Spindle-shaped viruses infect marine ammonia-oxidizing thaumarchaeaJong-Geol Kima, So-Jeong Kimb, Virginija Cvirkaite-Krupovicc, Woon-Jong Yua, Joo-Han Gwaka, Mario López-Pérezd,Francisco Rodriguez-Valerad, Mart Krupovicc, Jang-Cheon Choe, and Sung-Keun Rheea,1

aDepartment of Microbiology, Chungbuk National University, Heungduk-gu, 361-763 Cheongju, South Korea; bGeologic Environment Research Division,Korea Institute of Geoscience and Mineral Resources, 34132 Daejeon, Republic of Korea; cDepartment of Microbiology, Institut Pasteur, 75015 Paris, France;dEvolutionary Genomics Group, Universidad Miguel Hernandez, San Juan, 03540 Alicante, Spain; and eDepartment of Biological Sciences, Inha University,22212 Incheon, Republic of Korea

Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved June 21, 2019 (received for review April 3, 2019)

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeotaare ubiquitous in marine ecosystems and play a prominent role incarbon and nitrogen cycling. Previous studies have suggested that,like all microbes, thaumarchaea are infected by viruses and that viralpredation has a profound impact on thaumarchaeal functioning andmortality, thereby regulating global biogeochemical cycles. However,not a single virus capable of infecting thaumarchaea has beenreported thus far. Here we describe the isolation and characterizationof three Nitrosopumilus spindle-shaped viruses (NSVs) that infectAOA and are distinct from other known marine viruses. AlthoughNSVs have a narrow host range, they efficiently infect autochtho-nous Nitrosopumilus strains and display high rates of adsorptionto their host cells. The NSVs have linear double-stranded DNA ge-nomes of ∼28 kb that do not display appreciable sequence simi-larity to genomes of other known archaeal or bacterial viruses andcould be considered as representatives of a new virus family, the“Thaspiviridae.” Upon infection, NSV replication leads to inhibitionof AOA growth, accompanied by severe reduction in the rate of am-monia oxidation and nitrite reduction. Nevertheless, unlike in the caseof lytic bacteriophages, NSV propagation is not associated with de-tectable degradation of the host chromosome or a decrease in cellcounts. The broad distribution of NSVs in AOA-dominated marineenvironments suggests that NSV predation might regulate the diver-sity and dynamics of AOA communities. Collectively, our resultsshed light on the diversity, evolution, and potential impact of thevirosphere associated with ecologically important mesophilic archaea.

spindle-shaped virus | ammonia-oxidizing archaea | viral predation |chronic infection

Members of the phylum Thaumarchaeota are widespreadand abundant in marine ecosystems and play key roles in

nitrogen cycles by mediating ammonia oxidation (1, 2). Ammoniaoxidation is implicated in controlling the availability of nitrogenspecies, production of N2O (3, 4), and is associated with carbonfixation in the deep ocean. Thus, information on key factors af-fecting abundance and composition of the communities ofammonia-oxidizing archaea (AOA) is crucial for understandingthe biogeochemical processes of nitrogen cycling in the oceans.The relative contribution of resource competition (bottom-up)and predation (top-down control) are the key drivers of bio-geochemical cycles, affecting microbial activity and communitystructures. To understand the abundance and composition of theAOA communities, the effects of physicochemical factors andmetabolic traits of AOA ecotypes on the efficiency of resourceutilization have been thoroughly assessed (2, 5).Predation pressure can also influence AOA communities but

has been rarely studied. Flagellate grazing was proposed to affectthe distribution and abundance of AOA in planktonic microbialassemblages (6, 7). Danovaro et al. (8) suggested that viral in-fection represents a key mechanism controlling the turnover ofarchaea, especially AOA, in surface deep-sea sediments. Putativethaumarchaeal proviruses related to tailed bacterial and archaeal

viruses of the order Caudovirales have been previously identified inthe genomes of the soil thaumarchaeon Nitrososphaera viennensis(9) and the extremely thermophilic thaumarchaeon CandidatusNitrosocaldus cavascurensis (10). Furthermore, several meta-genomic and single-cell genomic studies have resulted in the as-sembly of putative AOA virus genomes, all related to members ofthe order Caudovirales (11–13). Notably, some of these assembledvirus genomes were found to carry putative genes encoding theammonia monooxygenase subunit C (amoC), a key component ofammonia monooxygenase (AMO) (13, 14), suggesting an active roleof viruses in nitrogen cycling in the oceans. Nevertheless, not a singlethaumarchaeal virus–host system has been isolated or cultivated thusfar, precluding functional studies on the virus–host interactions andthe effect of viruses on the metabolic activity of thaumarchaea.Archaea are associated with a remarkably diverse virosphere,

which is characterized by unique morphotypes not observedamong viruses infecting Bacteria and Eukarya. These includevirions with spindle-shaped, bottle-shaped, droplet-shaped, coil-shaped, and other morphologies (15–18). Among these archaea-specific morphotypes, spindle-shaped viruses are among themost widely distributed (19) and were found not only in extremegeothermal and hypersaline habitats, but also in marine environments,

Significance

Ammonia-oxidizing archaea (AOA) are major players in globalnitrogen cycling. The physicochemical and metabolic factors af-fecting the composition of AOA communities and their efficiencyof resource utilization have been studied extensively. However,viral predation on AOA remains unexplored due to lack of iso-lated virus–host systems. Here we report on the isolation andcharacterization of three Nitrosopumilus spindle-shaped viruses(NSVs) that infect AOA hosts. NSVs represent a potentially im-portant group of marine viruses with a chronic infection cycle,providing important insights into the diversity and evolution ofthe archaeal virosphere. The wide spread of NSVs in AOA-containing marine environments suggests that NSV predationmight regulate the diversity and dynamics of AOA communities,thereby affecting the carbon and nitrogen cycling.

Author contributions: J.-G.K. and S.-K.R. designed research; J.-G.K., V.C.-K., W.-J.Y., andJ.-H.G. performed research; S.-J.K. and M.L.-P. contributed new reagents/analytic tools;S.-J.K., V.C.-K., M.L.-P., F.R.-V., M.K., and J.-C.C. analyzed data; and J.-G.K., F.R.-V., M.K.,and S.-K.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: DNA sequencing data have been deposited in the GenBank databasewith the identifiers MK570053 to MK570059. Accession number of the Nitrosopumilusstrain SW genome is CP035425.1To whom correspondence may be addressed. Email: rhees@chungbuk.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1905682116/-/DCSupplemental.

Published online July 16, 2019.

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including coral surfaces (20), particulate matter-rich bays (21), and theoceanic basement (22), although their hosts and genome sequenceswere not determined.In this study, we isolated and characterized spindle-shaped

viruses infecting AOA from coastal seawater and revealedproperties of their life cycles. We show that virus infection has adramatic effect on ammonia oxidation and is likely to affect thepopulation structure and functioning of the AOA community. Ourresults shed light on the diversity, evolution, and potential impact ofthe virosphere associated with ecologically important archaea.

Results and DiscussionIsolation, Morphology, and Stability. Three virus strains, designatedNitrosopumilus spindle-shaped viruses 1, 2, and 3 (NSV1, NSV2,and NSV3, respectively), were isolated from suspended partic-ulate matter (SPM)-rich seawater samples taken from the west-ern coast of the Korean Peninsula using as a host the axenicAOA strain SW (SI Appendix, Fig. S1), which was isolated fromsurface water (20 m deep) at the Yellow Sea, Korea (23) (seebelow for further information). Since AOA could not form lawnson agar plates, the dilution-to-extinction method was used toisolate the viruses from enrichment cultures. General features ofNSVs are summarized in Table 1. The sizes of the spindle-shaped NSV virions were similar (Fig. 1 and SI Appendix, Fig.S2), measuring 64 ± 3 nm in diameter and 112 ± 6 nm in length,with a short tail at one pole (Fig. 1A). The morphological fea-tures of NSVs were very similar to those of viruses in the familyFuselloviridae and in the genus Salterprovirus, which infecthyperthermophilic and hyperhalophilic archaea, respectively (19,24, 25). A large fraction of produced virions remained at-tached to the cell surface (Fig. 1B). Elongation of virions intoarrowhead-shaped particles with long tails was observed 6 h postinfection (Fig. 1C), suggesting that flexibility of virions might beimportant to the infection process—as has been observed forspindle-shaped virions of the fusellovirus SSV1 (26) and thebicaudavirus ATV (27). The adsorption of NSVs to AOA cellswas rapid, with ∼50% of virions bound to cell surfaces within 10min (SI Appendix, Fig. S3). Certain hyperthermophilic archaealviruses exhibit comparably rapid adsorption kinetics (28); con-versely, halophilic archaeal viruses—including those with spindle-shaped virions—generally exhibit slow adsorption kinetics (29).Spindle-shaped viruses are frequently observed in extreme

environments, and it is suggested that this morphotype has beenselected for its robustness under a wide range of extreme envi-ronmental conditions (24, 30, 31). To study how the isolatedNSV virions respond to physicochemical fluctuations in the en-vironment, their stabilities were tested under varying regimes ofpH, salinity, and temperature. NSV virions remained stable andinfectious between pH 3 and 9, salinities between 0.1 and 20%,

and temperatures up to 55 °C (SI Appendix, Fig. S4). These re-sults showed that NSV virions were well-adapted to both surviveenvironmental fluctuations and interact with the unique archaealcell surface, which consists of a cytoplasmic membrane andproteinaceous S-layer (32–35). Notably, mesophilic AOA arebelieved to have evolved from a (hyper)thermophilic ancestor(36, 37). Thus, the observed resilience of the NSV particles couldhave been inherited from an ancestral extremophilic virus.

Host Specificity. Based on the comparison of average nucleotideidentities (ANIs), strain SW belongs to the genus Nitrosopumilus,but represents a species that is most closely related to Nitro-sopumilus maritimus SCM1 (SI Appendix, Fig. S5). Thus, hostspecificities of the three NSVs were tested using strains ofNitrosopumilus species closely related to the strain SW (>98%sequence similarity of the 16S rRNA gene; SI Appendix, Fig. S1)SCM1 (33), DDS1 (23), HCA1 (33), and BC. In the presence ofNSVs, neither virus production nor inhibition of ammonia oxi-dation by these AOA strains was observed, indicating that thehost range of NSVs might be rather narrow. The sensitivity ofclosely related strains to viral infection could be potentially af-fected by the presence of host defense mechanisms or a lack ofspecific receptors on the cell surface. To date, there is no reportof a CRISPR-Cas–dependent viral defense system in the thau-marchaeal group I.1a (38). Similarly, homologs of the Dnd de-fense system could not be identified in the available genomes ofAOA strains (i.e., strains SCM1, DDS1, and SW) (39) used in thisstudy. These findings suggest that resistance might instead be dueto the absence or modification of cell surface receptors. Notably,comparison of the closely related Nitrosopumilus maritimus SCM1and Nitrosopumilus sp. SW genomes revealed that genomic island 2predicted to be involved in cell surface modification had differentgene content in the two strains, which might explain the differentsusceptibility to NSVs (SI Appendix, Fig. S6 and Dataset S1).

Table 1. General features of NSVs and scaffolds related to NSV

Isolation site

Virus/putativeviral

scaffolds*Adsorption

rate, 50% (min)Latent

period (h)Attached

fraction (%)Genomesize (kb)

Numberof ORFs G+C mol%

AOA-likegenes

Accessionnumber

Bulcheon(36°57′N, 126°20′E)

NSV1 5 6 69 27.5 48 29.8 5 MK570053

NSV2 10 6–8 80 28.9 51 29.8 4 MK570055Scaffold83 — — — 14.6 30 27.1 1 MK570056Scaffold98 — — — 13.4 20 31.3 1 MK570057Scaffold261 — — — 7.1 18 29.7 0 MK570058Scaffold342 — — — 6.0 13 27.2 2 MK570059

Daecheon(36°58′N, 126°20′E)

NSV3 <5 6 70 27.5 48 29.8 5 MK570054

—, not applicable.*Scaffold is obtained from early phase enrichment culture for NSV2.

A B C

Fig. 1. Transmission electron microscopy images of negatively stained NSV1virions. (A) NSV1 virions. (Scale bar, 50 nm.) (B) NSV1 particles attached tothe surface of an SW cell. (Scale bar, 200 nm.) (C) Elongated NSV1 particlesattached to the surface of an SW cell after 12 h of infection. The arrowsindicate elongated NSV1 particles. (Scale bar, 200 nm.)

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Genome Analysis. The genomes of NSV1, NSV2, and NSV3consist of linear dsDNA molecules of ∼27, ∼29, and ∼27 kb,respectively, containing 176-bp terminal inverted repeats (Table 1).The numbers of ORFs in the genomes of NSV1, NSV2, andNSV3 were predicted to be 48, 51, and 48, respectively. TheNSV1 and NSV3 genomes were highly similar (ANI = 99.8%;Fig. 2), whereas the NSV2 genome was slightly more divergent(ANI = 95%; Fig. 2). Divergent partial NSV genomes were alsoobtained as scaffolds 83, −98, −261, and −342 from meta-genomes of the initial NSV2 enrichment culture, indicating agreater diversity of NSVs (Fig. 2). Despite being closely relatedto each other, NSVs did not display appreciable sequence simi-larity to other known archaeal or bacterial viruses in BLASTPsearches (E value cutoff: 0.001; Dataset S2). Indeed, none of theNSV ORFs gave BLAST best-hits to known viral proteins, andonly 9 out of 48 (18.7%) NSV1 ORFs yielded significant matchesin the nonredundant sequence databases to known cellularproteins—a common trend in archaeal viruses (15). Five of thenine hits were to proteins encoded by various marine thau-marchaea, and four were to bacterial proteins (Dataset S2). Thethaumarchaea-like ORFs encode putative genome replicationproteins (ORF3 and ORF43), glycosyltransferases (ORF20 andORF39), and a DNA-binding protein (ORF44) (see below).Accordingly, NSVs might be considered as representatives of anew archaeal virus family with the proposed name “Thaspiviridae”(for Thaumarchaeal spindle-shaped viruses).More sensitive sequence analysis based on profile hidden

Markov model (HMM) comparisons allowed for functional an-notation of 15 putative NSV1 genes (Dataset S2). ORF3 andORF43, respectively, encode predicted protein-primed family BDNA polymerase (pPolB) and a DNA sliding clamp known asproliferating cell nuclear antigen (PCNA) that are likely to beinvolved in NSV genome replication. ORF15 encodes a pre-dicted Cdc6-like AAA+ ATPase that may also be involved ingenome replication. However, given the broad functional di-versity of AAA+ ATPases, involvement of ORF15 in other steps

of the infection cycle cannot be ruled out. Similarly to manyother archaeal viruses (40), NSV1 encodes two predicted gly-cosyltransferases (ORF20 and ORF39), with the closest homo-logs present in thaumarchaeal genomes, and one predicted DNAmethyltransferase (ORF33)—apparently recruited from bacteriaby horizontal gene transfer. ORFs 6, 7, 29, 31, 32, and 37 encodeshort proteins with predicted zinc-binding domains; ORFs 18, 26,and 44 encode putative DNA-binding proteins with wingedhelix-turn-helix, looped-hinge helix, and ribbon-helix-helix do-mains, respectively (Dataset S2). To gain further understanding onthe proteins encoded by NSVs, purified NSV1 virions weresubjected to proteomic characterization by liquid chromatographycoupled to tandem mass spectrometry (LC-MS/MS). Ten NSV1proteins were detected by proteomic analysis (Dataset S3).

Evolutionary Relationships to Other Archaeal Viruses. Despite thelack of direct sequence similarity, general features of the genomeorganization and proteome of NSV1 are reminiscent of those ofother archaeal viruses. In particular, the pPolB of NSVs is sharedwith several groups of archaeal viruses and nonviral mobile ge-netic elements, which, like NSV, have linear genomes with terminalinverted repeats. These include the haloarchaeal spindle-shaped(genus Salterprovirus) and pleomorphic (family Pleolipoviridae, ge-nus Gammapleolipovirus) viruses His1 (41) and His2 (30), re-spectively; hyperthermophilic bottle-shaped (family Ampullaviridae)(25) and ellipsoid (family Ovaliviridae) (42) viruses; and casposons,which integrate into the genomes of diverse thaumarchaea (43, 44).To understand the relationship between NSVs and other pPolB-

encoding archaeal and bacterial mobile genetic elements, a maxi-mum likelihood phylogenetic analysis of their respective pPolBsequences was performed. When midpoint rooted, a well-supportedphylogeny splits between bacterial and archaeal sequences, with theonly exception being the ovalivirus SEV1, which groups with bac-terial rather than archaeal homologs (Fig. 3A). The pPolB se-quences from NSVs form a sister group to the clade that includeshalophilic viruses His1 and His2, as well as sequences from marine

Fig. 2. Comparative genomics of NSVs. Genomic maps of NSVs and related scaffolds. Shared ORFs are connected by color-coded shaded areas based onsequence identity. Genomes of NSVs are flanked by terminal inverted repeats. %GC represents mol% G+C content of DNA.

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sediment metagenomes. At the base of this clade are casposonsfrom the marine Thaumarchaeota. Interestingly, G+C mol% valuesof His1 (39%) and His2 (40%) are different from that of their host,Haloarcula hispanica (63%), but close to those of AOA (∼34%).Collectively, these results suggest horizontal exchange of the pPolBgenes between casposons, NSVs, and haloarchaeal viruses. Fur-thermore, phylogenetic analysis suggests that pPolB genes ofthaumarchaeal mobile elements are ancestral to those of His1-likeviruses of halophilic archaea. However, many more viral genomesof both His1-like and NSV-like viruses are needed to substantiatethis hypothesis.NSVs and the salterprovirus His1 are the only known spindle-

shaped viruses with linear dsDNA genomes carrying pPolB genes,suggesting a specific evolutionary connection between the two virus

groups. Notably, despite the lack of detectable sequence similarity,the two viruses possess a similar gene repertoire (Fig. 3B), includingmany genes encoding zinc-binding proteins, AAA+ ATPase, gly-cosyltransferases, and a putative terminal protein which is foundby LC-MS/MS in the virions of both NSV1 (Dataset S3) and His1(45). Furthermore, all known spindle-shaped viruses—includingHis1—encode relatively short (70–140 aa) major capsid proteinscontaining two highly hydrophobic α-helical regions predicted toform transmembrane domains (19), but with little sequence sim-ilarity to each other. Among NSV1 proteins, only one, encoded byORF12, fits these characteristics (SI Appendix, Fig. S7) and wasdetected in the virions by LC-MS/MS (Dataset S3). Based on theseshared properties, NSVs appear to be distantly related to His1 and,more generally, to other spindle-shaped archaeal viruses.All previously characterized archaeal viruses with unique vi-

rion morphologies not observed among viruses of bacteria oreukaryotes infect extremophilic hosts (15, 25), suggesting thatthese archaea-specific morphotypes have evolved as an adap-tation to extreme environments. The fact that genomes ofthaumarchaeal viruses previously discovered by metagenomicsare all related to those of tailed bacteriophages (11–13) is con-sistent with this possibility. However, the identification of spindle-shaped viruses infecting mesophilic marine thaumarchaea stronglysuggests that the spread of unique archaeal morphotypes extends tomesophilic archaea and possibly encompasses other archaeal line-ages. Furthermore, these results suggest that spindle-shaped viruseshave a deep evolutionary history within the domain Archaea, whichlikely dates back to the last archaeal common ancestor.

Viral Impact on Host Metabolism. The effects of NSVs on thephysiology and metabolism of their AOA host were examinednext. During the first 2 d post infection (dpi), viral DNA repli-cation occurred concurrently with AOA growth and was ac-companied by a normal rate of ammonia oxidation (Fig. 4 and SIAppendix, Fig. S8). However, host cell growth ceased 2 dpi, whileammonia oxidation continued until 4 dpi at a rate similar to thatin the uninfected AOA (Fig. 4 and SI Appendix, Fig. S8). Pre-sumably, during this period, the energy obtained from ammoniaoxidation was directed to virus replication, consistent with theobserved increase in the viral titer until 6 dpi. However, after 5dpi, the rate of ammonia oxidation and nitrite production de-creased dramatically, indicating that NSVs had a severe effect onmetabolic activity in their AOA hosts. Notably, virus productionwas not associated with detectable degradation of the hostchromosome determined by quantification of archaeal 16SrRNA gene (Fig. 4B) or a decrease in cell counts measured usingepifluorescence microscopy (SI Appendix, Fig. S9). Consistentwith this observation, cells with damaged cell envelopes, as ob-served for some lytic archaeal viruses (46), were not detectable bytransmission electron microscopy (TEM). Instead, virions wereobserved in abundance on the cell surface without obvious asso-ciated perturbations (Fig. 1B), suggesting that viral replication andrelease did not lead to cell lysis. It has been previously shown thatspindle-shaped viruses of hyperthermophilic and halophilic ar-chaea are also released from their hosts without causing cell lysis(26, 47). In the case of Sulfolobus spindle-shaped virus SSV1, vi-rions are assembled during the budding of the viral nucleoproteinthrough the cell membrane, which remains intact, in a processhighly similar to the egress of enveloped eukaryotic viruses (26).We hypothesize that NSV virions are assembled and releasedfrom the cell by a similar mechanism without lysing the host cells.As suggested by the TEM analysis (Fig. 1B) and the adsorp-

tion assays (SI Appendix, Fig. S3), high proportions (>60%) ofthe produced NSVs remained cell-associated, presumably bothas adsorbed virions on the host cell surface and as intracellularreplicated genome copies (Fig. 4C and SI Appendix, Fig. S8 Cand D). In resource-poor oceans, a nonlytic mode of replication

(phi29-like phages)

(monoderm hosts)(diderm hosts)

"Autolykiviridae"ATY46514.1_Sulfolobus ellipsoid virus 1

MGYP000338936204MGYP000401327420

candidate archaeal division MSBL1metagenomic sequencesAmpullaviridae

Thaumarchaeal casposons (marine)

NSV1_gp3NSV2_gp4NSV3_gp3scaffold98_gp4

scaffold83_gp11

Ovaliviridae

YP_529524.1_His1YP_529644.1_His2MGYP000367844654

MGYP000577203607MGYP000041105224MGYP000220981334

Marine sediment metagenome

A

B

Nitrosopumilusspindle--shaped viruses

Gamma- pleolipovirus

Salterprovirus

Picovirinae

TectiviridaeTectiviridae

94

6396

97

88

10099

100100

100100

100

100

100 100

100100

100100

10099

99

99

99

99

99

92

Fig. 3. Phylogeny of pPolB and comparative genome maps of NSV1 andHis1. (A) Maximum likelihood phylogenetic analysis of pPolB sequences frombacterial and archaeal viruses. In this tree, archaeal viruses comprise thefollowing taxa: Ampullaviridae, Pleolipoviridae,Ovaliviridae, and Salterprovirus.Sequences originating from marine environments and hyperhalophilicarchaeal viruses are highlighted with light blue and green backgrounds,respectively. Sequences from metagenomic datasets are indicated withgray font. (B) Comparison of the NSV1 and His1 genome maps. Function-ally equivalent genes are indicated with matching colors. Genes encodingproteins detected in the purified virus particles are shown in cyan. Genesencoding small proteins containing Zn-binding domains are shown ingreen. Abbreviations: pPolB, protein-primed family B DNA polymerase; MCP,major capsid protein (putative); wHTH, winged helix-turn-helix; GTase, gly-cosyltransferase; MTase, DNA methyltransferase; PCNA, proliferating cellnuclear antigen; RHH, ribbon-helix-helix. The question mark next to theputative MCP denotes the uncertainty of this prediction.

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and high adsorption rate might represent an optimal strategy forsurvival of viruses infecting chemolithoautotrophic AOA hosts.The total number of NSV particles produced under laboratory

conditions was ∼298 ± 18 virions per AOA cell at 6 dpi and∼103 virus particles per micromole of NH3 oxidized, with slightstrain-specific variations (Fig. 4 and SI Appendix, Fig. S8). Thenonlytic replication strategy of NSVs and other spindle-shaped ar-chaeal viruses (26, 47), which allows for continuous virion productionand release, is radically different from the lytic life cycle of tailedviruses of the order Caudovirales (48) but, in certain aspects, re-sembles the nonlytic production of filamentous bacteriophages of theInoviridae family (49). Consequently, conventional ecologicalmodels which are tailored to the mode of bacterial predation bylytic head–tail phages (50, 51) might benefit from revision ac-counting for alternative virus life cycles—exemplified here byNSVs and the Nitrosopumilus sp. SW.

Environmental Distribution. Distribution of NSVs in marine envi-ronments was analyzed using quantitative real-time PCR of theNSV-specific pPolB gene. NSVs were detected in abundance invarious marine sediments (104–106 NSV genome copies per gram ofsediment), coastal seawaters (104–106 NSV genome copies per literof seawater), and coral-rich seawater (∼104 NSV genome copies perliter of seawater) (SI Appendix, Table S1). By contrast, NSVs werebelow the detection limit in a liter of typical oligotrophic seawater,consistent with a previous study showing that spindle-shaped virusparticles corresponded to <1% of virions in tested seawater samples(52). A comparison of the ratio of AOA counts to NSV counts(Pearson’s correlation coefficient 0.557; P value 0.015) indicatedthat NSV levels might be positively correlated with the abundance ofAOA present. However, the correlation of counts of strain SW-specific gene slp2 encoding an S-layer protein to those of NSVcounts was weak, suggesting that viral host specificity might be as-sociated to the different versions of this protein (53).Metagenomic recruitment analysis showed that NSVs were not

detected in any of the available metaviromes from marine envi-ronments including those from sediments. A high adsorption rate ofNSV particles to host cells and particulate matter (Figs. 1B and 4C)might have caused low recovery of NSVs from the viral fraction ofthe sediments during virome preparations, which could explain thelack of reads matching NSVs in the public metaviromes. Indeed, thispossibility was confirmed experimentally: only ∼1% of NSV particlespresent in marine sediments could be extracted into the viral frac-tion using the approach commonly used for virome preparation (SIAppendix, Fig. S10). The adsorption of NSV to particulate matter isalso evidenced by frequent observation of NSV-like morphotypes inSPM-rich bays (21) and surface microlayers of corals (20). Thus,NSV-like genomes could be underrepresented in the viral fractionsextracted by conventional means from marine environments.In addition to resource competition (bottom-up control), predation

(top-down control) can act as a key driver of biogeochemical cycles byaffecting microbial activity and community structures. Thus far, avirus capable of infecting thaumarchaea had not been isolated, whichhad limited the fundamental knowledge of the impact of viral in-fection on the functioning and mortality of AOA. In this study,spindle-shaped viruses were found to infect a marine ammonia-oxidizing thaumarchaeon. The genome architecture and life cycle ofthe examined NSVs indicate that they are distantly related to spindle-shaped viruses that infect hyperthermophilic and hyperhalophilic ar-chaea but represent a distinct new viral family. Characterization ofthe infection strategy employed by NSVs suggests they might beadapted for efficient infection of chemolithoautotrophic AOA hostsand survival in resource-poor oceans. This study provides evidencethat viral predation severely affects the metabolism of infected AOAcells, with a potential impact on global carbon and nitrogen cycling.

0.8

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0.0

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0 2 4 6 8 10 12 14Time (days)

)%(

surivdedne psus

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enegANRr

S61l

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ypoceneg

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etirtindna

ainom

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AOATotal NSV1

1.0

Ammonia (NSV1-infected)Ammonia (Non-infected control)Nitrite (NSV1-infected)Nitrite (Non-infected control)

A

B

C

Fig. 4. Properties of the NSV1 infection cycle. Strain SW cells were in-fected with NSV1. Error bars represent SDs for three biological repli-cates. (A) Comparison of ammonia oxidation by NSV1-infected andnoninfected control cells. (B) Virus production by strain SW cells infectedwith NSV1. AOA growth and virus production were measured by qPCRquantification of 16S rRNA and pPolB genes, respectively. (C ) Fraction ofnonadsorbed NSV1 virions, estimated by qPCR of viral pPolB gene. Viralgenomic DNA of nonadsorbed virions was prepared from the culturesupernatant.

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Materials and MethodsIsolation of AOA viruses, analysis of infection cycle of NSVs, and sequencingand annotation of viral genomes are described in SI Appendix, SI Materialsand Methods. Details of DNA extraction from seawater and marine sedi-ment, quantification of AOA and NSV from marine environments, andmetagenomic read recruitments are provided in SI Appendix, SI Materialsand Methods. Adsorption, host range, and stability of NSVs were tested asdescribed in SI Appendix, SI Materials and Methods. Phylogenetic analysisof pPolB, comparative genomic analysis of AOA strains and NSVs, andproteome analysis of NSV1 are described in SI Appendix, SI Materials andMethods.

ACKNOWLEDGMENTS. This research was supported by National ResearchFoundation of Korea (NRF) grants (Mid-Career Researcher Program [NRF-2018R1A2B6008861], Basic Research Laboratory Program [NRF-2015R1A4A1041869],and C1 Gas Refinery Program [NRF-2015M3D3A1A01064881]) funded by theMinistry of Science, Information and Communication Technology, and FuturePlanning.M.K. was supported by a grant from l’Agence Nationale de la Recherche(#ANR-17-CE15-0005-01). The authors would like to thank Thibault Chaze andMariette Matondo (Pasteur Proteomics Platform) for help with the proteomicsanalyses. M.L.-P. was supported by a postdoctoral fellowship (Juan de la Cierva)from the Spanish Ministerio de Economía y Competitividad (IJCI-2017-34002).F.R.-V. was supported by grant CGL2016-76273-P (Agencia Estatal de Investiga-ción/European Development Regional Fund [FEDER], EU), (cofounded with FEDERfunds) from the Spanish Ministerio de Economía, Industria y Competitividad.

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