Post on 26-Jun-2015
description
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