Experimental fossilisation of viruses from extremophilic Archaea

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Experimental fossilisation of viruses from extremophilic
Archaea
Fran¸cois Orange, Annie Chabin, A. Gorlas, S. Lucas-Staat, C. Geslin, M. Le
Romancer, D. Prangishvili, Patrick Forterre, Franc`es Westall
To cite this version:
Fran¸cois Orange, Annie Chabin, A. Gorlas, S. Lucas-Staat, C. Geslin, et al.. Experimental fos-
silisation of viruses from extremophilic Archaea. Biogeosciences, European Geosciences Union,
2011, 8, pp.1465-1475. <10.5194/bg-8-1465-2011>. <insu-00615454>
HAL Id: insu-00615454
https://hal-insu.archives-ouvertes.fr/insu-00615454
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Biogeosciences,8,1465–1475,2011
Biogeosciences
www.biogeosciences.net/8/1465/2011/
doi:10.5194/bg-8-1465-2011
©Author(s)2011. CCAttribution3.0License.
Experimental fossilisation of viruses from extremophilic Archaea
F.Orange1,2,A.Chabin1,A.Gorlas3,S.Lucas-Staat4,C.Geslin3,M.LeRomancer3,D.Prangishvili4,P.Forterre4,
andF.Westall1,2
1CentredeBiophysiqueMole´culaire,UPR4301,CNRS,RueCharlesSadron,45071Orle´ansCedex2,France
2ObservatoiredesSciencesdel’Universenre´gionCentre,UMS3116,1ARuedelaFe´rollerie,45071Orle´ansCedex2,France
3Universite´ deBretagneOccidentale,UMR6539,CNRS–InstitutUniversitaireEurope´endelaMer,
TechnopoˆleBrest-Iroise,RueDumontd’Urville,29280Plouzane´,France
4MolecularBiologyoftheGeneinExtremophilesUnit,InstitutPasteur,25rueduDocteurRoux,
75724ParisCedex15,France
Received: 16February2011–PublishedinBiogeosciencesDiscuss.: 4March2011
Revised: 20May2011–Accepted: 26May2011–Published: 9June2011
Abstract. Theroleofvirusesatdifferentstagesoftheorigin 1 Introduction
oflifehasrecentlybeenreconsidered. Itappearsthatviruses
may have accompanied the earliest forms of life, allowing The role of viruses in the early evolution of life has re-
the transition from an RNA to a DNA world and possibly cently been reevaluated. Viruses were long considered as
beinginvolvedintheshapingoftreeoflifeinthethreedo- non-living by-products of cellular activity and thus unable
mains that we know presently. In addition, a large variety to have played a role in the origin and evolution of their
of viruses has been recently identified in extreme environ- hosts. Overthelastdecade,however,ourviewsontheorigin
ments, hosted by extremophilic microorganisms, in ecosys- andnatureofviruseshavedramaticallychanged. Structural
tems considered as analogues to those of the early Earth. similarities between viruses infecting phylogenetically dis-
Traces of life on the early Earth were preserved by the pre- tanthostssuggestthatviruseswerealreadyactiveatthetime
cipitation of silica on the organic structures. We present oftheLastUniversalCommonAncestor(LUCA)andprob-
the results of the first experimental fossilisation by silica of ablybefore(Bamfordetal.,2005; KooninandDolja,2006;
virusesfromextremophilicArchaea(SIRV2–Sulfolobusis- Krupovic and Bamford, 2008). Comparative genomics has
landicusrod-shapedvirus2,TPV1–Thermococcusprieurii revealedthatmostgenesencodedbyviruseshavenocellular
virus1,andPAV1–Pyrococcusabyssivirus1). Ourresults homologues,testifyingtothecreativityofvirocells(Forterre,
confirmthatvirusescanbefossilised, withsilicaprecipitat- 2011)ininventingnewproteins.Severaltheoriesanddiscov-
ingonthedifferentviralstructures(proteins,envelope)over erieshavebeenmaderegardingtheancientnatureofviruses
several months in a manner similar to that of other experi- andtheirpossiblekeyrolesintheearlyevolutionoflifeand
mentallyandnaturallyfossilisedmicroorganisms.Thisstudy intheoriginofmoderncells(Forterre,2002;Bamford,2003;
thussuggeststhatviralremainsortracescouldbepreserved Kooninetal.,2006;forrecentreviewsandalternativeview-
intherockrecordalthoughtheiridentificationmaybechal- points, see Moreira and Lo´pez-Garc´ıa, 2009 and Forterre
lengingduetothesmallsizeoftheviralparticles. andPrangishvili,2009a,b). Forinstance,viruseshavebeen
evoked to explain the origin of DNA cells. Starting from
anRNAworld(cellswithRNAgenomes),DNAcouldhave
appearedinviruses(DNAviruses)andthenhavebeentrans-
ferredtocells(Forterre,2002). Onehypothesisalsosuggests
that interactions between DNA viruses and RNA cells (fu-
sion,genetransfers)couldhaveresultedintheformationof
Correspondenceto: F.Orange thethreedomains(Eukaryotes,Bacteria,Archaea)(Forterre,
([email protected]) 2006). Finally, the discovery of Mimivirus, a giant DNA
PublishedbyCopernicusPublicationsonbehalfoftheEuropeanGeosciencesUnion.
1466 F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea
virus infecting Amoeba (La Scola et al., 2003), permits a isms are a sink for available silica. An additional source
virusgenometobeincludedintheuniversaltreeoflife.This of silica in the early oceans came from the very active hy-
suggests that giant viruses could be intermediates between drothermalprocessescyclingsilicaandotherelementsfrom
cells and previously known viruses and could form a new the crust back into the ocean (see review in Westall and
domainoflife(Raoultetal.,2004). Southam, 2006). In situ and experimental silicification of
Amajordevelopmentinrecentyearsinvirologyhasbeen microorganismsinthesehydrothermally-influencedenviron-
thediscoveryofverydiversenewvirusesinextremeenviron- ments has been studied in depth over the past two decades
ments.Suchenvironmentsareparticularlyinterestingasthey (Westall et al., 1995; Toporski et al., 2002; Konhauser et
areoftenconsideredasanaloguesoftheenvironmentsonthe al.,2004;Orangeetal.,2009andreferencestherein). These
earlyEarthinwhichlifecouldhaveoriginated. Microorgan- investigationshaveprovidedpreciousinformationregarding
isms found in these environments are also considered to be the processes involved and helped the identification of sili-
possibleanaloguestotheearliestformsoflifeonEarthand cified traces of life in ancient rocks (see review in Westall,
possiblyonMars(NisbetandSleep,2001;Konhauseretal., 2010). Recent studies have started to explore the ability of
2003). Recognitionoftheancientnatureofvirusessuggests virusestobemineralisedandthepossibilitythattheycould
that early life forms could have hosted viruses. The study bepreservedinthefossilrecord.Severalstudieshavestudied
ofvirusesofextremophilicmicroorganismsisarelativelyre- interactionsbetweendifferentvirusesandiron(Daughneyet
cent field. Nevertheless, it has already allowed the identifi- al., 2004; You et al., 2005; Templeton et al., 2006; Kyle et
cationofseveralthousandsofviruseshavingdifferentmor- al., 2008b). Daughney et al. (2004) have shown the abil-
phologiesandcharacteristics,andalsoofnewvirusfamilies ityofamarinebacteriophagetointeractwithdissolvedpro-
(seereviewinLeRomanceretal.,2007). Viruseshavebeen tons,andtoactasasiteforironbinding,duetothepresence
identified in all known extreme environments: hypersaline of negatively-charged functional groups in its capsid. Kyle
(Orenetal., 1997; Dyall-Smithetal., 2003; Pagalingetal., et al. (2008b) observed the iron mineralisation of viruses in
2007;Sime-Ngandoetal.,2010),alkalinelakes(Jiangetal., theacidwatersofRioTinto(Spain)anddiscussedtheinflu-
2004),deserts(Prigentetal.,2005),polarregions(Maranger enceofthismineralisationonthebiogeochemicalprocesses
et al., 1994; Kepner et al., 1998; Borriss et al., 2003; Gow- andonthepossibilityforvirusestobepreservedintherock
ing, 2003), acid mine drainages (Kyle et al., 2008a), deep record. PoinarandPoinar(2005)reportedthepossiblepres-
subsurfacerocks(Birdetal., 2001; Kyleetal., 2008b), and ence of preserved viruses in 15 to 100Ma insect preserved
inhydrothermalenvironments.Thesearchfornewvirusesin inamber. LaidlerandStedman(2010)monitoredtheexperi-
the latter environment has been especially fruitful, follow- mentalfossilisationofbacteriophageT4forafewdaysunder
ing the pioneering work of Wolfram Zillig on the viruses simulated hot spring silicifying conditions and showed that
of hyperthermophilic Archaea (Martin et al., 1984; Rice et silica could precipitate around viral structures and preserve
al., 2001; Rachel et al., 2002). Many new viral families them.
thatinfecthyperthermophilicArchaeainterrestrialandma- Our study presents the results of the first long-term ex-
rine hot springs have been identified (Geslin et al., 2003b; perimentalfossilisationofthreevirusesandthefirstexperi-
Ortmann and Suttle, 2005; Geslin et al., 2005; Prangishvili mental fossilisation of viruses hosted by hyperthermophilic
et al., 2006a; Ortmann et al., 2006; Le Romancer et al., Archaea. Among the great variety of viruses in hyperther-
2007).Althoughthesevirusesallhavedouble-strandedDNA mophilic Archaea, we chose for this study viruses produc-
genomes, theyproducevirionswithverydiversemorpholo- ingvirions(viralparticles)withverydifferentstructuresand
gies(e.g.rod-shaped,filamentous,spindle-shaped,ellipsoid, morphologies: the rod-shaped virus SIRV2 (Sulfolobus is-
head and tail) and most proteins encoded in their genomes landicus rod-shaped virus 2) and the spindle-shaped TPV1
have no homologues, except sometimes in other viral lin- (Thermococcus prieurii virus 1) and PAV1 (Pyrococcus
eages(Prangishvilietal,2006b). abyssi virus 1) viruses. These morphologies appear to be
Althoughstructuralandcomparativegenomicspointtothe specificforvirusesinfectingorganismslivinginextremeen-
antiquity of viruses, their fossil remains have yet to be de- vironments,eitherathottemperatures(rod-shaped,spindle-
tected in the rock record. Although challenging, consider- shaped) or with high salt concentrations (spindle-shaped),
ingthesizeofviralparticles,detectionofpossiblefossilised withtheexceptionofsomeplantviruseswhichalsoproduce
viral remains in the vicinity of putative fossilised microor- rod-shapedparticles.
ganisms could be a direct proof of the antiquity of viruses Our objectives were (1) to determine whether viruses
andanadditionalcluetoassessingthebiogenicityoftheob- couldbefossilisedbysilicaandwhethertheycouldbepre-
served structures. The oldestknown fossilmicroorganisms, servedoverrelativelylongperiodsoftime(severalmonths),
dating back to almost 3.5 billion years (Ga) ago, were pre- and(2)toevaluatewhetherrecognizablefeaturesintheviral
served as silicified remains (see review in Westall, 2010). strains used could still be identified once silica had precipi-
The preservation by silica was due to the fact that the early tatedaroundthem.
Earth’s oceans were silica enriched compared to the under-
saturatedoceansofthepresentEarth,wheresiliceousorgan-
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F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea 1467
2 Materialsandmethods
2.1 Descriptionofthevirusesusedforthisstudy
SIRV2 (Sulfolobus islandicus rod-shaped virus 2) belongs
to the Rudiviridae family (Prangishvili et al., 1999; Bize et
al.,2009)andisalyticvirus(i.e.whichcausesthedeathof
the host cell) that infects a strain of the Crenarchaeota Sul-
folobusislandicus,anacidophilicandhyperthermophilicAr-
chaea that was originally isolated from samples taken from
solfataric fields in Iceland (Zillig et al., 1994). SIRV2 par-
ticles are stiff rods up to 900nm long and ∼20nm wide
(Fig. 1a; Prangishvili et al., 1999) and consist of a super-
helix including a protein with double-stranded linear DNA.
Incontrasttootherviruses, suchasrod-shapedmembersof
theLipothrixviridae,SIRV2virionsarenotenveloped.
TPV1 (Thermococcus prieurii virus 1) and PAV1 (Pyro-
coccusabyssivirus1)wereisolatedfromtheEuryarchaeota
Thermococcus prieurii strain Bio-pl-0405IT2 and Pyrococ-
cus abyssi strain GE23, respectively (Geslin et al., 2003a;
Gorlasetal., 2009). Thelatteraretwoneutrophilicandhy-
perthermophilic Archaea of the Thermococcales order (Er-
auso et al., 1993; Gorlas et al., 2011). TPV1 and PAV1
are non-lytic, spindle-shaped viruses (TPV1: 140×80nm;
PAV1:120×80nm),characterisedbydouble-strandedcircu-
larDNA,andarefoundeitherisolatedoringroups(Fig.1c
andd). TPV1andPAV1haveanenvelopecomposedofviral
proteins and lipids from the host. These viruses morpho-
logicallyresemblemembersoftheFuselloviridaefamilybut
havenotyetbeenclassified.
2.2 Virusisolation
Thevirusesusedinthisstudywereharvestedfromfreshcul-
turesofthehoststrains.
For the production of SIRV2, the Sulfolobus islandicus
Fig.1. (A–D):TEMmicrographsshowingexamplesoftheviruses
strain LAL14/1 was grown until the late exponential phase
used for this study. (A) SIRV2 particle; note the central cavity,
wasreached,asdescribedbyZilligetal.(1994). Cellswere sometimediscontinued.(B)FragmentsofSIRV2particleonanun-
removed by low-speed centrifugation (3500g in a Sorvall stainedgrid;notetheabsenceofvisiblefeatures,andaviralDNA-
GS3 rotor). The viruses were precipitated from the super- proteinfilamentthatlinksthetwofragments(arrow). (C)Aggre-
natant by the addition of 1MNaCl and 10% polyethylene gateofTPV1particles. (D)PAV1particles(arrows);notethepres-
glycol6000(PEG6000)andincubatedovernightat4◦C.A ence of remnant P. abyssi flagellas in the sample. (E) TEM mi-
pelletofviruseswascollectedbycentrifugationinaSorvall crographshowingexampleofthesilicaprecipitateformedsponta-
neouslyinthecontrolsample,whichcontainednoviruses,observed
GSArotorat23000gfor30minandsuspendedinTAbuffer
after 4days; unstained grid. All TEM micrographs were made at
(20mM Tris-acecate, pH 6). SIRV2 were purified by cen-
trifugationinaCsClbuoyantdensitygradient(0.45gml−1) 200kVongridsnegativelystainedwithuranylacetate,unlessoth-
erwisestated.
in a Beckman SW41 rotor centrifuge at 250000g for 48h.
Fractionscontainingtheviralparticleswerecollectedwitha
syringe then dialyzed against TA buffer for further analysis 80◦C and 85◦C, respectively, up to the late exponential
(Bettstetteretal.,2003). Theviruseswerestoredat4◦Cina phase. The cells were pelleted by low-speed centrifuga-
20mMTris-acetatebuffer(pH6)untilused. tion at 6000g for 15min. TPV1 and PAV1 were precip-
Thermococcus prieurii strain Bio-pl-0405IT2 (Gorlas itated from the supernatant in 1MNaCl with 10% PEG
et al., 2011) and Pyrococcus abyssi strain GE23 (Er- 6000 overnight at 4◦C with gentle stirring. The pre-
auso et al., 1993; Marteinsson et al., 1995) were grown cipitate was pelleted by centrifugation at 13000g for 1h
in the medium described by Geslin et al. (2003a), at 15min, then drained and resuspended in a specific buffer
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1468 F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea
for each virus (TPV1: TPV1-buffer–10mMTris-HCL, intheSIRV2medium,whileitdidnotchangethepHofthe
100mMNaCl,5mMCaCl ;PAV1:TEbuffer–10mMTris- TPV1andPAV1media.
2
HCl and 1mM EDTA, pH 8). A second precipitation with
PEG6000(10%)and1MNaClwasmadeduring1.5hand 2.4 Electronmicroscopy
theprecipitatewascollectedasdescribedabove. Aftercen-
trifugation(5000gfor10min),thesupernatantwaskeptand The vials were sampled at different times (between 2 and
thepelletwasextractedtwomoretimesunderthesamecon- 60days for the SIRV2 experiment; between 1 and 180days
ditions with reduced volumes of TPV1 or TE buffer. The for the TPV1 and PAV1 experiments; 4days for the control
virus-containingsupernatantswerepooledandstoredforone sample) by collecting ∼1µl of the sample and immediately
nightat4◦C.Aftercentrifugation(5000gfor15min)tore- preparingitfornegatively-stainedtransmissionelectronmi-
move residual cell debris, the supernatant was concentrated croscopy(Geslinetal.,2003a).
by ultracentrifugation at 33000g for 1h 45min (Beckman For negative staining, a droplet of sample (either unsili-
OptimaLE-80K70.1Tirotor)andthepelletwasresuspended cified or silicificed) was placed on a carbon-coated copper
inTPV1orTEbuffer. Viruseswerepurifiedfromthesesus- TEMgrid. Thesamplewasallowedtoabsorbtothecarbon
pensionsbycentrifugationinaCsClbuoyantdensitygradi- layerfor2minbeforeremovingtheexcessliquidwithapiece
ent(TPV1: 1.32gml−1;PAV1: 1.298gml−1)inaBeckman offilterpaper. Somesampleswerestainedtoincreasedcon-
Optima LE-80K 70.1Ti centrifuge rotor (TPV1: 180000g trastbyplacingadropletofsaturateduranylacetateethanolic
for6h; PAV1: 220000gfor24h). Fractionscontainingthe solutionfor40sandthenremovingtheexcessliquid. Some
nucleicacidsweredetectedat254nmandcollectedusinga grids were also observed unstained to better distinguish sil-
densitygradientfractionator(model185,ISCO).Thesefrac- icadepositionontheviralstructures. Thepreparedsamples
tionswerethendialyzedagainstalargevolumeofTPV1or werethenairdried. Theywereobservedandanalysedwitha
TEbufferandwerestoredat4◦Cuntilused. PhilipsCM20TransmissionElectronMicroscope(Centrede
MicroscopieElectronique,UniversityofOrle´ans),equipped
withanEDXdetector(OxfordInstruments).
2.3 Silicificationprocedure
The fossilisation experiments were made directly in the
bufferingmediaoftheviruses(SIRV2: Tris-acetatebuffer– 3 Results
20mMTris-acetate,pH6;TPV1:TPV1buffer–10mMTris-
3.1 ExperimentalfossilisationofSIRV2
HCl,100mM,5mMCaCl ,pH8;PAV1:TEbuffer–10mM
2
Tris-HCl, 1mM EDTA, pH 8). This was done for practical
SIRV2 particles appear as stiff rods of several hundreds of
reasonsaswellastomaintainthevirusesinafavourableen-
nanometersinlengthand∼20nminwidthandcontainacen-
vironment so that their evolution could be followed during
tralcavity(Fig.1a),notvisibleonunstainedgrids(Fig.1b).
fossilisationoverseveralmonths.
This virion is naturally fragile and readily breaks up into
A stock silica solution (3200ppmSi) was prepared
fragments that may be linked to each other by filaments,
from a pure sodium silicate solution (Riedel de Hae¨n)
identified as the result of the degradation of the viral helix
containing ∼27% SiO and ∼10% NaOH (Na Si O ,
2 2 3 7 andmadeofDNAwithremnantsoftheviralprotein(Fig.1b,
M=242gmol−1). ItspHwasadjustedto8beforeinjection
arrow) (Prangishvili et al., 1999; Vertergaard et al., 2008).
intothevirussuspension.
Apart from fragmentation, the preservation of SIRV2 frag-
19µl of a suspension of purified SIRV2 were mixed with ments during fossilisation was generally good. In the first
1µl of the stock silica solution in a sealed glass vial to ob- daysofthefossilisationexperiment,agranularandalveolar-
tain a final concentration of ∼160ppmSi. For TPV1 and textured silica precipitate formed spontaneously in both the
PAV1, 180µlofsuspensionsofpurifiedvirusesweremixed control experiment without viruses (Fig. 1e) and in the ex-
with20µlofthestocksilicasolutiontoobtainafinalconcen- periment with the viruses (Fig. 2a, b), indicating that nei-
trationof∼320ppmSi. TheSIRV2fossilisationexperiment thervirusparticlesnorthebufferingmediumhadanyinflu-
vialswerestoredatroomtemperature(duetothesmallvol- ence on precipitation or on the structure of the silica. Ini-
ume, high temperatures led to evaporation). The TPV1 and tially,afteroneweekoffossilisationonlyafewSIRV2par-
PAV1 fossilisation experiment vials were placed in an oven ticles were observed in direct contact with, and sometimes
at 60◦C. A control sample without viruses, consisting of a trappedwithin,thesilicaprecipitate(Fig.2a)butmostwere
∼160ppmSi silica solution in distilled water, was also pre- not(Fig.2b). Whereembeddedinthesilicadeposit,thevi-
paredandleftatroomtemperature. ral helix appeared to have been compacted or decayed and
Thesmallvolumesinvolvedintheexperimentsmeantthat the virion was only visible as a faint, dark outline (Fig. 2a,
wewerenotabletomakeprecisemonitoringofthepHand arrows) that probably represents degraded viral DNA and
silicaconcentrationovertime. Weassumedthattheinjection protein. Silica nucleation on the virion was observed af-
of the silica solution at pH 8 only slightly increased the pH ter 1 week (Fig. 2c, arrow) and, after 30days, nanometric
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F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea 1469
Fig. 2. TEM micrographs showing progressive steps of the experimental fossilisation of SIRV2 at a ∼160ppmSi silica concentration.
(A)48h,SIRV2particlepartiallytrappedinsidethesilicaprecipitate(Si);thearrowsunderlinetheshapeofthevirioninsidetheprecipitate.
(B)7days,fragmentsofSIRV2particlesinthevicinityofasilicaprecipitate(Si);thetwoseparatedfilamentsofviralDNA-proteinarevisible
(arrow). (C)7days,unstainedgrid,fragmentofSIRV2particle;adark∼10nmparticleisseenattachedonthevirion(arrow). (D)30days,
SIRV2particle;notethenumerousdarksilicaparticlesinthecentralcavity. (E)60days,unstainedgrid,fragmentsofSIRV2particle;note
the dark silica particles attached on the outer surface of the virion, and filling the central cavity. (F) 60days, EDX spectra made on the
SIRV2particlesof(E),showingaSisignalslightlyhigherthanonthebackground;theCssignalcomesfromthecesiumchlorideusedfor
viruspurificationandtheCusignalcomesfromthecoppergrid. (G)60days,unstrainedgrid,viralDNA-proteinfilamentcoveredbysilica
particles.AllTEMmicrographsweremadeat200kVongridsnegativelystainedwithuranylacetate,unlessotherwisestated.
dark particles had formed in the central cavity of some of the viral particles was also observed. EDX analyses made
the virions (Fig. 2d). This phenomenon was more evident ontheseparticlesshowedasiliciumsignalslightlystronger
after 60days of fossilisation with virions showing, on un- thanthesignalfrombackgroundnoise(Fig.2f). Finechains
stained grids, a strongly contrasted central cavity (Fig. 2e), ofsilicaparticles(Fig.2g)mayrepresentsilicadirectlypre-
suggesting that the individual particles observed previously cipitatedonisolatedviralDNA-proteinfilaments,ortherem-
had continued their growth and eventually merged. More nantsofvirionfragmentscompletelycoveredbysilica.
pronounced silica nucleation and binding on the outside of
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1470 F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea
Fig. 3. TEM micrographs showing progressive steps of the experimental fossilisation of TPV1 at a ∼320ppmSi silica concentration.
(A)1week,twoTPV1particles(arrows)nearasilicaprecipitate(Si). (B)1month,TPV1particle(arrow)attachedtothesilicaprecipitate
(Si). (C) 75days, TPV1 particles with dark nanometric particles within. (D) 75days, unstained grid, TPV1 particles on which a dark
precipitate has formed. (E) 75days, EDX spectra obtained on the virions of (D) and on the background, showing Si and Cs signals on
theprecipitate;theClsignalcomesfrombufferingmediumandtheCusignalcomesfromthecoppergrid. (F)180days,deformedTPV1
particlenearasilicaprecipitate(Si);notethedarkparticlewithin.(G)180days,unstainedgrid,TPV1particlecoveredandfilledwithadark
precipitate.AllTEMmicrographsweremadeat200kVongridsnegativelystainedwithuranylacetate,unlessotherwisestated.
3.2 ExperimentalfossilisationofTPV1andPAV1 of some of the virus particles (Fig. 3d). In the latter case,
EDXanalyses(Fig.3e)indicatethatthisprecipitateconsists
of both silica and cesium, the latter being derived from the
Althoughagranular,alveolarsilicaprecipitateformedspon-
purificationbuffer. Suchaprecipitatehadnotbeenobserved
taneously within one day of the experiment, similar to that
inearlierstepsoftheexperimentnoronunfossilisedviruses
observed in the SIRV2 and control experiments (Fig. 3a),
particles. After 180days, the virions were significantly de-
it was only after 30days that the TPV1 particles were ob-
graded (Fig. 3f). Some of the viral particles contained only
served in direct contact with the precipitate (Fig. 3b). No
a few nanoparticles of silica within their structure (Fig. 3f)
virions trapped in the silica precipitate were observed. The
whilesomeotherswereheavilycoatedandimpregnatedwith
first signs of possible silica precipitation on the viral struc-
theapparently-progressingSiO -Csprecipitate(Fig.3g).
turesoccurredonlyafter75dayswhennanometersized,dark 2
particleswereobservedwithin(Fig.3c)andontheenvelope
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F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea 1471
Fig. 4. TEM micrographs showing progressive steps of the experimental fossilisation of PAV1 at a ∼320ppmSi silica concentration.
(A)1day,PAV1particle(arrow)attachedtothesilicaprecipitate(Si). (B)60days,unstainedgrid,numerousPAV1particlesnearthesilica
precipitate.(C)60days,unstainedgrid,aggregateofPAV1particlestrappedinafinelygrainedsilicaprecipitate.AllTEMmicrographswere
madeat200kVongridsnegativelystainedwithuranylacetate,unlessotherwisestated.
MonitoringofthePAV1fossilisationexperimentwascom- initial polymerisation at a concentration close to the satura-
plicatedbythepresenceofnumerousfragmentsofP.abyssi tionconcentration(62ppmSiindistilledwater;Gunnarsson
flagella as well as numerous artefacts in the preparation of and Arno´rsson, 2000; Lalonde et al., 2005). In support of
purified PAV1 (Fig. 1d). PAV1 particles could be observed this, the silica precipitate formed in the SIRV2, TPV1 and
in direct contact with the silica precipitate after only 1day controlexperimentswassimilartothatobservedinprevious
(Fig.4a). Thisprecipitatewasdifferentfromthatformedin fossilisationexperiments(Orangeetal.,2009).
theSIRV2andTPV1experimentsandconsistedofveryfine The fact that the silica precipitates in the SIRV2 experi-
particles (compare Figs. 4c and 3b). The reason for this is ment were invariably linked to viral DNA-protein filaments
unknown,althoughitpossiblycouldbeaconsequenceofthe (Fig. 2b) suggests that the spontaneously-formed silica de-
differentcompositionofthePAV1bufferingmedium(which positprobablyrapidlyentrappednumerousSIRV2particles,
includes EDTA, a chelating and binding agent). No precip- aswasobservedbyLaidlerandStedman(2010).Someparti-
itates formed inside or on free PAV1 particles after 60days cleswerebarelyobservableasdarkoutlineswithinthemin-
(Fig. 4b), although several virus particles were seen at the eral precipitate (Fig. 2a). The viral particles that remained
edgeofthesilicaprecipitate(Fig.4c),suggestingthatanim- free in the medium showed progressive silica binding with
portantnumberofPAV1particleshadbeentrappedinit. time on their outer surface and in their central cavity. Ini-
tialsilicanucleationwithinthecavityoftheSIRV2particles
did not appear to damage the body (Fig. 2d, e) although, at
4 Discussion later steps of the fossilisation process, strings of silica par-
ticles observed on DNA-protein filaments (Fig. 2g) suggest
Thevirusesusedinthisexperimentbelongtodifferentvirus that viral particles may be heavily damaged by the growth
familieshavingcompletelydifferentmorphologiesandstruc- ofsilica. TherarityofsilicifiedDNA-proteinfilamentsmay
tures. Thismaypartlyexplainwhytheresultsofthefossil- beduetothefactthatDNAdegradesrapidlywhenexposed
isation experiments were different. In the case of SIRV2, a totheenvironment(Schelbleetal.,2008),whiletheremains
non-enveloped rod-shaped virus, silica binding on the viral ofDNAtrappedinsilica(Fig.2a,g)maybepreserved. Sil-
particles was significant and progressive whereas only lim- ica binding to the outer surface of the virions appears to be
ited silica binding occurred on TPV1 and PAV1, enveloped the result of two mechanisms: (i) the direct nucleation of
spindle-shapedviruses. silica (Fig. 2c) followed by continuing polymerisation, re-
sultingintheformationofhemisphericalsilicaparticlesthat
4.1 Fossilisationoftheviruses progressively grow and merge (Fig. 2c, e); (ii) the binding
of already formed silica particles on the virions, or on pre-
Weassumed(butcouldnotverify,duetothesmallvolumes viouslyboundsilicaparticles(e.g.themiddlesilicaparticle
of the samples) that the silica behaviour during the experi- amongthethreeattachedtoSIRV2particleinFig.2e,which
mentswassimilartothatdescribedinpreviousexperimental has a similar aspect to the free particles present around the
fossilisationstudies(reviewinKonhauseretal.,2004).Upon virion). These mechanisms have previously been described
injection into the vials, silica was in monomeric (Si(OH) ) in studies of the fossilisation of microorganisms (Birnbaum
4
orslightlypolymericformandquicklyspontaneouslypoly- etal.,1989; Schultze-Lametal.,1995; Westalletal.,1995;
merised as a colloidal amorphous silica precipitate. Dis- Toporski et al., 2002; Orange et al., 2009). Silica proba-
solved silica must have remained in the medium after this blybindsviacovalentorhydrogenbondsbetweenthesilica
www.biogeosciences.net/8/1465/2011/ Biogeosciences,8,1465–1475,2011
1472 F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea
particlesandthehydroxyland/orcarboxylfunctionalgroups physical integrity can quickly be affected due to their small
containedintheproteinofthehelix(seereviewofthefossil- sizeandconstitution. Ourresultsshowthat,ifviralparticles
isationprocessesinKonhauseretal.,2004). canbeconservedoverseveralmonths,theycantheoretically
FossilisationofthevirusesTPV1andPAV1differedfrom becomefossilised. Inaddition,ourresultssuggestthatviral
that of SIRV2. The former have an envelope that contains particlesmayhavebeentrappedinthesilicaprecipitate,with
lipids that derive from the hosts (the Archaea P. abyssi and viralremainsbeingpossiblypreserved,althoughthequality
T. prieurii, respectively) (Geslin et al., 2003a; Gorlas et al., ofthepreservationcouldnotbepreciselyascertainedinthis
2011). Previous results from the experimental fossilisation case.
of Archaea showed that the simple cell wall of these mi- The fact that viruses can be fossilised suggests that natu-
croorganisms(S-Layer+plasmamembrane)hasonlyalim- rally fossilised viruses may occur in rocks in the same way
itedabilitytobindsilica(Orangeetal.,2009). Itwasthere- asfossilisedmicrobialcells. Thequestionis,howcouldwe
fore to be expected that, having a similar composition, the identify them? Morphology is a criterion for their identifi-
envelopes of TPV1 and PAV1 would not bind significant cation but their simple nanometric-sized structures may be
amounts of silica. This was indeed observed for TPV1, al- confusedwithmineralsorotherartifactsofthesamesize(of
thoughsomevirionsoccasionallyoccurredindirectcontact theorderof∼100nm).Ontheotherhand,itmaybepossible
with silica (Fig. 3b), and, after 75days of exposure to the toidentifyfilamentousorrod-shapedstructures,suchasthe
silica solution, nanometer-sized particles were ubiquitously about 500×20nm-sized SIRV2 particles, in ancient rocks.
observed inside TPV1 (Fig. 3c, f). Only at this advanced These viruses are about the same size and shape as the so-
stageinthefossilisationprocedure(75days),werenanopar- called“nanobacteria”identifiedin3.9Gaoldcarbonatecon-
ticlesofaSiO -Csprecipitatefirstobservedonthevirionen- cretionsintheMartianmeteoriteALH84001(McKayetal.,
2
velopes(Fig.3d). ThesourceoftheCswasthecesiumchlo- 1996–notethatitisnowacceptedthatthelatteraresimply
rideusedforharvestingpurifiedTPV1. Chelatedmetalions mineralprecipitations;GibsonJr.etal.,2001). Althoughsil-
mayactasbridgesinthefixationofmineralionstoorganic icaprecipitatedinthecentralcavityofSIRV2(Fig.2e)asa
materials. ThishasbeendemonstratedwithFe(III)chelated structureoflessthan10nminwidthandseveralhundredsof
toBacteriaandArchaea(BeveridgeandMurray,1976;Bev- nanometers long, it would be difficult to distinguish such a
eridge and Koval, 1981; Orange et al., 2011). However, as feature from polymeric fibrils or filaments of other biologi-
amonovalentcation,Cs+ cannotactasadirectintermediate caloriginthathadbeensilicified. Viralorganiccompounds,
betweensilicaandthevirionenvelopeorinternalmaterials. such as lipids and proteins, could also be preserved in very
SilicabindingtotheTPV1particlesmostlikelyoccurredin small amounts but they would rapidly be degraded, hence
the same way as with the SIRV2 particles, namely through renderingtheirdetectionandidentificationasviralbiomark-
covalent or hydrogen bonding of the silica. Similar results ers difficult. Thus, even though it is theoretically feasible,
for TPV1 and PAV1 were expected, since these two viruses detection of virus remains in rocks will be highly challeng-
have similar structures. Instead, no silica precipitation was ing.
observed on free PAV1 particles (Fig. 4b) while numerous
virions were trapped in the silica precipitate (Fig. 4c). The
5 Conclusions
unknownfactorsthatledtotheformationofadifferentsilica
precipitatemayalsoberesponsibleofthesedifferencesinthe
This study is one of the first attempts to fossilise viruses
silicificationprocess.
andthefirstmonitoringofthefossilisationofvirusesofex-
tremophilic Archaea over a period of several months. Our
4.2 Implicationforthepreservationandidentification
results show that the viruses of extremophilic microorgan-
ofviralremainsinrocks
ismscanbefossilisedbysilicaandthatthestructureshavea
chanceofbeingpreservediftheprocesscantakeplaceovera
The three viruses used in this artificial fossilisation study
periodofseveralmonths. Thevirusstructureandthemolec-
were well preserved, apart from a slight deformation pos-
ular composition of their outer surface (lipid envelope, pro-
sibly due to the length of the experiment rather than to the
tein) clearly play a role in the rate and manner of fossilisa-
exposure to silica. The fossilisation was made directly on
tion. Although faithful preservation of the viral structures
virions stored in buffered solutions, which is obviously not
over geological time is probably unlikely, some traces may
a natural condition. The rationale for making the experi-
neverthelessremaininrocks. Ourstudythuscontributesim-
ments on the microorganisms in a buffered solution is the
portantinformationtothisnewfieldofresearch. Duetothe
previously noted importance for the cell to remain alive, or
greatdiversityofvirusesintermsofmorphologyandstruc-
at least not to lyse, during the time necessary for silica to
ture, additional studies are needed to complete these results
formadepositaroundthecellsthickenoughtoallowforfos-
withthefossilisationofotherkindsofvirusinordertobetter
silisationandfurtherpreservation(Orangeetal.,2009). This
understandthemechanismsinvolved.
periodoftimevariesdependsonthestrainorthetypeofmi-
croorganism. Viral particles obviously do not lyse but their
Biogeosciences,8,1465–1475,2011 www.biogeosciences.net/8/1465/2011/
F.Orangeetal.: ExperimentalfossilisationofvirusesfromextremophilicArchaea 1473
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