Jumping
species—a
mechanism
for
coronavirus
persistence
and
survival
Vineet
D
Menachery,
Rachel
L
Graham
and
Ralph
S
Baric
Zoonotictransmissionofnovelvirusesrepresentsasignificant
threattoglobalpublichealthandisfueledbyglobalization,the
lossofnaturalhabitats,andexposuretonewhosts.For
coronaviruses(CoVs),broaddiversityexistswithinbat
populationsanduniquelypositionsthemtoseedfuture
emergenceevents.Inthisreview,weexplorethehostandviral
dynamicsthatshapetheseCoVpopulationsforsurvival,
amplification,andpossibleemergenceinnovelhosts.
Address
DepartmentofEpidemiology,UniversityofNorthCarolinaatChapelHill,
ChapelHill,NC,UnitedStates
Correspondingauthor:Baric,RalphS([email protected])
CurrentOpinioninVirology2017,23:1–7
ThisreviewcomesfromathemedissueonViralpathogenesis
EditedbyRaulAndinoandMichaelDiamond
ForacompleteoverviewseetheIssueandtheEditorial
Availableonline31stMarch2017
EditedbyMichaelDiamond
http://dx.doi.org/10.1016/j.coviro.2017.01.002
1879-6257/ã2017TheAuthors. Thisisanopenaccessarticleunder
theCCBY-NC-NDlicense(
http://creative-commons.org/licenses/by-nc-nd/4.0/).
Introduction
Inthepastdecade,moleculartechniqueshaveexpanded identificationofzoonoticviruses,includingcoronaviruses (CoVs)[1].Traditionally,approachesforviralidentification haveincludedculturing,antigenstaining,electron micros-copy, and serology[2];however, these techniqueswere inherentlybiasedtowardsknownviralfamiliesandwere largelyinsensitivetouncharacterizedspecies.Incontrast, moleculardiagnostics rapidlyidentifiedunknown patho-gens starting with SinNombrevirusin the late 20th century, continuingwithSARS-CoVintheearlypartofthiscentury, andmostrecentlywithMERS-CoV[3–5].Asthemolecular approachesimproved,thesetechniqueshavebecome stan-dard in identifying infectious agents in both acute and chronicdiseasesettings.Coupledwithreducedcost,these newapproacheshavepermittedapplicationforpathogen discovery;thenumberofknownCoVshasincreased sub-stantially,aidedbybothsurveysofanimalpopulationsand infrastructureinvestmentstoimprovediagnosticcapacity indiseasehotspots[6].Importantly,theresultinginventory illustratesthe broad diversityharboredin zoonotichosts and the presence of quasi-species that may serve as a
reservoirforCoVpersistence.Inthisreview,weexamine howbothbathostsandtheCoVsthattheyharbormaybe uniquely positioned to seed future emergence events, especially as human populations increase and penetrate theundevelopedregionsoftheworld.
overcome species barriers without sacrificing the form or function of other important elements. This dichot-omy inCoVs is governed bytwo distinct mechanisms: fidelityandgeneacquisition(Figure1).Amajor limita-tion to RNA virus capacity is the need to minimize sequencelengthtosurviveerrorcatastrophe[24]. How-ever, CoVs, as some of the largest members of the Nidovirales order, have overcome this barrier by pro-ducing a large replication complex with known RNA synthesis and modification activities that include a proofreading machine, mediated primarily via the 30– 50 exoribonuclease activity of non-structural protein (nsp) 14 [25]. As such, this large and complex RNA replication machinery has allowed CoVs to achieve upwards of 32kb in size while maintaining the func-tional componentsrequiredfor viability. Coupled with robust fidelity, CoVs have also used recombination, horizontalgene transfer,geneduplication, and alterna-tive open reading frames to expand the functional capacity for its current and new hosts [26]. Together, both fidelity and gene acquisition have honed and refined CoV proteins, whichcan be dividedinto three broad groups based on selective pressure: spike, con-served, and variable proteins (Figure 1). For a novel CoV to emerge, these three groups must function in harmony,providingsufficientchangestoovercome spe-cies barriers while maintaining key viral functions.
Keying
in:
spike
drives
emergence
Chargedwithbindingthehostreceptor,thespikeprotein ofCoVsgovernsspeciesspecificityandisacriticaltarget humans [20]. Together, these factors likely work in
combination and indicate how diverse pools of CoV quasi-speciescansurviveinbatpopulations.
While bat species maintain factors that permit virus persistence,theuniquehostenvironmentalsopromotes broaddiversityinCoVquasi-speciespools.Asaresultof flight,accumulationofROSspeciesmayoccurfor short periodsoftimeandhavebeenshowntohavemutagenic effects, potentially overwhelming CoV proofreading repair and/or altering viral polymerase fidelity and increasingspeciesdiversity,apossible keyto cross-spe-ciestransmission[21].Similarly,theconstitutive expres-sionof type IIFN in bathosts mayselectfor advanta-geous viralmutations that enhance resistance to innate immuneantiviraldefensepathwaysandprovidea repli-cationadvantage,especiallyaftercrossspecies transmis-sion[14]. Conversely,the absenceof key inflammatory mediatorsinbatspeciesprovidesnoselectivepressureto minimizetheseresponses[13];subsequently,infectionof anewhostcouldresultinmassiveandpathogenic inflam-mation responses, as seen with both SARS-CoV and MERS-CoV infections in humans [22,23]. Overall, the uniqueaspectsthatpermitquasi-speciespoolsofviruses inbatsalsocontributetotheirdiversityandpotentialto emergein newspecies.
Balancing
act:
honing
CoV
survival
and
emergence
Whilebats provideacritical foreground, emergence of CoVs requires that key viral factors be altered to
Figure1
Genetic Conservation
Genetic Diversity
Spike S2
Viral Enzymes
Structural Proteins
CoV Fidelity Gene Modulation
Spike S1 Accessory Proteins Non-Structural
Current Opinion in Virology
Balancingcoronavirusemergence.Batpopulationsmaintainauniqueenvironmentthatfacilitatessurvivalandmaintenanceofdiversepoolsof
viruses.Toovercomespeciesbarriers,CoVmustmodifysomekeyviralfactorswhilemaintainingothers.Twomechanismsgovernthisbalance:
fidelityandgenemodulation.Usingtheseprocesses,CoVsshapetheirproteinsconservingsome(viralenzymes,structuralproteins,spikeS2)
whilemodifyingothers(non-structuralproteins,accessoryproteins,spikeS1).Theresultingpoolsthereforemaintainviabilitywhilealsopossessing
for host immunity [27].Divided into two parts, the S1 portion forms the globular head of the spike trimer (Figure 2a), drivesreceptorengagement,andisvariable across and within CoV groups (Figure 2b) [28,29]. In contrast, theS2 domain maintains theentry machinery and requires more conservation across the CoV family (Figure2a,b).Withbindingrequiredforinfection, muta-tionswithinS1, and mostnotably,thereceptor-binding domain(RBD),havebeenthoughttobecriticalforCoV emergence[30].Usingchimericvirusesemployingcivet, early, and middle-phase spike proteins demonstrated viability for the closely related strains in human cells [31,32].However,forsomestrains,suchasSZ16and bat-derived HKU3-CoV,theclosestknownSARS-CoV pro-genitorsatthetime,progenyvirionswerenotrecoverable inVeroorprimaryhumanairwayepithelialcells,despite evidence of RNA replication [30,32].Toovercome this barrier, single humanizing mutation K479N was intro-ducedintoSZ16andachimericHKU3 viruscontaining the RBD of SARS-CoV was designed and permitted replication, likelydueto itscapacitytobindthehuman ACE2receptor[30,31].Asimilarapproachwasusedwith group 2C CoV HKU5; substitution of the entire ecto-domainfromSARS-CoVspikeresultedinanHKU5virus that was ableto infect humancells [33]. Together,the dataarguethattheabilityofthespiketobindreceptoris requiredfor viabilityinnovelhosts.
However,morerecentadvancesidentifiedbatCoVspike proteins that could produce robust infection without manipulation [34,35]. Building from sequences closely relatedtotheepidemicSARS-CoVstrains[36],chimeric virusesemployingthespikesequencesfromSHC014and WIV1 clusters produced CoVs capable ofreplicatingin humancellsandcausingdiseaseinvivo[34,35].Coupled withthediscoveryofsequencesevenmorecloselyrelated totheepidemicSARS-CoVstrainsandevidenceofrobust S1recombination[37],theresultssuggestthatextensive mutationofthespikeRBDmaynotbetheonlycorrelate for infection of human hosts. Notably, both chimeric viruses wereattenuated relative to theepidemic strain, suggesting thatadaptationwithinthenew host contrib-utes todiseaseandpathogenesis[34,35].Yet, itremains unclearifthesemutationsoccurexclusivelywithintheS1 portion of spike or if subtle changes in the S2 region contribute to enhanceddisease byinterfacing with sur-faceandintracellularproteasesthatfunctioninentryand egress[38,39].
Mainstays
and
accessories:
adding
tools
but
keeping
a
base
The CoV spike protein captures a critical dichotomy necessary for emergence,employing enough novelty in itsS1regiontobindnewhostreceptorswhileconserving functionalentryactivityinitsS2portion.However,while
Figure2
(a) (b)
1a
1b
2a
2b
2c
2d 1
2
3 4
PEDV
HCoV-229E HCoV-NL63
HCoV-OC43 HCoV-HKU1 BtCoV-HKU3
BtCoV-HKU5 BtCoV-HKU9 BtSCoV-SHC014 BtSCoV-WIV1 SARS-CoV MERS-CoV
AIBV PoDelta-CoV
Spike S1 Spike S2
vs.MERS vs.SARSvs.MERS vs.SARS
8 7
0 0
0 0
0
20
40
60
80 100 0
0 33 35
35 76 88 37
37 36 36 35 33 30
31 33
99 99 90
98 58 58
58 59 50 49 49 49
49 49
100 100
100 100 70
42 36
62 62 62 62 62 61
51 51 50 48 58 64
83
Current Opinion in Virology
Conservationandmodificationofspikeprotein.TheCoVspikeproteiniscriticalreceptorbindingandentry.Therefore,whilemodificationislikely
requiredforinfectionofnewspecies,thespikeproteinmustalsomaintainitsentrymechanism.(a) StructureofMHV-CoVspiketrimer(adapted
fromRef.[53]),dividingtheproteinintoS1globularheadportions(blue),andS2conservedstalk(green).(b)Heatmapswereconstructedfroma
setofrepresentativecoronavirusesfromallfourgenogroupsusingalignmentdatapairedwithneighbor-joiningphylogenetictreesbuiltinGeneious
critical for infection of new hosts, changing the spike proteinaloneisnotsufficientto causeepidemicdisease [34,35];therefore,changeswithinthebackbonearealso necessarytospeedemergence.Yet,thesamedichotomy seenwiththespikeglycoproteinsisnecessaryin balanc-ingchangewithintheCoVbackbone.Certainelements, mostnotablyaccessoryproteins,maybeaddedor modi-fiedto enhance infection withinnew hosts.In contrast,
other viral motifs and proteins must be conserved to maintain virus functionality. For each, CoV fidelity, recombination,andevolutionarypressurehoneandrefine thesegenes, providingaframework for emergencein a newspeciestooccur.
Forhighlyconservedviralfunctions,thepresenceofCoV fidelity machinery provides an important means to
Figure3
(a)
(b)
0 10 20
25
30 Kb
ORF1a
ORF6 nsp2 nsp14
ORF1b
S
1 2 3 4 5 6-11
12 13 14 15 16
Nucleocapsid
3a 7a
8a 9b
b b
b M
E 6 N
1a
1b
2a
2b 1
2
PEDV
HCoV-229E HCoV-NL63
HCoV-OC43
HCoV-HKU1
BtCoV-HKU3
BtSCoV-SHC014
BtSCoV-WIV1
SARS-CoV
MERS-CoV 2c
2d
3
4
BtCoV-HKU5
BtCoV-HKU9
AIBV
PoDelta-CoV
vs.MERS vs.MERS
vs.MERS
vs.SARS vs.SARS vs.SARS vs.SARS
0
20
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100
0 0
0 0
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100 36
36 45
45
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38 96
98
98
41
37 31
33 25
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74 73
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31
45
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93
98
98 55
58
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39
37 37 47 70
71 73 79 78
78
78 78 78 75
68 66
66
67 67
68
69
59 60
76
32 57
57
99
97
Current Opinion in Virology
MaintenanceandchangetheCoVbackbone.ChangestotheCoVbackbonecanaidemergence,butmustbebalancedagainstconservationof
otherelements.(a) GenomicstructureofSARS-CoVwithproteinspredictedtobeconserved(blue),variable(red),orinbetween(purple).(b) Heat
mapswereconstructedfromasetofrepresentativecoronavirusesfromallfourgenogroupsusingalignmentdatapairedwithneighbor-joining
phylogenetictreesbuiltinGeneious(v.9.1.5)andvisualizedinEvolView(evolgenius.info).TreesshowthedegreeofgeneticsimilarityofORF6,
maintain theseactivitiesin thecontext ofan expansive genome. Broadly, theseconserved viralproteins canbe categorized into structural and enzymatically active groups (Figure 3a). For structural proteins, including thenucleocapsid(N),matrix(M),andenvelope(E),high within-groupconservationismaintained,withmore mod-est similarity seen across the entire CoV family (Figure 3b). This level of conservation, similar to the S2portionofspike,suggeststheneedtomaintain func-tional interaction for the formation of viral particles. Similarly,ORF1ab polyproteingenesfinda distinction, withgenesinvolvedinproteasecleavageandthe replica-tioncomplexhavinghighlevelsofsimilarityacrossCoV families.Forexample,enzymaticallyactiveproteins,such as nsp14 and nsp16, maintain very high conservation, likely due to their specific functions in proofreading and20OmethylationofnascentRNA[25,40](Figure3). For both groups, some mutational space is available, accounting for differences across the family; however, function mustbemaintainedto ensureCoVsurvival.
Incontrast,accessoryproteinsdistinguishCoVinfections from eachother,with highvariability across thefamily, allowingvirusestoadapttocurrentandnovelhosts.The majority of these genes have beencharacterized in the context of antagonizing host immune responses, most notably type IIFN pathways[41]. However, the func-tionsoftheseproteinsmayextendbeyondhostimmunity andmaybespecies-specific.Forexample,theSARS-CoV accessoryproteinORF6wasinitiallycharacterizedbased onitscapacitytointerferewithSTAT1nuclear localiza-tion[42].Furtherstudyindicatedthatmodulationofthe IFNresponseswasabyproductofkaryopherintransport andhadasignificantimpactonhostmodulation beyond typeIIFNatlatetimespost-infection[42,43].Notably, protein-codingsequencessimilartoSARS-CoVORF6are not readily detected beyond thegroup 2B CoV family, suggestingamorerecentacquisition(Figure3).Similarly, SARSORF8hasundergonesignificantmodification,with a29-nucleotidedeletionfoundinepidemicstrains result-ingin twonovelproteins (ORF8aand8b)[44];coupled withreportsofhumanisolateswithlargerdeletions,these resultssuggestthattheepidemicstrainmayberemoving aproteinonlynecessaryforsurvivalinbats[45].Evenfor viral genes within the ORF1ab polyprotein, significant changescanbenotedacrossviralfamilies.Nsp2,cleaved co-translationallyfromnsp3andpresentinsomeformin allCoV,isresponsibleforawidevarietyofactivitiesand has minimal cross-genus sequence homology, although withingroups,similaritiesarevariable(Figure3)[46–48]. Together,theseresultsarguethatacrosstheCoVfamily, significantdifferencesinaccessoryproteinscanmodulate andchangeinfectionaspects,includingkinetics,severity, and species.
Yet, evenwithin more closely related subgroups, novel genes can appear from diverse sources and potentially
fuelemergence.The recentdiscoveryand characteriza-tionoftwocloselyrelatedSARS-likeviruses,WIV1and WIV16, revealed a novel acc.essory protein, ORFX, whichwasnotfoundin theepidemicSARS-CoVstrains [49]. Containing no sequence homology to any known proteins, the novel gene modulates type I IFN and activates NFkB signaling pathways, suggesting a role in modulating host immunity. While the majority of accessory proteins are thoughtto beacquired from the host, recent worksuggeststhat novelCoV proteins can evenbetakenfromotherpathogens[50].Identificationof anovelcoronavirus(Ro-BatCoVGCCDC1)alsorevealed thepresence of aunique30 proteinwith homologyto a knownreovirusgene;asimilarfindingwiththe hemaglu-tinin-esterase in a subset of CoV further suggests the possibility of recombination events occurring between viral families [8,51].Together, the results indicate that CoVscansample,acquire,andmaintainarangeofdiverse proteins thatmay becriticalfor maintenance in natural hosts andemergenceinnew species.
Conclusion
Withpermissivenaturalhosts andinherenttoolsto bal-ance gene modulation/maintenance,CoVs are uniquely positioned to emerge in novel hosts. For both the epi-demicstrains(SARSandMERS-CoV)andcontemporary human strains (HCov 229E, NL63, OC43), significant human disease may be the outcome of cross-species transmission. Importantly, opportunities exist to utilize metagenomics data to prepare and possibly mitigate future emergence events. In seeking these goals, researchersneedtoconsiderthefactorsthatdrive emer-gence. In determinationsof potentialthreats, exploring the variable spike S1 portion of bat CoVs to identify viruses capable of binding to human receptors is key. Similarly, targeting highlyconserved genes like the S2 region of spike has allowed for the development of therapeutics with broad efficacy against current and potential future CoVsthat emerge [28,52]. In addition, understanding the mechanisms and impact of highly variable genes provides another metric for threat and identifiestargetsforthegenerationofattenuatedvaccine strains.Together,theseapproachesprovideaplatformto leverageourunderstandingofhowCoVsemergefrombat sources to prepare and potentially stem future disease outbreaks.Withglobalization,habitatlossindeveloping nations, and uneven public health infrastructures, the survival andamplification of novel CoVsin bat popula-tions is now a lurking threat that requires immediate attention andpreparation.
Acknowledgements
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