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Supporting Information Supporting Information Legendre et al. 10.1073/pnas.1510795112 SI Methods Infected Cells and Virion Imaging. Sample Recovery and Radiocarbon Dating. Sterility controls of the Infectious cycle observations by TEM. A. castellanii-infected cell cul- samples were performed during their collection as previously tures were fixed by adding an equal volume of PBS with 2% glu- described (16, 32, 33). The samples of buried soils were taken taraldehyde and incubated for 20 min at room temperature. Cells × from the frozen outcrop walls in Chukotka, on the Stanchikovsky were recovered and pelleted for 20 min at 5,000 g. The pellet was yar (GPS coordinates: 68.370155, 161.415553, 68°22′13″N and resuspendedin1mLofPBSwith1%glutaraldehyde,incubatedat 161°24′56E), 23- to 24-m height from the Anui River. The melting least 1 h at 4 °C, and washed twice in PBS prior coating in agarose material was first cleaned out from the wall surface, and the un- and embedding in Epon resin. Each pellet was mixed with 2% low- melting agarose and centrifuged to obtain small flanges of ap- thawing layer was exposed; the frozen rock was excavated to make 3 a hollow of 30–40 cm deep, and a sample was taken from this proximatively 1-mm containing the sample coated with agarose. hollow. After treating with 95% ethanol, the sample was placed in These samples were then embedded in Epon resin using a standard a sterile plastic bag and stored frozen. In the laboratory, the method: 1-h fixation in 1% osmium tetroxide, dehydration in in- samples were stored in freezers at −20 °C. creasing ethanol concentrations (50%, 70% including uranyl ace- tate 2%, 90%, and 100% ethanol), and embedding in Epon-812. Virus Isolation and Production. Mollivirus was isolated from a piece Ultrathin sections of 70 nm were poststained with 4% uranyl ac- of the buried soil sample P1084-T as previously reported (16). etate and lead citrate and observed using a Zeiss EM 912 operating Briefly, 400 mg of P1084-T were resuspended in 6 mL of Prescott at 100 kV. and James medium (27). The infection trials were performed twice Scanning electron microscopy observations of Mollivirus purified particles. and produced identical results. Each 3 mL were supplemented with A suspension of purified Mollivirus particles in PHEM buffer 300 μL of Amphotericin B (Fungizone), 250 μg/mL, and 1.65 mL (240 mM Pipes, 100 mM Hepes, 8 mM MgCl2, 40 mM EGTA, – of this 10% Fungizone solution was left overnight at 4 °C under pH 6.9) was adsorbed on a poly-L-lysine coated silica slide for stirring. After decantation, the supernatant was recovered and centri- 10 min at room temperature, and then fixed with 2.5% glutaral- fuged at 800 × g for 5 min. Acanthamoeba castellanii (Douglas) Neff dehyde in PHEM buffer for 20 min. After three washes of 5 min in (ATCC 30010TM) cells adapted to resist Fungizone (2.5 μg/mL) PHEM buffer and two of 2 min in ddH2O, the silica slide was put in were inoculated with 100 μL of the supernatant and with the pellet 50% acetone for 5 min. Serial increasing acetone baths (75%, 85%, resuspended in 50 μL of Tris (20 mM), CaCl (1 mM), pH 7.4. The 95%, 100%) of 5 min were done, followed by two more baths in 2 100% acetone for 5 min. Samples were then placed in the chamber cells were cultured at 32 °C in microplates with 1 mL of protease of a critical point dryer filled with 100% acetone. After cooling to peptone–yeast extract–glucose (PPYG) medium supplemented with 10 °C, the acetone was replaced by carbon dioxide before heating at antibiotics [ampicillin, 100 μg/mL, and penicillin–streptomycin, the critical point under pressure. Samples were sputter coated with 100 μg/mL (Gibco); Fungizone, 2.5 μg/mL (Life Technologies)] 80 Å of gold and observed on a Jeol JSM-6320F at 15 kV. and monitored for cell death. Visualization of 5-ethynyl-2′-deoxyuridine-labeled Mollivirus particles. A. castellanii cells were infected by Mollivirus at a MOI of 0.25 Virus Purification. The wells presenting an infection phenotype μ ′ were recovered, centrifuged for 5 min at 500 × g to remove the and grown in the presence of 100 M 5-ethynyl-2 -deoxyuridine cellular debris and used to infect four T-75 tissue-culture flasks (EdU) until the infectious cycle was complete. The virions were recovered and used to infect A. castellanii cells at a MOI of 20. plated with fresh Acanthamoeba cells. After completion of the Cells were recovered after 30, 60, and 90 min of infection and infectious cycle, the cultures were recovered, centrifuged for fixed with formaldehyde (3.7%), permeabilized with Triton 5 min at 500 × g to remove the cellular debris, and the virus was X-100 (0.5%), and labeled with Alexa Fluor 488 picolyl azide in pelleted by a 30-min centrifugation at 3,000 × g prior purifica- a copper buffer according to the manufacturer protocol (Click-it tion. The viral pellet was then resuspended and washed twice in Plus EdU 488 Imaging Kit; Molecular Probes). Images were PBS and layered on a discontinuous sucrose gradient [30%/40%/ recorded on a Zeiss Axio Observer Z1 inverted microscope using × g 50%/60% (wt/vol)], and centrifuged at 5,000 for 15 min. The a63× objective lens associated with a 1.6× Optovar. virus produced a white disk, which was recovered and washed − twice in PBS and stored at 4 °C or 80 °C with 7.5% DMSO. Mollivirus Virion DNA Extraction. The genomic DNA was recovered × 10 2 from 1.8 10 purified particles using the PureLink Genomic Virus Cloning. A. castellanii cells (70,000/cm ) were seeded on a DNA Extraction Mini Kit (Life Technologies) according to the 12-well culture plate with 1 mL of PPYG. After adhesion, viruses manufacturer’s protocol. were added to the well at a multiplicity of infection (MOI) of 50. After 1 h, the well was washed several times with 1 mL of PPYG Mollivirus Genome Sequencing. Five hundred nanograms of ge- to remove the excess of viruses. The cells were then recovered by nomic DNA were sheared to a 150- to 700-bp range using the gently scrapping the well, and a serial dilution was performed in Covaris E210 instrument (Covaris). Sheared DNA was used for the next three wells by mixing 200 μL of the previous well with Illumina library preparation by a semiautomatized protocol. 500 μL of PPYG. Drops of 0.5 μL of the last dilution were re- Briefly, end repair, A-tailing, and ligation of Illumina compatible covered and observed by light microscopy to verify that there adaptors (Bioo Scientific) were performed using the SPRIWorks were less than two cells. The 0.5-μL droplets were then distrib- Library Preparation System and SPRI TE instrument (Beckman uted in each well of a 24-well culture plate. Thousand uninfected Coulter), according to the manufacturer’s protocol. A 300- to A. castellanii cells in 500 μL of PPYG were added to the wells seeded 600-bp size selection was applied to recover most of the frag- with a single cell and monitored for cell death. The corresponding ments. DNA fragments were amplified by 12 cycles of PCR using viral clones were recovered and amplified prior purification, Platinum Pfx Taq Polymerase Kit (Life Technologies) and DNA extraction, proteome analysis, and cell cycle characteriza- Illumina adapter-specific primers. Libraries were purified with tion by electron microscopy. 0.8× AMPure XP beads (Beckman Coulter). After library profile Legendre et al. www.pnas.org/cgi/content/short/1510795112 1of7 analysis by Agilent 2100 Bioanalyzer (Agilent Technologies) and Metagenomic data analysis. Reads containing sequences of low com- qPCR quantification, the libraries were sequenced using 151 plexity were filtered out to avoid spurious matches using the build-in base-length read chemistry in paired-end flow cell on the Illu- “dust” software tool from the SGA assembler (34) with the fol- mina MiSeq (Illumina). About 2 × 1.5 million useful reads were lowing parameters: minimal length = 50 and dust threshold = 2. obtained. This resulted in a total of 368,474,026 usable reads. These reads were mapped to the Mollivirus genome as well as to the genomes Transcriptome Preparation. of the following other giant viruses: Pandoravirus salinus (Refseq Mollivirus-infected A. castellanii cells. Adherent cells were infected ID: NC_022098.1), Pandoravirus dulcis (Refseq ID: NC_021858.1), by Mollivirus with a MOI of 50 and distributed in 30 flasks (1.4 × Pithovirus sibericum (Refseq ID: NC_023423.1), Mimivirus (Refseq 107 cells/flasks of 175 cm2) containing 20 mL of PPYG and left at ID: NC_014649.1), Megavirus chilensis (Refseq ID: NC_016072.1), 32 °C for 30 min, after which viruses in excess were removed. For and the cellular host A. castellanii (GenBank Assembly ID: each time point, 12 mL were recovered to make three pools GCA_000313135.1) using bowtie2 with the “very sensitive” pa- (1: 30 min, 1, 2 h; 2: 3, 4, 5 h; 3: 6, 7, 9 h) for transcriptomic rameter (35). The genomes from Mollivirus and Pithovirus ex- analysis, 3 mL for the quantitative temporal proteomic study and hibited small but significant numbers of permafrost metagenomics 3 mL for TEM observations. mapped reads (Table 1). Cumulative distributions of these mapped RNA extraction. RNA was extracted using the RNeasy Midi kit reads against their positions in their respective target genomes (catalog no. 75144; Qiagen) using the manufacturer’s protocol. were plotted (Fig. 7). Briefly, the cells were resuspended in 4 mL of RLT buffer sup- plemented with 0.1% β-mercaptoethanol and disrupted by sub- Mollivirus Genome Assembly and Annotation.
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