchwork of predicted functions amidst a larger reservoir of genes of unknown functions. Finally, the phylogenetic reconstructions not only revealed gene transfers between cells and viruses, but also between viruses from different families.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

Nucleosome Structures Built from Highly Divergent Histones: Parasites and Giant DNA Viruses.

In eukaryotes, genomic DNA is bound with histone proteins and packaged into chromatin. The nucleosome, a fundamental unit of chromatin, regulates the accessibility of DNA to enzymes involved in gene regulation. During the past few years, structural analyses of chromatin architectures have been limited to evolutionarily related organisms. The amino acid sequences of histone proteins are highly conserved from humans to yeasts, but are divergent in the deeply branching protozoan groups, including human parasites that are directly related to human health. Certain large DNA viruses, as well as archaeal organisms, contain distant homologs of eukaryotic histone proteins. The divergent sequences give rise to unique and distinct nucleosome architectures, although the fundamental principles of histone folding and DNA contact are highly conserved. In this article, we review the structures and biophysical properties of nucleosomes containing histones from the human parasites Giardia lamblia and Leishmania major, and histone-like proteins from the Marseilleviridae amoeba virus family. The presented data confirm the sharing of the overall DNA compaction system among evolutionally distant species and clarify the deviations from the species-specific nature of the nucleosome.

Vaccinia virus H7-protein is required for the organization of the viral scaffold protein into hexamers

Viruses of the giant virus family are characterized by a structurally conserved scaffold-capsid protein that shapes the icosahedral virion. The vaccinia virus (VACV) scaffold protein D13, however, transiently shapes the newly assembled viral membrane in to a sphere and is absent from the mature brick-shaped virion. In infected cells D13, a 62 kDa polypeptide, forms trimers that arrange in hexamers and a honey-comb like lattice. Membrane association of the D13-lattice may be mediated by A17, an abundant 21 kDa viral membrane protein. Whether membrane binding mediates the formation of the honey-comb lattice or if other factors are involved, remains elusive. Here we show that H7, a 17 kDa protein conserved among poxviruses, mediates proper formation of D13-hexamers, and hence the honey comb lattice and spherical immature virus. Without H7 synthesis D13 trimers assemble into a large 3D network rather than the typical well organized scaffold layer observed in wild-type infection, composed of short D13 tubes of discrete length that are tightly associated with the endoplasmic reticulum (ER). The data show an unexpected role for H7 in D13 organization and imply that formation of the honey-comb, hexagonal, lattice is essential for VACV membrane assembly and production of infectious progeny. The data are discussed with respect to scaffold proteins of other giant viruses.

Giant virus biology and diversity in the era of genome-resolved metagenomics.

The discovery of giant viruses, with capsids as large as some bacteria, megabase-range genomes and a variety of traits typically found only in cellular organisms, was one of the most remarkable breakthroughs in biology. Until recently, most of our knowledge of giant viruses came from ~100 species-level isolates for which genome sequences were available. However, these isolates were primarily derived from laboratory-based co-cultivation with few cultured protists and algae and, thus, did not reflect the true diversity of giant viruses. Although virus co-cultures enabled valuable insights into giant virus biology, many questions regarding their origin, evolution and ecological importance remain unanswered. With advances in sequencing technologies and bioinformatics, our understanding of giant viruses has drastically expanded. In this Review, we summarize our understanding of giant virus diversity and biology based on viral isolates as laboratory cultivation has enabled extensive insights into viral morphology and infection strategies. We then explore how cultivation-independent approaches have heightened our understanding of the coding potential and diversity of the Nucleocytoviricota. We discuss how metagenomics has revolutionized our perspective of giant viruses by revealing their distribution across our planet's biomes, where they impact the biology and ecology of a wide range of eukaryotic hosts and ultimately affect global nutrient cycles.

The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

COVID-19: epidemiological data of 1263 patients from southern Minas Gerais, Brazil

Background: COVID-19 is an easily contagious and high-speed viral disease that is directly affecting people's lives and exposing the fragility of health systems around the world, especially in Brazil. In this context, the present research sought to observe, through medical records, the conduct of positive patients with COVID-19.Methods: The methodology used was the analysis of medical records of patients with COVID-19 in five hospitals in Minas Gerais. The following data were collected from patients who were hospitalized from March 2020 to October 2020: personal characteristics; pathological antecedents and evolution of the clinical picture.Results: The results found describe the number of patients in each of the analysed hospitals. In addition, the predominant age, the sex of the patients in percentage, the number of patients with subarachnoid hemorrhage (SAH), chronic obstructive pulmonary disease (COPD), diabetes, dyslipidemia, altered D-dimer, anemia and hematologic change are described. Intensive care unit (ICU) and ward admissions are also addressed, as well as the type of respiratory complication, percentage of individuals who required intubation, intubation time and outcome. The results were used in statistical analysis for the association between variables with the Chi-square test to verify the significance between them. The outcome was compared with age, sex, diabetes, dyslipidemia, D-dimer biomarker, anemia, hematological changes and type of hospitalization. With the type of hospitalization, diabetes and dyslipidemia were compared.Conclusions: Faced with all this context of public health urgency, this work helps in the standardization of variables to collaborate in the fight against this giant and persistent virus that public health is trying to fight.

The Astounding World of Glycans from Giant Viruses.

Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed.

Metabolic arsenal of giant viruses: Host hijack or self-use?

Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.