Clandestinovirus: A Giant Virus With Chromatin Proteins and a Potential to Manipulate the Cell Cycle of Its Host Vermamoeba vermiformis.

For several decades, the vast world of DNA viruses has been expanding constantly. Various discoveries in this field have broadened our knowledge and revealed that DNA viruses encode many functional features, which were once thought to be exclusive to cellular life. Here, we report the isolation of a giant virus named "clandestinovirus," grown on the amoebal host Vermamoeba vermiformis. This virus was discovered in a mixed co-culture associated with another giant virus, Faustovirus ST1. Clandestinovirus possesses a linear dsDNA genome of 581,987 base pairs containing 617 genes. Phylogenetically, clandestinovirus is most closely related to Acanthamoeba castellanii medusavirus and was considered a member of the proposed Medusaviridae family. However, clandestinovirus genome is 65% larger than that of medusavirus, emphasizing the considerable genome size variation within this virus family. Functional annotation of the clandestinovirus genes suggests that the virus encodes four core histones. Furthermore, clandestinovirus appears to orchestrate the cell cycle and mitochondrial activities of the infected host by virtue of encoding a panel of protein kinases and phosphatases, and a suite of functionally diverse mitochondrial protein homologs, respectively. Collectively, these observations illuminate a strategy employed by clandestinovirus to optimize the intracellular environment for efficient virus propagation.

Generation of Infectious Mimivirus Virions Through Inoculation of Viral DNA Within Acanthamoeba castellanii Shows Involvement of Five Proteins, Essentially Uncharacterized.

One of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography-mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins-L442, L724, L829, and R387-and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein-DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role.

Marseilleviruses: An Update in 2021.

The family Marseilleviridae was the second family of giant viruses that was described in 2013, after the family Mimiviridae. Marseillevirus marseillevirus, isolated in 2007 by coculture on Acanthamoeba polyphaga, is the prototype member of this family. Afterward, the worldwide distribution of marseilleviruses was revealed through their isolation from samples of various types and sources. Thus, 62 were isolated from environmental water, one from soil, one from a dipteran, one from mussels, and two from asymptomatic humans, which led to the description of 67 marseillevirus isolates, including 21 by the IHU Méditerranée Infection in France. Recently, five marseillevirus genomes were assembled from deep sea sediment in Norway. Isolated marseilleviruses have ≈250 nm long icosahedral capsids and 348-404 kilobase long mosaic genomes that encode 386-545 predicted proteins. Comparative genomic analyses indicate that the family Marseilleviridae includes five lineages and possesses a pangenome composed of 3,082 clusters of genes. The detection of marseilleviruses in both symptomatic and asymptomatic humans in stool, blood, and lymph nodes, and an up-to-30-day persistence of marseillevirus in rats and mice, raise questions concerning their possible clinical significance that are still under investigation.

A virophage cross-species infection through mutant selection represses giant virus propagation, promoting host cell survival.

Virus adaptation to new hosts is a major cause of infectious disease emergence. This mechanism has been intensively studied in the context of zoonotic virus spillover, due to its impact on global health. However, it remains unclear for virophages, parasites of giant viruses and potential regulators of microbial communities. Here, we present, for the first time to our knowledge, evidence of cross-species infection of a virophage. We demonstrated that challenging the native population of Guarani virophage with two previously unidentified giant viruses, previously nonpermissive to this virophage, allows the selection of a mutant genotype able to infect these giant viruses. We were able to characterize the potential genetic determinant (deletion) carried by the virophage with the expanded-host range. Our study also highlights the relevant biological impact of this host adaptation by demonstrating that coinfection with the mixture containing the mutant virophage abolishes giant virus production and rescues the host cell population from lysis.

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Virophages of Giant Viruses: An Update at Eleven.

The last decade has been marked by two eminent discoveries that have changed our perception of the virology field: The discovery of giant viruses and a distinct new class of viral agents that parasitize their viral factories, the virophages. Coculture and metagenomics have actively contributed to the expansion of the virophage family by isolating dozens of new members. This increase in the body of data on virophage not only revealed the diversity of the virophage group, but also the relevant ecological impact of these small viruses and their potential role in the dynamics of the microbial network. In addition, the isolation of virophages has led us to discover previously unknown features displayed by their host viruses and cells. In this review, we present an update of all the knowledge on the isolation, biology, genomics, and morphological features of the virophages, a decade after the discovery of their first member, the Sputnik virophage. We discuss their parasitic lifestyle as bona fide viruses of the giant virus factories, genetic parasites of their genomes, and then their role as a key component or target for some host defense mechanisms during the tripartite virophage-giant virus-host cell interaction. We also present the latest advances regarding their origin, classification, and definition that have been widely discussed.