Investigating the effect of natural variation on an unusual H9 wild isolate strain’s viral fitnessIslam Hussein1, Brandt Meixell, Nichola J. Hill1, Eric J. Ma1, Jonathan Runstadler1,2

1Department of Biological Engineering, MIT; 2Division of Comparative Medicine, MIT

Future Directions• Testingmorecombinations,suchasD253N• TestingtheactivityofPB2mutantsinaviancellline(DF-1)• TestingtheinvitroandinvivoprofilesofPB2viralmutantsengineered

byreversegenetics

Results Synopsis• 8BM3470(8BM)viruswasgeneratedasaresultofareassortmentevent

thatinvolvedH9N2,H5N2,H7N3andprobablyotherviruses.• Phylogeneticanalysisofall8segmentsofthe8BMvirusrevealedthat

thePB2 segmentwasdonated fromanH5N2virus (A/snowgoose/Montana/466771-4/2006).

• H5N2PB2segmentacquisitionslightlyincreased8BMH9N2polymeraseactivitywhencomparedwithanon-reassortantH9N2strain.

• Aminoacidresidue#591 isan importantdeterminantofH9N2PB2function.

• Havingabasicresidue(RorK)atthispositionsignificantlyincreasespolymeraseactivity;K591hasbeenrecorded innaturallycirculatingH9N2virusesinChinaandSaudiArabia.

• Homology modeling indicates that replacing Q (neutral) with apositively chargedRorK changes theelectrostatic surfaceat thoseresiduessignificantly,andmightincreasethebindingaffinitytootherinteractingpartners.

• CombiningQ591KwithE627Kdoesn’tofferanysynergisticeffecttoH9N2polymerasefunction.

8BM3470 is likely a triple reassortant between H5N2, H9N2 and H7N3

Certain basic amino acids greatly enhance polymerase activity

A/egret/Hunan/1/2012 (H9N2)

A/northern shoveler/Interior Alaska/8B3470/2009 (H9N2)

A/snow goose/Montana/46671-4/2006 (H9N2)

A/snow goose/Quebec/17416/2006 (H9N2)

Virus isolation sitesNorthern shovelerSnow goose

Legend

GlobalInformationSystemsAnalysisPhylogeneticAnalysisandAffinityPropagationClustering

MO/06/H5N2

KO/07/H5N2OH/02/H6N2

MD/02/H6N2

MN/99/H4N6

NP PA

BC/07/H7N3

AL/07/mixed

AL/08/H3N8

AL/07/H4N6

AL/07/H1N1

AL/08/__N6

CA/08/H7N3 AL/08/H4N1

CA/08/H1N1

AL/08/H1N1

AL/08/H4N6

AB/09/H3N8

PB1

CA/08/H5N3

AB/08/H1N3

CA/08/H7N3AL/07/H7N3

CA/09/H10N7

CA/08/H2N8

CA/07/H7N3 CA/08/H4N6PB2

MO/06/H5N2

QC/06/H9N2

Reassortment between two or more viruses is animportant mechanism for generating new viralsubtypeswithnovelinternalandexternalcomponents.

Introduction

Wild birds are the natural reservoirs ofavianinfluenzaviruses,andcantransmitthemtomammalianspecies.

Theinfluenzavirusiscomposedof8RNAgenomesegmentsencoding11proteins.

The polymerase complex (RNP) is comprised of 4 of the 11 proteins, namely,Nucleoprotein (NP),PolymeraseBasic1and2 (PB1andPB2), andPolymeraseAcidic(PA).TheRNPisanimportantdeterminantinhostspecificityandviralreplicationrate.

Fig.1:Affinitypropagationclustering(top)andphylogeneticanalysis(bottom)providedacluetotheoriginsofthisvirus.Top:Centerclustercirclesrepresent8BM’saminoacidsequence;outercirclesrepresentco-clusteredsequences;blackcoloredcirclesarethemostgeneticallyrelatedasrepresentedinthephylogenetictree.Bottom:Maximum-likelihoodtreeofeachprotein’snucleotidesequence,with8BMhighlighted.Onlybranchescorrespondingtotheclustersontopareshown.

Fig.2:GISanalysisontheNorthernShovelerandtheSnowGooseindicatedthattheyintersect,lendingcredencetothereassortmenthypothesis.

Identificationofaminoacidstotest PolymeraseAssayDesignandResults

T S I A D M R M G PA D T T N I Q V S L

106 107 147 247 390 473 508 575 590 6798BM3470Consensus

Fig.3:Basedonamultiplesequencealignmentwith4otherclosely-relatedstrains,10aminoacidchangesawayfromconsensuswereidentified.Wehypothesizedthatthereassortmenteventconferredthesechanges.

Fig.4:Yamadaet. al. (2010) characterized theelectrostatic surface of the PB1 protein from(A) H1N1 (PDB: 3KHW) and (B) H5N1 (PDB:3KC6),findingthatE627KcompensatesforthechangeS590GandR591Q.Figurefromoriginalpaper.

590...FQSLVPKAARSQYSGFVRTLF... 8BM3470...FQSLVPKAARGQYSGFVRTLF... WT (majority H9 strains)...FQSLVPKAARSQYSGFVRTLF... Polymorphism

...FQSLVPKAARSRYSGFVRTLF... Test

...FQSLVPKAARSKYSGFVRTLF... Polymorphism

...FQSLVPKAARCQYSGFVRTLF... Polymorphism

...FQSLVPKAARGLYSGFVRTLF... Polymorphism

...FQSLVPKAARRQYSGFVRTLF... Polymorphism

...FQSLVPKAARGRYSGFVRTLF... Test

...FQSLVPKAARGKYSGFVRTLF... Test

Fig.5:Naturalpolymorphismsexist at position 590 and591. Based on this set ofpolymorphisms, we mademutations in the PB2polymerase of 8BM andintroduced them back intothe8BMpolymerasecomplex.

8BM3470 PB2 WT

S590 Q591

8BM3470 PB2 GK

G590 K591Fig. 6: Homology modellingusing SWISSMODEL of thePB2 protein from 8BM. AnH1N1 sequence (PDB: 3KHW)was used as the basemodel.Coulombic surface coloringwas performed in UCSFChimera.Thesurfacecoloringindicates that the charge at

thesurfacewhereGKreplacesSQchangesdramatically.WehypothesizethatthismaychangethebindingaffinityofPB2withitsinteractingpartners.

Gaussia Luc

NPPA

PB1PB2

UTR UTR

Fig. 7: A Gaussia luciferase-based assayfor measuring polymerase activity wasusedtoassesstheeffectofmutationsonthepolymerase complex. Fiveplasmids,

housingtheRNPcomplexcomponentsandthereporterplasmid,weretransientlytransfectedintoHEK293Tcells,andluminescencewasmeasured48hourslater.

WT

1

2

3

Q591R

Fold

Cha

nge

10BM

1

2

3

8BM

Fold

Cha

nge

10BM vs 8BM Q591R mutation

Fig.8:The8BMPB2polymerasehasa two-foldactivityovera related10BM16764R0(10BM)polymerase.TheQ591Rmutationinthe8BMPB2polymeraseincreasesthepolymeraseactivityby3fold.

Q

R

K

G S10BM

G S8BM

10X

1X

E627K fold change vs. WT

Fig. 9: E627K enhances the fold change of 591Qcompared toWTwhen combinedwith 590G/S. NosynergismdetectedbetweenQ591KandE627K.

8BMGK4.5X

SK4.0X

GR3.0X

SR3.0X

SL1.3X

RQ1.0X

SQ1.0X

GQ0.87X

GL0.86X

CQ

10BM5.6X

4.0X

2.9X

2.8X

0.7X

1.2X

1.3X

1.0X

0.7X

0.6X0.68X

**

Fig.10:Themutations591Kor591RhavethegreatesteffectonthepolymeraseactivitycomparedtoWT(starred).591KalreadyexistsinH9N2virusescirculatinginChinaandtheMiddleEast.

Acknowledgments