Subnanometer structure of medusavirus capsid during maturation using cryo-electron microscopy.

Medusavirus is a giant virus classified into an independent family of Mamonoviridae. Amoebae infected with medusavirus release immature particles in addition to virions. These particles were suggested to exhibit the maturation process of this virus, but the structure of these capsids during maturation remains unknown. Here, we apply a block-based reconstruction method in cryo-electron microscopy (cryo-EM) single particle analysis to these viral capsids, extending the resolution to 7-10 Å. The maps reveal a novel network composed of minor capsid proteins (mCPs) supporting major capsid proteins (MCPs). A predicted molecular model of the MCP fitted into the cryo-EM maps clarified the boundaries between the MCP and the underlining mCPs, as well as between the MCP and the outer spikes, and identified molecular interactions between the MCP and these components. Several structural changes of the mCPs under the fivefold vertices of the immature particles were observed, depending on the presence or absence of the underlying internal membrane. In addition, the lower part of the penton proteins on the fivefold vertices was also missing in mature virions. These dynamic conformational changes of mCPs indicate an important function in the maturation process of medusavirus.IMPORTANCEThe structural changes of giant virus capsids during maturation have not thus far been well clarified. Medusavirus is a unique giant virus in which infected amoebae release immature particles in addition to mature virus particles. In this study, we used cryo-electron microscopy to investigate immature and mature medusavirus particles and elucidate the structural changes of the viral capsid during the maturation process. In DNA-empty particles, the conformation of the minor capsid proteins changed dynamically depending on the presence or absence of the underlying internal membranes. In DNA-full particles, the lower part of the penton proteins was lost. This is the first report of structural changes of the viral capsid during the maturation process of giant viruses.

A long-term prospecting study on giant viruses in terrestrial and marine Brazilian biomes.

The discovery of mimivirus in 2003 prompted the search for novel giant viruses worldwide. Despite increasing interest, the diversity and distribution of giant viruses is barely known. Here, we present data from a 2012-2022 study aimed at prospecting for amoebal viruses in water, soil, mud, and sewage samples across Brazilian biomes, using Acanthamoeba castellanii for isolation. A total of 881 aliquots from 187 samples covering terrestrial and marine Brazilian biomes were processed. Electron microscopy and PCR were used to identify the obtained isolates. Sixty-seven amoebal viruses were isolated, including mimiviruses, marseilleviruses, pandoraviruses, cedratviruses, and yaraviruses. Viruses were isolated from all tested sample types and almost all biomes. In comparison to other similar studies, our work isolated a substantial number of Marseillevirus and cedratvirus representatives. Taken together, our results used a combination of isolation techniques with microscopy, PCR, and sequencing and put highlight on richness of giant virus present in different terrestrial and marine Brazilian biomes.

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 amoebae infected with the medusavirus showed four types of particles, i.e., pseudo-DNA-empty (p-Empty), DNA-empty (Empty), semi-DNA-full (s-Full), and DNA-full (Full). Time-dependent changes in the four types of particles and their intracellular localization suggested a new maturation process for the medusavirus. Viral capsids and viral DNAs are produced independently in the cytoplasm and nucleus, respectively, and only the empty particles located near the host nucleus can incorporate the viral DNA into the capsid. Therefore, all four types of particles were found outside the cells. The cryo-EM of these particles showed that the intact virus structure, covered with three different types of spikes, was preserved 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 p-Empty particles. The results suggest that these 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. IMPORTANCE Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture. Time-course analysis and intracellular localization of the medusavirus in the infected cells suggested a new maturation process via the four types of particles. Like the previously reported pandoravirus, the viral DNA of medusavirus is replicated in the host's nucleus. However, viral capsids are produced independently in the host cytoplasm, and only empty capsids near the nucleus can take up viral DNA. As a result, many immature particles were released from the host cell along with the mature particles. The capsid structure is well conserved among the four types of particles, except for the open membrane structures in the empty particles, suggesting that they are used to exchange scaffold proteins for viral DNAs. These findings indicate that medusavirus has a unique maturation process.

Characterization of Mollivirus kamchatka, the First Modern Representative of the Proposed Molliviridae Family of Giant Viruses.

Microbes trapped in permanently frozen paleosoils (permafrost) are the focus of increasing research in the context of global warming. Our previous investigations led to the discovery and reactivation of two Acanthamoeba-infecting giant viruses, Mollivirus sibericum and Pithovirus sibericum, from a 30,000-year old permafrost layer. While several modern pithovirus strains have since been isolated, no contemporary mollivirus relative was found. We now describe Mollivirus kamchatka, a close relative to M. sibericum, isolated from surface soil sampled on the bank of the Kronotsky River in Kamchatka, Russian Federation. This discovery confirms that molliviruses have not gone extinct and are at least present in a distant subarctic continental location. This modern isolate exhibits a nucleocytoplasmic replication cycle identical to that of M. sibericum Its spherical particle (0.6 µm in diameter) encloses a 648-kb GC-rich double-stranded DNA genome coding for 480 proteins, of which 61% are unique to these two molliviruses. The 461 homologous proteins are highly conserved (92% identical residues, on average), despite the presumed stasis of M. sibericum for the last 30,000 years. Selection pressure analyses show that most of these proteins contribute to virus fitness. The comparison of these first two molliviruses clarify their evolutionary relationship with the pandoraviruses, supporting their provisional classification in a distinct family, the Molliviridae, pending the eventual discovery of intermediary missing links better demonstrating their common ancestry.IMPORTANCE Virology has long been viewed through the prism of human, cattle, or plant diseases, leading to a largely incomplete picture of the viral world. The serendipitous discovery of the first giant virus visible under a light microscope (i.e., >0.3 µm in diameter), mimivirus, opened a new era of environmental virology, now incorporating protozoan-infecting viruses. Planet-wide isolation studies and metagenome analyses have shown the presence of giant viruses in most terrestrial and aquatic environments, including upper Pleistocene frozen soils. Those systematic surveys have led authors to propose several new distinct families, including the Mimiviridae, Marseilleviridae, Faustoviridae, Pandoraviridae, and Pithoviridae We now propose to introduce one additional family, the Molliviridae, following the description of M. kamchatka, the first modern relative of M. sibericum, previously isolated from 30,000-year-old arctic permafrost.

Isolation of Yasminevirus, the First Member of Klosneuvirinae Isolated in Coculture with Vermamoeba vermiformis, Demonstrates an Extended Arsenal of Translational Apparatus Components.

The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis This giant virus, called Yasminevirus, has a 2.1-Mb linear double-stranded DNA genome encoding 1,541 candidate proteins, with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery, with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids (aa), 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic, with ∼34% of genes having their closest homologues in other viruses, followed by ∼13.2% in Eukaryota, ∼7.2% in Bacteria, and less than 1% in Archaea Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses.IMPORTANCE Yasminevirus is an icosahedral double-stranded DNA virus isolated from sewage water by amoeba coculture. Here its structure and replicative cycle in the amoeba Vermamoeba vermiformis are described and genomic and evolutionary studies are reported. This virus belongs to the Klosneuvirinae group of giant viruses, representing the second isolated and cultivated giant virus in this group, and is the first isolated using a coculture procedure. Extended translational machinery pointed to Yasminevirus among the quasiautonomous giant viruses with the most complete translational apparatus of the known virosphere.

In-depth analysis of the replication cycle of Orpheovirus.

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

Ubiquitous giants: a plethora of giant viruses found in Brazil and Antarctica.

BACKGROUND: Since the discovery of giant viruses infecting amoebae in 2003, many dogmas of virology have been revised and the search for these viruses has been intensified. Over the last few years, several new groups of these viruses have been discovered in various types of samples and environments.In this work, we describe the isolation of 68 giant viruses of amoeba obtained from environmental samples from Brazil and Antarctica. METHODS: Isolated viruses were identified by hemacolor staining, PCR assays and electron microscopy (scanning and/or transmission). RESULTS: A total of 64 viruses belonging to the Mimiviridae family were isolated (26 from lineage A, 13 from lineage B, 2 from lineage C and 23 from unidentified lineages) from different types of samples, including marine water from Antarctica, thus being the first mimiviruses isolated in this extreme environment to date. Furthermore, a marseillevirus was isolated from sewage samples along with two pandoraviruses and a cedratvirus (the third to be isolated in the world so far). CONCLUSIONS: Considering the different type of samples, we found a higher number of viral groups in sewage samples. Our results reinforce the importance of prospective studies in different environmental samples, therefore improving our comprehension about the circulation anddiversity of these viruses in nature.

Cedratvirus lausannensis - digging into Pithoviridae diversity.

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

The Large Marseillevirus Explores Different Entry Pathways by Forming Giant Infectious Vesicles.

UNLABELLED: Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. IMPORTANCE: Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.

A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.