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. 2021 Aug 25;95(18):e0091921.
doi: 10.1128/JVI.00919-21. Epub 2021 Aug 25.

Morphological and Taxonomic Properties of the Newly Isolated Cotonvirus japonicus, a New Lineage of the Subfamily Megavirinae

Haruna Takahashi  1 , Sho Fukaya  2   3 , Chihong Song  4   5 , Kazuyoshi Murata  4   5 , Masaharu Takemura  1   2
Affiliations

Morphological and Taxonomic Properties of the Newly Isolated Cotonvirus japonicus, a New Lineage of the Subfamily Megavirinae

Haruna Takahashi et al. J Virol. .

Abstract

Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae, excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae. Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae. Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae. IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae, subfamily Megavirinae, from the fresh water of a canal in Japan, named Cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae-lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.

Keywords: Cotonvirus japonicus; family Mimiviridae; giant virus; isolation.

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Figures

FIG 1
FIG 1
Electron micrographs of cotonvirus particles. (a, b) TEM images of a cotonvirus particle (a) and its stargate structure (b). (c) SEM image. (d) cryo-EM image showing an open stargate structure (white arrow). (e, f) TEM image of Mimivirus shirakomae (lineage A) (e) and Megavirus musashi (lineage C) particles (f). (g) cryo-EM image of Mimivirus shirakomae particles. Scale bars: black, 200 nm; white, 500 nm.
FIG 2
FIG 2
Host specificity of cotonvirus. Acanthamoeba comandoni, A. castellanii, A. culbertsoni, and Vermamoeba vermiformis cells were infected with cotonvirus, mimivirus (Mimivirus shirakomae), and megavirus (Megavirus musashi) at an MOI of 100. After 1 day, amoeba cells were observed using phase-contrast microscopy. Images in which cytopathic effect (CPE) of amoeba cells is shown are indicated by red squares. Noninfected amoeba cells were used as the control. Scale bar = 100 μm.
FIG 3
FIG 3
Circular representation of the cotonvirus genome. From the outside in, coding sequences (CDS) (blue) and tRNA (red), GC content (black), and GC skews (green and purple) are represented.
FIG 4
FIG 4
Best hits for predicted ORFs of cotonvirus. (a) Pie chart showing the best hits for amino acid homology between cotonvirus and the public sequence database. (b, c) The rhizomes show the relationship between cotonvirus genes and best hits in the family Mimiviridae (b) and among all species, including both living things and viruses (c).
FIG 5
FIG 5
Analysis of gene categories of cotonvirus. (a) Classification of cotonvirus genes based on the functional categories of genes. (b) Proportion of each functional category of genes in the subfamily Megavirinae that are best hits to cotonvirus.
FIG 6
FIG 6
Molecular phylogenetic analysis based on the nucleotide sequences of the nucleocytoplasmic large DNA virus (NCLDV) core genes, namely, B family DNA polymerase (a), major capsid protein (b), D5-like ATPase (c), mRNA-packaging enzyme (d), and virion-packaging ATPase (e).
FIG 7
FIG 7
Molecular phylogenetic analysis based on the concatenated NCLDV core genes, consisting of B family DNA polymerase, major capsid protein, D5-like ATPase, mRNA-packaging enzyme, and virion-packaging ATPase.
FIG 8
FIG 8
Proteomic tree based on the genomic sequences of NCLDVs. Each color represents the family or group of NCLDVs. The cotonvirus is in red.
FIG 9
FIG 9
Molecular phylogenetic analysis based on the nucleotide sequences of the aa-RS genes, namely, arginyl-RS (a), cysteinyl-RS (b), methionyl-RS (c), tyrosyl-RS (d), and isoleucyl-RS (e).
FIG 10
FIG 10
18S rRNA-like sequences of cotonvirus.
FIG 11
FIG 11
Putative evolutionary model of the subfamily Megavirinae. Our hypothesis is based on the molecular phylogenetic analyses of the proteomic tree and the aa-RS genes. Thick arrows (gray) indicate LGT.
FIG 12
FIG 12
Kinetics of cotonvirus-infected A. castellanii cells analyzed by the PKA3 algorithm. We measured the estimated cell number (a, b), average step (c, d), average size (e, f), and average circularity (g, h) of cotonvirus-infected A. castellanii cells from 0 to 36 hpi. The graphs on the right show the average value every 30 min. Mimivirus (Mimivirus shirakomae) and megavirus (Megavirus musashi) were analyzed for comparison with cotonvirus. Noninfected A. castellanii cells were used as the control. (f) Red, green, and blue arrows indicate the starting points of cell lysis in each virus-infected A. castellanii cell.
FIG 13
FIG 13
VF formation of cotonvirus. (a) Development of cotonvirus VF. First, the inner materials of cotonvirus particles are released and engulfed by amoeba cells into the host cytoplasm at 2 hpi. The cotonvirus core and membrane-like structures surrounding it are visualized at 4 hpi. Early VFs are visible at both 8 and 12 hpi, and Golgi apparatus-like structures are found around early VFs. Mature VF is formed, and the production of viral particles is initiated at 16 hpi. New virions are then produced and accumulate in the host cytoplasm at 20 hpi and 24 hpi, respectively. VF release by cell lysis is observed at 28 hpi. Scale bars: 2 hpi, 4 hpi, 500 nm; 8 hpi to 16 hpi, 28 hpi, 1 μm; 20 hpi, 24 hpi, 5 μm. (b) Four other views of early VFs surrounded by Golgi apparatus-like structures at 12 hpi.
FIG 14
FIG 14
STEM tomography of the VFs in cotonvirus-infected A. castellanii. (a) Tomographic slice. (b) 3-D model. Many flat and curved tubular structures (dark blue) are observed around early VFs (light blue). The area enclosed in pink shows the layered tubular-structures similar to the Golgi stack, whereas the area enclosed in orange shows a fusion of the early VF and the tubular structures. Scale bar = 500 nm.
FIG 15
FIG 15
Immunofluorescent staining of A. castellanii cells infected with cotonvirus. Blue signals represent DNA in VFs, virus particles, and host nucleus stained with DAPI. Green signals represent GM130 protein of Golgi apparatus labeled by anti-GM130 antibodies. (a) A. castellanii cells infected with cotonvirus. (b) Enlarged views of images in panel a at 24 hpi. (c) A. castellanii cells infected with mimivirus (Mimivirus shirakomae) and megavirus (Megavirus musashi) at 16 hpi. Scale bar = 20 μm.
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