Endogenous giant viruses shape intraspecies genomic variability in the model green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is an important eukaryotic alga that has been studied as a model organism for decades. Despite 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 numerous C. reinhardtii isolates, and in six strains we discovered endogenous genomes of giant viruses reaching over several hundred kilobases 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, one of the closest sequenced phylogenetic relatives of C. reinhardtii. Endogenous giant viruses add hundreds of new gene families to the host strains, highlighting their contribution to the pangenome dynamics and inter-strain genomic variability of C. reinhardtii. Our findings suggest that endogenization of giant viruses can have profound implications in shaping the population dynamics and ecology of protists in the environment.

A phylogenomic framework for charting the diversity and evolution of giant viruses

Large DNA viruses of the phylum Nucleocytoviricota have recently emerged as important members of ecosystems around the globe that challenge traditional views of viral complexity. Numerous members of this phylum that cannot be classified within established families have recently been reported, and there is presently a strong need for a robust phylogenomic and taxonomic framework for these viruses. Here, we report a comprehensive phylogenomic analysis of the Nucleocytoviricota, present a set of giant virus orthologous groups (GVOGs) together with a benchmarked reference phylogeny, and delineate a hierarchical taxonomy within this phylum. We show that the majority of Nucleocytoviricota diversity can be partitioned into 6 orders, 32 families, and 344 genera, substantially expanding the number of currently recognized taxonomic ranks for these viruses. We integrate our results within a taxonomy that has been adopted for all viruses to establish a unifying framework for the study of Nucleocytoviricota diversity, evolution, and environmental distribution.

Particle morphology of medusavirus inside and outside the cells reveals a new maturation process of giant viruses

Medusavirus, a giant virus, is phylogenetically closer to eukaryotes than the other giant viruses and has been recently classified as an independent species. However, details of its morphology and maturation process in host cells remain unclear. Here, we investigated the particle morphology of medusavirus inside and outside infected cells using conventional transmission electron microscopy (C-TEM) and cryo-electron microscopy (cryo-EM). The C-TEM of amoeba infected with the medusavirus showed four types of particles: empty, DNA-full, and the corresponding intermediates. Time-dependent changes in the proportion and following intracellular localization of these particles suggested a new maturation process for the medusavirus. Empty particles and viral DNAs were produced independently in the cytoplasm and nucleus, respectively, and only empty particles located near the nucleus incorporated the viral DNA into the capsid. All four types of particles were also found outside the cells. The cryo-EM of these particles showed that the intact capsid structure, covered with three different types of spikes, was conserved among all particle types, although with minor size-related differences. The internal membrane exhibited a structural array similar to that of the capsid, interacted closely with the capsid, and displayed open membrane structures in the empty and empty-intermediate particles. This result suggests that the open structures in the internal membrane are used for an exchange of scaffold proteins and viral DNA during the maturation process. This new model of the maturation process of medusavirus provides insight into the structural and behavioral diversity of giant viruses.

Paving the way towards understanding the inflammatory pathways triggered by giant viruses in mammalian cells: effect of mimivirus-cell interactions on IκBα degradation

Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 μm and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

Giant Viruses and the Tree of Life

When the first giant virus, the mimivirus, was discovered in 1992, it was misidentified as a bacterium because it was too large to have been a virus by the current understanding. Ever since, biologists have been debating how viruses should be categorized and described. Are they living? Are they something else? What is their place on the tree of life?

Cryo-Electron Microscopy of the Giant Viruses

High resolution study of the giant viruses presents one of the latest challenges in cryo-electron microscopy of viruses. Too small for light microscopy, but too large for easy study at high resolution by electron microscopy, they range in size from ~0.2-2 μm, from high symmetry icosahedral viruses such as Paramecium burseria Chlorella virus 1 to asymmetric forms like Tupanvirus or Pithovirus. To attain high resolution, two strategies exist to study these large viruses by cryo-EM: firstly, increasing the acceleration voltage of the electron microscope to improve sample penetration and overcome the limitations imposed by electro-optical physics at lower voltages, and secondly the method of “block-based reconstruction” pioneered by Michael G. Rossmann and his collaborators, which resolves the latter limitation through an elegant leveraging of high symmetry, but cannot overcome sample penetration limitations. In addition, more recent advances in both computational capacity and image processing also yield assistance in studying the giant viruses. Especially, the inclusion of Ewald sphere correction can provide large improvements in attainable resolutions for 300 kV electron microscopes. Despite this, the study of giant viruses remains a significant challenge.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells

Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii , which were previously unclear.

Comparative Analysis of Transcriptional Regulation Patterns: Understanding the Gene Expression Profile in Nucleocytoviricota

The nucleocytoplasmic large DNA viruses (NCLDV) possess unique characteristics that have drawn the attention of the scientific community, and they are now classified in the phylum Nucleocytoviricota. They are characterized by sharing many genes and have their own transcriptional apparatus, which provides certain independence from their host’s machinery. Thus, the presence of a robust transcriptional apparatus has raised much discussion about the evolutionary aspects of these viruses and their genomes. Understanding the transcriptional process in NCLDV would provide information regarding their evolutionary history and a better comprehension of the biology of these viruses and their interaction with hosts. In this work, we reviewed NCLDV transcription and performed a comparative functional analysis of the groups of genes expressed at different times of infection of representatives of six different viral families of giant viruses. With this analysis, it was possible to observe a temporal profile of their gene expression and set of genes activated in specific phases throughout the multiplication cycle as a common characteristic of this group. Due to the lack of information regarding the transcriptional regulation process of this group of pathogens, we sought to provide information that contributes to and opens up the field for transcriptional studies of other viruses belonging to Nucleocytoviricota.