In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba

Edited by James L. Van Etten, University of Nebraska, Lincoln, NE, and approved August 12, 2015 (received for review June 2, 2015)
September 8, 2015
112 (38) E5327-E5335

Significance

The saga of giant viruses (i.e. visible by light microscopy) started in 2003 with the discovery of Mimivirus. Two additional types of giant viruses infecting Acanthamoeba have been discovered since: the Pandoraviruses (2013) and Pithovirus sibericum (2014), the latter one revived from 30,000-y-old Siberian permafrost. We now describe Mollivirus sibericum, a fourth type of giant virus isolated from the same permafrost sample. These four types of giant virus exhibit different virion structures, sizes (0.6–1.5 µm), genome length (0.6–2.8 Mb), and replication cycles. Their origin and mode of evolution are the subject of conflicting hypotheses. The fact that two different viruses could be easily revived from prehistoric permafrost should be of concern in a context of global warming.

Abstract

Acanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of “omic” approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host’s ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses’ diversity remains to be fully explored.

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Data Availability

Data deposition: The Mollivirus genome sequence reported in this paper has been deposited in the GenBank database (accession no. KR921745). The transcriptomic data have been deposited in the Sequence Read Archive, www.ncbi.nlm.nih.gov/Traces/sra/ [accession no. SRX1078581 (SRR2084123 for the early class of expression, SRR2103267 for the intermediate one, and SRR2103268 for the late class of expression)]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, proteomecentral.proteomexchange.org, via the Proteomics Identifications partner repository [dataset identifiers <PXD002375> (Particule and Surfome) and <PXD002374> (Time Course)]. All data can be visualized on an interactive genome browser at the following link: www.igs.cnrs-mrs.fr/cgi-bin/gb2/gbrowse/Mollivirus/.

Acknowledgments

We thank Dr. J.-P. Chauvin, Dr. A. Kosta, F. Richard, and A. Aouane and Serge Nitsche for their expert assistance on the imaging platforms, Dr. Dorothée Murat for providing some transmission electron microscopy (TEM) images, and Miguel Ortiz Lombardia for his thorough reading of the manuscript. This work was partially supported by France Génomique Grant ANR-10-INSB-01-01, French National Research Agency Grant ANR-14-CE14-0023-01, the Provence-Alpes-Côte-d’Azur Région (2010 12125), ProFi Grant ANR-10-INBS-08-01, and Russian Scientific Fund 14-14-01115.

Supporting Information

Supporting Information (PDF)
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pnas.1510795112.st01.docx
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pnas.1510795112.st03.docx
pnas.1510795112.st04.docx

References

1
B La Scola, et al., A giant virus in amoebae. Science 299, 2033 (2003).
2
D Raoult, et al., The 1.2-megabase genome sequence of Mimivirus. Science 306, 1344–1350 (2004).
3
D Arslan, M Legendre, V Seltzer, C Abergel, J-M Claverie, Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. Proc Natl Acad Sci USA 108, 17486–17491 (2011).
4
J-M Claverie, C Abergel, Mimivirus and its virophage. Annu Rev Genet 43, 49–66 (2009).
5
Y Mutsafi, N Zauberman, I Sabanay, A Minsky, Vaccinia-like cytoplasmic replication of the giant Mimivirus. Proc Natl Acad Sci USA 107, 5978–5982 (2010).
6
N Yoosuf, et al., Related giant viruses in distant locations and different habitats: Acanthamoeba polyphaga moumouvirus represents a third lineage of the Mimiviridae that is close to the Megavirus lineage. Genome Biol Evol 4, 1324–1330 (2012).
7
MG Fischer, MJ Allen, WH Wilson, CA Suttle, Giant virus with a remarkable complement of genes infects marine zooplankton. Proc Natl Acad Sci USA 107, 19508–19513 (2010).
8
S Santini, et al., Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes. Proc Natl Acad Sci USA 110, 10800–10805 (2013).
9
M Moniruzzaman, et al., Genome of brown tide virus (AaV), the little giant of the Megaviridae, elucidates NCLDV genome expansion and host-virus coevolution. Virology 466-467, 60–70 (2014).
10
M Boyer, et al., Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Proc Natl Acad Sci USA 106, 21848–21853 (2009).
11
V Thomas, et al., Lausannevirus, a giant amoebal virus encoding histone doublets. Environ Microbiol 13, 1454–1466 (2011).
12
P Colson, et al., “Marseilleviridae,” a new family of giant viruses infecting amoebae. Arch Virol 158, 915–920 (2013).
13
G Doutre, N Philippe, C Abergel, J-M Claverie, Genome analysis of the first Marseilleviridae representative from Australia indicates that most of its genes contribute to virus fitness. J Virol 88, 14340–14349 (2014).
14
N Philippe, et al., Pandoraviruses: Amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science 341, 281–286 (2013).
15
MH Antwerpen, et al., Whole-genome sequencing of a pandoravirus isolated from keratitis-inducing acanthamoeba. Genome Announc 3, e00136-15 (2015).
16
M Legendre, et al., Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc Natl Acad Sci USA 111, 4274–4279 (2014).
17
J Besemer, A Lomsadze, M Borodovsky, GeneMarkS: A self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 29, 2607–2618 (2001).
18
M Clarke, et al., Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling. Genome Biol 14, R11 (2013).
19
NCBI Resource Coordinators (2015) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 43(Database issue):D6–D17.
20
P Renesto, et al., Mimivirus giant particles incorporate a large fraction of anonymous and unique gene products. J Virol 80, 11678–11685 (2006).
21
JM Claverie, C Abergel, H Ogata, Mimivirus. Curr Top Microbiol Immunol 328, 89–121 (2009).
22
UF Greber, A Fassati, Nuclear import of viral DNA genomes. Traffic 4, 136–143 (2003).
23
G Wu, L Nie, W Zhang, Integrative analyses of posttranscriptional regulation in the yeast Saccharomyces cerevisiae using transcriptomic and proteomic data. Curr Microbiol 57, 18–22 (2008).
24
R Michel, EN Schmid, R Hoffmann, KD Müller, Endoparasite KC5/2 encloses large areas of sol-like cytoplasm within Acanthamoebae. Normal behavior or aberration? Parasitol Res 91, 265–266 (2003).
25
KL Maxwell, L Frappier, Viral proteomics. Microbiol Mol Biol Rev 71, 398–411 (2007).
26
MP Weekes, et al., Quantitative temporal viromics: An approach to investigate host-pathogen interaction. Cell 157, 1460–1472 (2014).
27
FC Page A New Key to Freshwater and Soil Gymnamoebae (Freshwater Biological Association, Ambleside, UK, 1988).
28
V Dupierris, C Masselon, M Court, S Kieffer-Jaquinod, C Bruley, A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa. Bioinformatics 25, 1980–1981 (2009).
29
J Cox, M Mann, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367–1372 (2008).
30
J Cox, et al., Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13, 2513–2526 (2014).
31
YH Yang, et al., Normalization for cDNA microarray data: A robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15 (2002).
32
A Shatilovich, L Shmakova, A Mylnikov, D Gilichinsky, Ancient protozoa isolated from permafrost. Permafrost Soils. Soil Biology, ed Margesin R (Springer, Berlin), pp 97–115. (2009).
33
AV Shatilovich, LA Shmakova, SV Gubin, AV Gudkov, DA Gilichinskiĭ, Viable protozoa in late Pleistocene and Holocene permafrost sediments. Dokl Biol Sci 401, 136–138 (2005).
34
JT Simpson, R Durbin, Efficient de novo assembly of large genomes using compressed data structures. Genome Res 22, 549–556 (2012).
35
B Langmead, SL Salzberg, Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359 (2012).
36
R Luo, et al., SOAPdenovo2: An empirically improved memory-efficient short-read de novo assembler. Gigascience 1, 18 (2012).
37
JK Bonfield, A Whitwham, Gap5—editing the billion fragment sequence assembly. Bioinformatics 26, 1699–1703 (2010).
38
A Marchler-Bauer, SH Bryant, CD-Search: Protein domain annotations on the fly. Nucleic Acids Res 32, W327–W331 (2004).
39
J Shi, TL Blundell, K Mizuguchi, FUGUE: Sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol 310, 243–257 (2001).
40
A Marchler-Bauer, et al., CDD: A database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res 30, 281–283 (2002).
41
RL Tatusov, et al., The COG database: An updated version includes eukaryotes. BMC Bioinformatics 4, 41 (2003).
42
L Li, Jr CJ Stoeckert, DS Roos, OrthoMCL: Identification of ortholog groups for eukaryotic genomes. Genome Res 13, 2178–2189 (2003).
43
B Snel, P Bork, MA Huynen, Genome phylogeny based on gene content. Nat Genet 21, 108–110 (1999).
44
BL Cantarel, et al., MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res 18, 188–196 (2008).
45
M Stanke, A Tzvetkova, B Morgenstern, AUGUSTUS at EGASP: Using EST, protein and genomic alignments for improved gene prediction in the human genome. Genome Biol 7, S11.1–8 (2006).
46
A Lomsadze, V Ter-Hovhannisyan, YO Chernoff, M Borodovsky, Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res 33, 6494–6506 (2005).
47
I Korf, Gene finding in novel genomes. BMC Bioinformatics 5, 59 (2004).
48
DN Perkins, DJ Pappin, DM Creasy, JS Cottrell, Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).
49
A Conesa, et al., Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676 (2005).
50
J Krumsiek, R Arnold, T Rattei, Gepard: A rapid and sensitive tool for creating dotplots on genome scale. Bioinformatics 23, 1026–1028 (2007).
51
D Kim, et al., TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14, R36 (2013).
52
L Arike, et al., Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli. J Proteomics 75, 5437–5448 (2012).

Information & Authors

Information

Published in

The cover image for PNAS Vol.112; No.38
Proceedings of the National Academy of Sciences
Vol. 112 | No. 38
September 22, 2015
PubMed: 26351664

Classifications

Data Availability

Data deposition: The Mollivirus genome sequence reported in this paper has been deposited in the GenBank database (accession no. KR921745). The transcriptomic data have been deposited in the Sequence Read Archive, www.ncbi.nlm.nih.gov/Traces/sra/ [accession no. SRX1078581 (SRR2084123 for the early class of expression, SRR2103267 for the intermediate one, and SRR2103268 for the late class of expression)]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, proteomecentral.proteomexchange.org, via the Proteomics Identifications partner repository [dataset identifiers <PXD002375> (Particule and Surfome) and <PXD002374> (Time Course)]. All data can be visualized on an interactive genome browser at the following link: www.igs.cnrs-mrs.fr/cgi-bin/gb2/gbrowse/Mollivirus/.

Submission history

Published online: September 8, 2015
Published in issue: September 22, 2015

Keywords

  1. giant virus
  2. permafrost
  3. Pleistocene

Acknowledgments

We thank Dr. J.-P. Chauvin, Dr. A. Kosta, F. Richard, and A. Aouane and Serge Nitsche for their expert assistance on the imaging platforms, Dr. Dorothée Murat for providing some transmission electron microscopy (TEM) images, and Miguel Ortiz Lombardia for his thorough reading of the manuscript. This work was partially supported by France Génomique Grant ANR-10-INSB-01-01, French National Research Agency Grant ANR-14-CE14-0023-01, the Provence-Alpes-Côte-d’Azur Région (2010 12125), ProFi Grant ANR-10-INBS-08-01, and Russian Scientific Fund 14-14-01115.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Matthieu Legendre1
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Audrey Lartigue1
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Lionel Bertaux
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Sandra Jeudy
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Julia Bartoli
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Present address: Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Unité Mixte de Recherche 7255 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13009 Marseille, France.
Magali Lescot
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Jean-Marie Alempic
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Claire Ramus
Université Grenoble Alpes, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
INSERM, Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Christophe Bruley
Université Grenoble Alpes, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
INSERM, Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Karine Labadie
Commissariat à l’Energie Atomique, Institut de Génomique, Centre National de Séquençage, 91057 Evry Cedex, France;
Lyubov Shmakova
Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino 142290, Russia;
Elizaveta Rivkina
Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino 142290, Russia;
Yohann Couté
Université Grenoble Alpes, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Institut de Recherches en Technologies et Sciences pour le Vivant–Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
INSERM, Laboratoire Biologie à Grande Echelle, F-38000 Grenoble, France;
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Jean-Michel Claverie3 [email protected]
Information Génomique and Structurale, Unité Mixte de Recherche 7256 (Institut de Microbiologie de la Méditerranée, FR3479) Centre National de la Recherche Scientifique, Aix-Marseille Université, 13288 Marseille Cedex 9, France;
Assistance Publique–Hopitaux de Marseille, 13385 Marseille, France

Notes

3
To whom correspondence may be addressed. Email: [email protected], [email protected].
Author contributions: Y.C., C.A., and J.-M.C. designed research; M. Legendre, A.L., L.B., S.J., J.B., M. Lescot, J.-M.A., C.R., Y.C., C.A., and J.-M.C. performed research; K.L. performed sequencing; A.L., C.R., C.B., K.L., L.S., E.R., and Y.C. contributed new reagents/analytic tools; M. Legendre, A.L., M. Lescot, Y.C., C.A., and J.-M.C. analyzed data; M. Legendre, C.A., and J.-M.C. wrote the paper.
1
M. Legendre and A.L. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 38
    • pp. 11739-E5378

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