holeçular clonina of th. polymera.e gene. of influenza, b...
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1 -• Holeçular Clonina of th. Polymera.e Gene. of Influenza, B Vins.:
Complete Nucleot~de Sequence of the Virus Genome RNA Sepent
Bncoding the PBl Prote in
by
St;ella C. Xemdirlm
A th •• ts subIDittedo
to the Faculty of Graduat, Studies and Res.arch,
McGUI University, in partial fulfillment of the requirement
for the dearee of Ha.ter of Science.
Depart:MJlt of MicrobiololY and t.unolOIY
McGill University
Montrul, Quebec
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Holecular Clonina of the Polymer •• e Genes of Influenz, B Virus: ."
~ . Complete Nucliotlde Sequent,", of the Virus Genome RNA Sepent
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Encoding the PBl Protein
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Holecular 'Clonina ~f the Polymera.e Gen •• of Influenza B,V1rV.1
éompl~e'Nuëleotide Sequence' of the Virus Genome RNA S .... nt
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Encodina the PBI Proteine
by
" Stella C. lemdirim
Department of Hic'rC?QiololY and ImmunololY ,
,. McGUI- University
1 Abstract
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Influenza viruses have a segmented RNA se~ome consiatins of 8 di.tinct
RNA species. The three laraest of these code for t;he viru"polymerue . proteins. 'Â co~plete cDNA caPyof the influenza B/Lee/40 virus RNA segment
encodins the PBI polymerase protein was cloned into plasmid pBR322 and the
~leotide sequence of this cloned cDNA vas detenained.
~segment proved to"be 2368 nucleotides 'in lensth and .vas - '" '<l
The senome RNA
capable of encodins'
a polymera!!e (PBl) PFotein of 752 amine acids vith a calculated molecular , , ,
~eilht of 84-.407 daltons. ,The PBl protein Js a hilhly basic protei. vi~ a
net chara~ of +20 at pH 7 t. Sequence comparison betveen the influenza A
and B vlrus PBI proteins Jlvealed that they' shared 61% amine acid hoaololY. , ' )
An internaI bydrophobic domain and 90% of the proline re.ldue. found vithit o , .
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the influenza A virus PBI'protein were conaerved in the influenza B virua A <i '
molecule., The influenza i ànd B v~rus PBl proteins share the hiahe.t
homololY yet seen between prote in. encoded by theae di.parate virus ••. ~ -" ,... -
This remarkable conservation of primary structure arlue. for •• vere
functional con.traint on th. evolution of thi. influenza viru. pol,.ar ••• , J
. proteine
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.... --.. 1 would 'lilte to tbank 1111)' re •• arch supetvisor. Dr. Dalius J. Briedis
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for hi. au~danc. during my two y .. r. a. his student. ,
1 would also like to thanlt Anna Campana for her guidance "and 'great
technical asaistanée in pr,oducing 0 the electron micrographs preaented in ~
thia' tpsis.
Many th~. to Dr. Jàme. Coutton who supervised the electron
micro.copy course, for his advice and's~88estions.
" 1 wou1d especiall'Y like to thank IllY par!nts. Hr. and ptrs. Ignatiu~ - .
lemdirim. and my boyfriend Xenechukwu Nnagbo for tbeir support and
encouragement throughout the period of this work.
1 wou1d also 1ike to exp~ess iy gratitude to Gha1ib A1khatib for . , methodologic helpand advice and to Richard Glickman for help in computer-- -~s.i.ted d~ta .nalysis.
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1 also vant to thank Karen Ruthman. Suzanne Grothe and Ilizabeth Nowai \'" (q.
for excellent techbical asslst~ce.
linally, 1 would like.to thank Katia Sol for translating tne abstrabt ". ,
and Piona Lee. fçr typing this thesic. )
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Abstract
Res_
( . Table of Contenu
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Aèknowladgements ,
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1. ,INTRODUCTION
1. 2.
" 3. 4. 5.
Î 6. 7. 8. n
Influenza Vi-ruses and In~luenza ••••••••••••• '.' •••••••••••••• il 1 Classification ••••••••••••••••••••••• :: ••••••••••••••••••••• 2 Horphology .•••.••.••.•.•..•••.••••••••.•..•••••••••.•.• : ••.• Viral Polypeptides .. ) ...•.....•..•.•••.••.•..•..••.....•••.• Charact~ristics of the Nueleic Acid •••.••• : •••.•••••••.•.• i. Entry..-of Virus into Cells .••• r ................................. .
The Virion-Associated Polymerases and Transcription . . 0 •••••••
Replication of the Viral Genome •.•••••...••••.•.•....•••.••• F
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·6 17 18 19 27
II • ~TERIALS AND HETHODS ..
1.
. 2. ~-
.3. 4. 5.
A.
B.
Growth~ of Influenza B Virus and Isolation of V"rus-Specific RNA •••... 1- ............................................... :. 30 1>reparation of Influenza B Virus-Spec'ific Hessenger RNA ••••• 31 Electron Microscopie Examination of Influenza B Virus ..••••• 32 Prepara t ion of 1 125 -labeled Genollle RNA ....................... 33 Holecülar Cloning of .lnfluenza B Virus-Specifie RNA ..•.•. .-•. 34
Cloning (1) (H) (Ui)
Strategy I Cloninl - RNase B Hethod ............. !............................ 34 ,.. Plasmid Insertion and !acterial Transfo~tion· •.•• 36 Restli\iction Analys is .•..••••.••.•• " .••.•.•..• '. . • •• 37
Cloning Strategy II (1') Cloning Method - Hybridization of cDNAs
separataly derived fram Genome RNA and frOll mRHA
CiO (iii) 0
Civ) (v)
(vi) (vii) (viii)
to Yield ds-cDNA. •••••••••••••••••.•••••••• : •••••••• 38 Plasmid Insertion and !acterial Transformation •••• 39 Restriction An.lys is ••• '........................... 39 Putification of plasmid DNA ••••••••••••••••••••••• 40 Purification of Plasmid Insert DNA by A,ar~.e Ge'! Electrophoresls ................................ 41
\ End-labeling and Strand Separation of In.art DNA •• 41 !)NA Sequence Determination ••••••••••••••••.••••••• 42 Computer-Ass isted Analyais •••••••••••••..•.••••••• 44
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III. RBSULTS
1. 2.
Clonina Cloning
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Strategy l •••..•.• ~ • • • . • • • . . • . • • • • • • • • . . • • • • • • • • • • •. 4S Stratagy II ••...•••••••••••••••••••••••••••••••••••• 52
IV. DISCUSSION AND CONCLUSION 84
v~ BIBLIOGRAPHY "
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Influ.nz. i •• till • major epidemic di ••••• in man, and "influenza
'vi ru ••• remi.n unpar.lleled in their challenae ~o public health and-world
econollt!. tnflue ... ::.: ft and D ·,,rins. epidemic. occur yearly whil. major
pandutc. of r.ew immunotype. of 1n~luenza A -virus occur at 10-40 year
intarval:). ln batween the pandemie periods, less severe out breaks occur,
during \hich the virus slowly underaoe. chanaes in its major surface . ~ .ntigena. the hl'!llUlaalutinin and neuraminidase molecules. These surface
.ntigens acc~olate a series of mutational changes in a process known as ~
antiaenic drift (45, 53). Antigenic drift has been observed to occur in
influènza virus types A, Band C (3, 53). !nstead,of such point mutations
in the virus aenes encodina these surface antigens, entire hemagglutinin or
. neuraminidase aenes from hUman strains are felt to be exchanged for aenes
from animal virus strairis in an event known as ,.netic reassortment or
J&ntiaenic .hitt. Such "newu human viru.es, carryina surface protein aenes ,
d.rived from an~l .tr.in. are hiahly virulent because of lack of natural
~ity AIIOnl huaan. (56). S~ch antilenic .hift ha. not been ob.erved
a.on. influanz. B and C viru ... probably due to the lack of . an animal
r •• ervolr for the.e viru.e. (3,53).
-V.riation in the.e .urf.ce ~ti,en. (which are the major determinant.
for natural t.unity) in the fon of anUaenic drift or antiaenic .hift '
pre.ent ... jor obstacl.. to the effective control of influenza virus.. by
vaccin.tion. lndead, influenza viruse. are unique -.ona re.piratory
.viru ••• in that they c.use exces. IIOrtality. Uncomplicated iafluenza virus
,infecUon i. a tracMobronchiUs si.,.aled by heaelache., malai.e, fever,
chilI., .,al,i •• and dry coulh (53)~ The fever peak. within twenty four
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hours but start. de~lininl by 'the third da , and by' the sixth day ha.
diaappeared. Thi. i. vhat ma.t people vould di •• is. 'a',a particularly
.evere ,fo~ of the common cold. There are more .eriou. case. of influenza fJ.
durinl vhich the illne.. can progre.. to severe pneumonia characterized by
rapid respiration rate and often hypotension. Case •• uch as the.e
somet iJDe. prolre.s to death in one to four da). (S3).
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2. Clas.ificatioa
Influenza viruse. bel~nl tp the family of nelative stran~ RNA vi ru •••
known as Orthomyxoviridae. The' root name '''myxo" i. derived from the Gr.ek
vord "myxa" meaning mucus.. The term reflect. the' singular relation.hip
,influenza viruses have vith mur;opolysaccharides and Ilycoproteins (e ,1,.
neuraminic-acid containinl receptor~ on ce~l surfaces) (41).
Orthomyxoviruses have a segmented genome (41,45,49,56). The hilh rate
ri of genetie racombination as weIl as'evolution of these viruses is at lea.t
partly a' direct consequence of the nature of their genome orla~iz.tion.
Three immunologieal types of infruenza viruses, differing in the " 4
antigenie character of their ribonucleoprotein have baen defined. Th •• e
are the influenza A, B and C viruse.. Each of the •• "thr.e typ.. of
influenza viruses also po ••••• a number of ~~fferant v!~al surface
4ntigen.. Hovever, viruses of a .iJlUar type .bar. cOllaOn int.rnal
antilen. (49). , 1
Influenza virus •• po •• ess tvo surfac. antigan., the hemag.lutintn and
11 the neuruinida.e, vhich play ~ v.ry iIIportant role 'in viral patho,ene.i.
and viral epidamiololY. Influen.a A virus .. ara further divided tnto >
subtype. (.ubtype. of influenza,B or C viru ••• not havin. yet been found) f
ba.ed on th. nature of the .urface antt.en. and on ho.t ran,. (for various
specie. of lov.r mammals and of birds) (49,53). In the na..nclature of
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• -'taure 1: RNA ..... nt. of influenza A and B viru.s... Viral RNA. vare
'" an.lyeacl on a 4% po1yacrylutde S.l containinS'6 H ur... A, -
influenea A vina- RNA.' B. influenea' B viZ;U. RNA sa8JD8nt.; C,
., influenea B "viru. RNA .epaan~. run for a lonser period of time 0 on the .el. (Diasru is frOID Reference 8.)
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Influanu vlru.te., a. propo.ed by the World He.lth Orlaniz.tion, .n
anU,enlc de.cription of th. hual,lutinin .nd neur.inid •• e .ubtype. 1. , ~'~
ott.n incIud.d ln p.rentb •••• follovln, the .train de.ian.tion. ~
Influenz. A and B vinue. both po ..... elaht RNA ..... nt. (111. 1). , /
Influenz. C viru. ha •• ev.n RNA .e.-nt., and I.ekl • gene codinl for,
n.ur_inld •••. " Hovever , it. linlle aurfaee slycoprot.in <IP 88) po ..... e. o
• n.urulnid •• a-lik. Ictivity in addition to hema88lutin.tinl aetivity
(84) •
3. MoJ)!holop
Influenz. vi.r«s pÀrtiela. axhibit Ir •• t v.riation in .hap •. ~ 80th \,
irrelular, .pliarieal partiela. <Fil. 5) and Ion. fllllllel1toUi ~irwt ~ <>
p.rtiel •• have been ~Ti.uaIiz.d. FUamentoua p.rticl.s of .~proxiJDItaly
80-120 DIlI di_ter predOlll~.ta in nevIy i.olat.d virus or virus th.t has
underlone only one pB'.'le in 'ess'S or tissue cultur.. On the other hand,
multiple p •••• Sinl of virus favors spherie.l fonas (53,45, 49,66). Whether
~ virion will he .ph.r~cal or fUamentou. .ppur. to he determined by •
nu.ber of f.etor.. It ha. been ,ull •• ted that th. fUamentoUB
charaet.ri.tic .. y he s.netir.:_Uy dete1'llined and tlYt, in nature, hWDÀn
.• tr.ina of viru.Ï .re preda.inantly fl1~toUi (66). Hovav.r, .dapt.tion o
of • nevly 1.ol.ted huaan .train of virus to ,rovth in _bryonated 81S5 or
in eell culture. r.duoed tIN n'umNr of fil&MJltou. fonu. Mutation _y be ,r;
re.pon.ibl. for th. chan,e fre. filuentoua to .pheric.l fora; but it i • ~ -, ,
.lao po.libl. that .pheriul fonu hava better cbance. of .urvivin, in
l.boratory arovth and stora.e condition. (66). Stor.,. of virus i. knovn
to produce chanat. ln the viral envelope. Huch of the inf01'll&tion
.vailable on influenza virus ItOrphololY bal c~ fra. el.ctron .icl'o,copy.
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particle. may he a con.equence of artifact. or distortion. induc.d dur!nl
the preparation of the virus for eleetron mieroscopy.
Nagatively .tainad influanza virus partiele. are covered vith" ev.nly l ,
spac.d radial projections on their .urface (Fig. 6). The radial
projection. ara s,?ikes of glycoprotein (hamagglutlnin and neuraminida •• )
that project out from the viral envelope. Th'è Upoprotain env.lope b
_ :' eompo.ad of hôst-derived lipid bilayer and the two surface glycoprotein.
and proteet .. the virus fro~ environmental stresse.. The lipid bilayar with
spike projections of typical influenza virus partieles is Ulustrated in
the eleetron mierographs .hown in Figure 5 and 6, as well as in the
schamatte drawtng of the structure of the influenza virus shown in Pigure
2. Influenza virions have a high content of prote1n and lipide Purifiad,
preparations of influenza virus contain approximately 70% ~rotein, 22%
lipid, 7% carbohydrate and' only 1% RNA (49,56).
l'_ Viral Polzpepticta. \
Proteirr eoding assignmenta have heen made for the ganome RNA ..... nt.
of influenza A virus (1.29,46,56) and influenza B virus (8.56,61) by a
nUilber of .trategies. Higrational differenee. betve8n the RNA".egment. ",d
polypeptides of differing .trains of influenza virua. coupled vith the ~e
of, temperature-.ensitive (ts) mutants or lN-irradiated mutants carryin,
defect. in aIl but one gene proved helpful in the a.signDant of viral
polyp~ptide8 to RNA segment. (l,56). By cro •• ing a vUd type viru strain , '
" . vith a parUaUy UV-inaetivated .train that po •••••• d, on average, only on.
out of eiaht undamaged RNA segments, recombinants ariling throu,h ,en.Ue
r .... ort:Jaent could be analyzad to e.tablbh the parental or t,in of their
RNAs and proteina. The RNA and polypeptide .,bllity ot th. recOllbinant.
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"-Pliure 2) Â .chamatie diagram of the influenza virion. The lipid
bUayer as well as the .pilee layer (compo.ed of the ..,
hemalslutinin [HA] and neuraminida8e [NA]) are illustrated. The ,
structures within the virus (numbered 1-8) repre.ent the 8 RNA
.egments complexed to nucleoprotein and polymera.e proteins to
fona the ribollucleoprotein (RNP) complexe (Diagram i. from
Reference 4S.) . ./
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wer. compar.d vith the mobilitie. of the parent .trains~ If the
r.c~binant acqulred .even I~nome .• egment. from one parentàl .train and one '-'1
'-amant trom -the part~ally inactivated parental .trai~ and"analy.i. of
polypeptide compo.ition identifies the one polypeptide to have come from
the .econd parent,' then the 'coding function of this one l'ne would he
a.tabl ithed,
The development of .ID !ll!:2 translation sy.tems facUftated the
de.ignation "of codin, assianments. Briefly~ individual 8eqome RNA segments
were purified 4nd separàtely hybridized to total poly A-coniaining ,
cytoplasmic RN~ from influenza virus- infected cells (29). The RNAs were .
then translated in a wheat I~rm cell-free system" ~. In this system,
, .' }nfluenza virus-specifie miNAs direct synthesis ot aIl known influenza
virus 8ene-produèts. However, when these mRNAs are hybridi~ed to an exeess ,
, -o~ individual ,enome RNA segments to form mRNA:aenom~ RNA hybrids, each
genome RNA segment specifi'cally bl,oclts the transla/io'n of the; prO~ein that
, it enèodes. Comparison of the products of eell-fr~tr}nSlation of total
I,infected cel~ mRNA with that of genome RNA:mRNA hybrids id~tified whic)t
o "gment coded for a particular viral polypeptide, . .
In thi. wiy, .the eilht RNA .segments of influenza A and, B viruses {Ît'ig',
1) vere a •• iined to .pecific polypeptide.. It was cqncluded'that RNA (l
.egment. l, 2 and 3 coded (in varying orders depending"Ofl the yiru~ .train)
for the polymera.e protein. J'82, PBl and PA; RNA segment -4 c:oded for t~e
~lll~tlnin' (BA); .eamant 5 for the nucleoprotein (NP); sagment 6 for the
neur .. inida •• (NA), .e..-nt 7 for the membrane prote in (H), and segment 8
for the .. Jor non.tructural prote!n (NS 1J (1,41,56).
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Polymerase Proteins
Influenza virions carry'thtee polymera.e proteins vhich Ar. a •• oclated
with the RNA'and the nucleoprotein (NP),in a ribonucleoprotein (RNP) ~
cOlllplex (12,62,66). The pol)'1D8rase proteins have heen .hown to po ........ , . o
, transèriptase activity (36t38,42.,~4). They catalyze the process of viral
transcription, .which includes the' endonucleol~ic cleavaae, of heteroloaou.
RNAs containing cap'i (m'GpppNm) structures to aenerate capped primera
10-13 nucleotid~s long, the initiation of transctiption via the / -\
incorporation 'of guanosine residues onto the primers, and elona~tlon of the <1
r~; ,', viral mRNAs. These processes- will be discussed in areater detail latar • • ,ll,.oo' \ .........
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The polymerase proteins are poorly resol~ed using one-dimensional
sod,ium dodec:Yl sulfate polyacryl8..ll!ide gel electro~horesis (SDS-PAGE) and
vary in relative mobility amona different vrrus strains. Therefore, a two
dimensional gel system based on isoelectric focusina in the first dimen.ion --- . and 00 conventional SDS-PAGE in. the second dimension was successful1y used
to ~na1yz'; the~e proteins Cl6,27). Tvo.of the proteins provéd to he ba.ic
~hile,one was acidic." A nomenclature for these proteins vas devisad ba.ed
~n their, miara~ory be~v;C;;r on twoJi~n.ional aaIs. Th~ slUllar, fa.ter
migrating basic protein encoded by RNA segment 1 va. dasignated a. PB2
'while the 1araer, IDOra slovly miarating basic protei" wa. called PBI.. The
aCidi,c polymera .. protein vas designàted PA (lia. 3). ~ l!! vitro ~ransc'riptase assays have aeneraUy btt,en performed us Ina
purlfied,vi~ cores from detera,ent ~~.rupted influenza virions (37,59,62, (
77,78). Such cores contain RNA, ~, PA, PBI ana PB2. Att~t. to purify
polymer.se prote ln-RNA complexe. deprivad of Ducleoproteln have re.ult.d in • , <
-loss of tr~scripta.e activity. Racently, however, luch polymera.e-RNA , ,
'complexes composad'of onlY,RNA, PBl, PB2 and PA and vbicb .ra .till
tran.criptio~ally activa have .ucc ••• fully been purlfi.d (34). The
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P18~re 3: Tvo-dimensional gel analysis of tbe proteins of the'influen~a B
virùs transcriptase' complex (virus cores). ~o percent <)
ampholines pH 8-10.5 vere used for hon-equilibrium pH gradient
èlectrophorésis (NEPBGB) in the first dimQnsion with sodium
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'dodecyl sulfate pol~àcrylamide gel eleétrophoresis (SDS-PAGE) in 0 the second dimension. The proteins of the transcriptase Qomplex
. . vere isol.ted from influenza B/Bong Xong/5/71-infeétéd cells.
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(Di.gram i. from Reference ,26.} , "
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purificat~on procedure involve4 a cesium trifluoroacetate centrifugation
follov.d by ·phosphocelluloa. column chromatography. The polymeràse
proteina co-pur1fied vith the viral RNA and appeâred to, be more tightly ~
bound to RNA than was the NP.
H8IIIIIIAgglutinin
The h~gglutinin, an integral membrane glycoprotein, is the fourth . .
largest of the influenza virus,polypeptides. lt has been sa named because'
of its ability ta a8glu~inate erythrocytes by attaching ta sialic l ' l .cid-containing glycoproteins on erythrocytes. Su ch receptors also '~xist
J~ tarset cell. and t~. ~sslutinin madiate. ~ec.ptor-bindins and virus
attschment ta cells (45,53).
The hemagglutinin~ is the' major antigenic determinant in' natural .
immunity. Aa has previously been mentloned, i~~an un~ergo ~xtensiv~ , .ntigenie variation, thu~ aiving rise to recurring epidemics. The
, hemagglutinin mediates the two important funetions of ~eceptor-binding and
cell attachment as weIl as cell penetration and uncoating.
VIrus .penetration and entry into hast cell'occurs via a fusion event
between the viral envelope and cellular endosomal and lysosomal membranes. ,
Tbe fusion aetivity requires the cleavage of the hemagglutinin (BA) in ta
tvo subunits, 'BAl ànd HA 2 , ~hat r~in linked by disulfide bridges .. The
amino terminus of BA~carries, a hydrophobie signal peptide of thirty five
amino acids (45). This signal pépt~de is proteolytieally ~emoved during HA
.biosynthesis and ID8JDbrane insertion. The carboxyl terminus of HA eon'tains 1 a resion of 25 hy~rophobie residues vhich Jlachor the molecule 1i'ithin the
pl ....... bran •• On cleavage of BA ta HAl and BA 2 , a nev~hydrophobic amino .. tel'1Dinu. ts cruted on HA 2 and the membranè fusion activity of ,the protein
18 .cUvated.
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The three dimensional .truc~ure of aA has been determined to 3A
re.olution by X-ray cry~t~llography (80). ~. heœagllutintn has besn shown . ,
to ba • trimer consisting of a tripl~-stranded coiled region of a-h~lices ,.
o extending 76A f~om the membrane and a globular region of anti-par~llèl
~-sheet that contains the receptor binding site. The variable antigenic
determinants are positioned on this globular,domain (53;80). These
structural'studies suggest that receptot-binding and f~sion activities'are
separated into different physical domain. of the hemagglutinin. The
receptor-binding sitéS are located at the tips of the spikes (the globular , " (
,region), while the presumed fusion-active site (the hydrophobic. N~termin~s
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of HA 2 ) is located in the stem region, in close proximity to the site of
anchorage of HA in the plasma membrane (79).
Nucleoprotein
The n~leop!otein is the/type spécific antigen that distinguishes
types A, Band C influenza viruses'. The nucleoprotein forma the backbone , '
~f the helica! nuc!eocapsid·(5~). The nucleoprotein is phosphorylated at a \
\ serine residues. Bot~ phosph~ryla~ed ana nonphosphorylated forma of the
nucleoprotein have baen ~isualized in infected'cells (2,45,58,72). The " ,
rote of the phosphorylation is not,yet known. The nu~feoprotsin i. a basic
pr~tein. However, cluster. of basic residues fpr interaction vith RNA are
~ot apparent so ft is possible that the RNA -.y he associated vith numerous' . , . - reaion,f of the molecule 02,41,45). '
Neuraminiuse
The neuraminid.se is an Integral membrane protein, appearln. in \
èlectron lDi.crog~aphs .s a mushrOOII sb.ped spUte- (53). vhich is a tetr ... r
of four NA monomers.· It is believed to function ta prevent •• 1f-
'.
o
l
15
.aar.a.tion .nd ~o promot. r.l •••• of virus p.rticles by hydrolysis of 2~3'
Ilyco.idic lint.ae. betw •• n t.~in.l neuraminic .cid residues~d N-acetyl
'.l.cto.amin. in .1ycoprot.in. (53,66). In this w.y, the virion is able to
fr •• it •• lf fram n.uraminic .cid-cont.ininl structures present in the host
c.ll membrane fram which it is buddinl' .. f
The neuraminid •• e, unlike the hemagllutinin, does not underlo post . tran.lational proteolyti?processinl' The neuramlnidase,(NA) possesses
only a single large hydrophobie region at its __ amino te~inus. Unlike the
sign.l sequence of HA, this amino-te~inal hydrophobie sequence is not
removad from the mature protein by proteolytic cleavage, but ramains
embedde4 in the membrane and serves as an anchor (6,19). In addition to -
its anchor function, the amino-te~inus of ~ has also been posulated to \
provide the signal function in· translocation across membranes (6). Thus,'
it appears that signal peptide cleavage is not an absolute requir~nt f
tran'sport aèross membranes since m can be transported from the rough •
endoplasmic reticulum (RER) via the lolgi apparatusJmd into the p 8sma
pembrane ~e'Pi~a ~he-t.ct that it. hydrophobie .. ~.rminu. doe. not
underlo p,oteolytic cle.vage. 9
In influenza B viruses, RNA segment 6 codes for two proteins derived
from • bici.tronic miNA; the neuraminidase and • .econd integr.l membrane
protein found only in infected cells which has been desilnated the NB
polypeptide (69).
H .. bran. Prot.in
1 Thr •• ~.par.t. ~ .r. tran.cribed fra. RNA •• pent 7. One (Hl
.aNA) is an unint.rrupted n.arly compl.t. tran.cript of RNA •• gment 7.
Thi. Kl miNA is tran.l.t.d ta produce Hl prot.in (9). H2 protein i.
trandat.d frOll •• plic.d IÎRNA derived frOll the Hl IIRNA. The H2 mRNA
•
o
o
o
-------.~
1~
t
contains an interrupted rel ion of 689 nucleotide. followinl nucleotida.
encoding the.N-terminal nine amino acid. of the Hl prote in (45,53). The
271 nucleotide body region of H2 mRNA is J'-coterminal with the Hl mRNA but
is translated in a different reading frame. H, mRNA also conta in. an
interrupted region. lt contains a 5' end sequence prior to the splice t
point which is identical to the 5' ends of Hl and H2 mRNA.. H, maNA ha.
the potential to encode an 8-residue peptide that would be identical to the
carboxyl-terminus of Hl (45,53). Havever, such a product nas not yet baen
identified.
Hl is the major membrane protein found in virions. lt forms the
electron dense shell that encloses the ribonucleoprotein (66). Hl is a
cytoplasmic protein coating the inside of the virus envelope, while H2 is a .. ,
nonstructural i~egral membrane protein expressed at the surface of
infected cells but not of virions. lts function is unknown (46).
, ... Nonstructural Protein
~ .1 Genome RNA segment 8 encodes two nonstructural polypeptide.. NS 1 and
, NS 2 , that are translatèd from different miNAs. The NS 2 miNA i. producad by
splicing of the NS l maNA ~hich i. colinear with the genome RNA segment.
•. The reading frames for NS l and NS 2 overlap by 70 amino .cid. that are
translated from different reading frames '(10,45,53). NS 1 ,and NS 2 .hara 9
&mino acid. at their amino-termini, but after thi. aequence the miNA for
NS 2 has a deletion of 423 nucleotidas add then rejoins tha ra.t of the aRNA
in the +1 reading ~frame (10,53).
HS l and HS 2 are found only in infeéted calI.. HS l 'ia .ynthe.ized
early in infection and accumulate. in the nucleus. NS 2 i •• yntha.tzed lata
in infection and i. fouhd in the cytopla .. (17,53). NS l i. a
ph08phoptotein with phosphate r •• idue. attachad to ona or .ara thraonlna
residuà. (45). The funct ion. of HS 1 and NS 2 ara unknovn.
17
s. Cbaracurt.tica of the NUcleic Acid
Influenza virua i. termed a neaative strand RNA virus .ince its
sinale-stranded aenaa., RNA i. of oppo.ite .ense ta .. ssen,er RNA (mINA).
The lenome RNA, unlike the maNA, is uncapped at-it. 5'-terminus. Instead,
the S'-termini consist of pppUp •••
The aenome of influenza virus has baen shown ta contain eiaht .inale-
stranded RNA segments (Fia. 1). However, at least eleven'gene products
have Deen identified •. Genome ,segments ~ and 7, encodina the nonstructural
(NS) and membrane (H) proteins, respectively, of influenza A and B viruses
and RNA segment 6 encodina the NA and NB proteins al influenza B virus are
multicistronic (8,9,46,69). The influenza genome RNA has baen estimated ta
'have a total molecùlar wejaht of 6 x la' ta 7 x la' daltons (12).
The pandemic impact of influenza viruses (influenza A viruses in
particular) il attributed ta the nature of their segmented genome. This r-
allows strains of a similar virus type ta participate in high frequency
genetic recombination. The recombinational event does not occur by the
.usual cto.sover or copy choice mechanism. The term genetic reaslortJDent
(antiaenic shift) more aptly de.cribes the sort of recombination that
occurs in influenza viruses. It refera to the reassortment of RNA .egments
betveen proaeny virion.. It is po.tulated that when such reassortment
occur. between human and animal virus strains .uch that the resultina
proseny virus acquire. aenome RNA. enccdin, .urface proteins from the
parental animal .train which human. lacl&: antibody aaainlt, a pandemic may "
da,velop. The aena.e RNA. codina for .urface antiaena also underao '. more
" aradual chanae mown a. antiaenie drift. This' involve. the slow
accbaulation oropoint mutation. (3,45,53).
Another characteri.tic of influen~a virus ~ is the fadt tbat n.ted
RNA).~ not infectious. Influenza virua RNA i. complexed to a nucleoprotein
o
(
,
o
'.
o
18
and,thr •• polymer •• e protein~. If th. RNA'is di •• oci.ted fra. thi. co.ple. t, •
it la not infectious bec!.~se the virfon-... oc,iated RNA tran.cripta.e
activity is responsible for transcribin. viral miNA. fro. virion •• nODe
RNA. follovJnS attachment'.nd p.netr.tion of the virus into th~ ho.t cell~
The facr-°that infectivity and RNA polymer •• e activity .re lo.t .t the ._
rate SUIS.sts that tAe RNA polymer.s •• ctivity i. r.quired for inf.ctivity
(12). The nucleocapsid structure of influenz~ virus .ffords .oma ) , ,
protection for the RNA segments. Hovever. the associ.ted proteine
(nucleoprotein and polymerase proteins) do not provide comelete nucl ••••
protection. They are arransed in a manner that le.vos .oma of the pho.pho-•.
diester bonds of the RNA exposed beeause "the RNA. in the nueleopla_ida .r.
sensitive t~ ribonuclease {12.41).
6. Intry of Virus into Cells'
The entry of mQst enveloped viruses into cella is mediated by • fusion
event of the viral membrane vith cellul.r membrane.. In.oma virus •• (such
,as paramyxoviruses) ~he viral membrane fuse. directly vith the pl .....
membrane at physiolosieal pH and the vJral RNP. are directly rel •••• d into ( .
, th. cytoplasm of the cell (12,79). In influ.nza viruses. fu.ion ean oeeur
only .t lov pH and is felt to oecur vith th. membran •• of .ndocyti~
v.sicles vhich internally develop th. neces.ary4rop in pH by union vith
lysosomal vesicles (82). o
J_ -
The cl.avale of the heaaillutinin precursor to RAl .nd HA 2 i. an ,
absolute requirement for infactivity and fu.~on vith calI ... b~Anas. Once -
cleaved, • nev .. ino terminus, comprisad of ~ hi,hly con.e~.d re.fon of
tan r •• idu •• i. ,enerat.d on RAZ. Thi. r.sion ia nor.mally buried in the
virus meabran.. At lov pH, cleav.sa indue .. a confor.mational chan,e that
\
._.~---.
c
"
\ 19
expos.s th. amine ~.l'IIinu of RAz. This leads to' a ,chana. in antiaenicity r-\ ~
of the mol.cul.j- two n.v antiaenic site. are acquired and the molecule
acquir •• the capacity to interact vith ~d fuse lipid membranes (53 .. 80).,
lt ~s oeen shown that, HA-must he anchored to a lipid matrix for
" fusion to tate plac~ (79). The carboxyl terminus of HA 2 anchors the
protein to the. viral membrage. The fusion peptide at the N-te~inus of .. . ,
HA 2 , exposed by a conformational change at low pH,' in a yet unknown manner ~
forma a bridge between the viral membrane and the endosomal membranè. The . ~
virus partiele binds to receptor and is then internalized by'endoeytosis.
The endocytosed vestcle is 'then believed to fuse with lysosomes followed by • 1
fusion of the viral'envelope with-the endocytosed vesicle-lysosome ~brane ,
at low lysosomal pH (79,82). This final fusion ~vent causes release of the
viral RNP. into the cytoplasm.
7.' The Viripn-Ûsociated PolP!r.... aacI 'l'rU8Criptioa . - , ~
Upon entry ~f the influenza virus RNP into the cell, thf first event
,is the synthesis of viral ~s by ~he virionJa.sociated polymerase . ~
proteina. virus ~ranscription ha. been shown to occur early ln infection
and is unaff.ctad by prote in synthesis Inhibitors (12,52).
The fact that the NP protein accumula tes in the nucleus (17 .. 24)'plus
the findina that influenza virus transcription i •• ensitive to actinomycin
D (an inhibitor of callular DNA-depandent RNA transcription), rai.ad the '- ~.
pos.ibility that nuclear event. played a role in influ~z. virus RNA ;0
.ynthe.i.. UV-irradiation of cella prlor to infection. host-cell DNA
da.aling drug., or anythina that had deleterioua effect on cellular ~ . . ~ . tran.cription vere found to advers.ly affect influenza virus transcrip~ion.
Th ••• finding_ ~.r. put in a new dimension when~ it va. discovered that
œ-aaanitin.. a .~cific inh{bitor of cellular DNA-dependent RNA polymerase ,
...
o
o
20
-'
II. inhibi~ed the in !!!2, but not ~h. in vitro, viral RNA transcription
process (42,45). ~eplication of v,irus.s Irown in mutant c.ll. po ..... .in' .
a-amanitin·resistant RNA polymera.a II was not inhibited by a-amanitin
(42). Thus, it vas estabHshed that influenza virus !!! ~ mRNA .ynth.sta .
required a host function, presumably RNA polymerase'II transcription .
products.
Sinee cellular maNA synthesis takes place in the nucleu., and viral
Jti trftnscription seems to require the products of cellular transcription, it
h
was proposed that influenza virus miNA synthesis must"alSo take place in o
tHe nucleus. This vas confirmed by pulse~1abe1~nl and cell fractionation
experiments (17,24,28,52,74). In addition, splieing iB a nue1ear event and l
the H2 and NS 2 proteins ~f influenza virus are s~lieed products. maNA. of \ . ~
viruses that replicate in the cytoplasm do not contain intern.1 modifièd A
residues, (e. g. m'A) whereas sueh residues ,..have been found in cellular ~_~ \.D' ~
weIl as influenza virus mRNAs (38,41). These findings support, the current
belief that influenza virus transcription occurs in the nucleus . .
A set of bri11iant experiments by Krug and a~sociates demonstrated
" tbat new synthesis of host cell maNAs was the host function required' by
influenza-viruses for their transcription (42,59).
Studies on influenza virus A in ~ transcription revea1ed Ipw. , .
levels of influenza virus transcriptase activity as compared to the in !!!2 'II • ,.. _ • ,
levaIs. Experiments shoved that dinucleotide primers Ireatly stimtilated !!!
~ tran~c~iption. Wh'ile the dinueleotide GpC stiJIIUlated the reacUon,
ApG was by far the IIIOst efficient primer. These molecule. were , j \
ineorporated into the 5.' 'end of newly synthesized RNA chains vhich w.eu. t 'b '-
noncapped ineomp1elte transcripts of genolDe RNA .egment. (42). Hes •• nler
RNA. (mRNAs) iso1ated from infected cella contaln a S'-terminal methy1atéct . \
type 1 cap structure. In aadition, primer exten.ion .equancinl of
c ..
c--
.. -
21
populations of virus-,pecific mRNAs showed that viral IDRNAs contained,
S' -terminal heteroganeous sequence. which .pp •• rad no t, to he ahcoded by th~' . - '
a.nome lUtA (68). Horeovar, netther'methylating npr'capping enzyme
act'ivit~es could be detacted in influenza viri'Ons. Thus, it was proposéd • r
that the 5 t cap structure observed 9l1der .!!! .1d!2 conditions came from a
primer syntheaized by'host RNA polyme~a~e I;z
~-alo~in mINA as 'well as capped alfaIfa mbsaic viru~ (AI..HV) RNAs we~e -' • t \
-uaed to identify'the S'-terminal host-derived fragments that participated
, " . )---
in ini1~enz~ virus mRNA synthesis._, ~-a10~in p~imed RNA Synt)e~i~-~as po obaerved ~o be, on a mo1ar basis, a thousand-fold more effifient a primer
'- than ApG (42). In the presence of yiral cor~s. ~-g1obin ~)tW$S 'used t •
• f
prime influenza virus tran~ori,.Ptio~ in an in -'§itro t~~nscr,p ion system.
Analysis of the reaction products showed product RNA. that mi 'rated more
,slowly than did the ApG-primed resetion products, such that they appe~red ,
: to be approximàtely 11-15 nucleotides longer (42,68). Thfs suggssted, that
the ~-globfn mRNA had contributed 11-15 nucleotides to the naseent
jlDOlecules. ~-alobin mRNA eon~aining a chemically introduced l2P-Iabel in
the 5' -term~n.l methylated câp structure was then used as primér in th~
tran.criptase assay. The resulting mRNA segœents were found,to contain .
• s2P-labeled c;ps, thus demonstrating the transfer of the ~-globin
~-d&riv.d caps to the viral mRNAs. ' It dppeared that 11-15 ~-globin l_
specific nuc1eotides, ift additi~n to the cap structure, were transferred to
th~ influenza vJ.ru;s mRNAs. Theae !lndings lent crede~èe to th. propàsll
th~t capped heterologoua host mRNAs.were eannibal~z~d t;y infl~enza viruses
for their mRNA synthesis. Severa! studies have in,~icatect thàt these, host .
, IBRNA. 'Ar. cleaved at purine residues (preferably A residues) 11-15
nuc'leoUdes downstream from their 5 J -terminal capJ to produce the primat
fragments that lead to initi.tion of influenza virus transcription
(42,59,68) . - , ' 1
1
o·
-..
o
o
.... - -
The effect of different type. of cap structures on viral mRNA , "
synth .. i. has all~ baen .tudied (38,42,59,68). It has heen damonstrated 1 ~
, that a fully methylateci cap 1 st'ructure (m 7 GpppGm) h absolutely requtred: ~ ( . .
mRNAs wlth Cap 0 (m 7 Gppp) structures and cap core (GpppG) structuz::e. are
not cleaved ta primer fragments. Uncapped ribopolymers such as poly(AG) . ~
inhibited mRNA''=p'dmed as weIl as ApC-primed viral ,RNA tr.anscription. S~~ce
the ApG prim~d reaction was also inhibited, ft is pcsBible that.. both the
,cleavage and elongation steps were inhibtted (5'9).
UV-crosslinkins, photoaffinity labeling and temperature sensitive
mutants have helped to el~cidate the role of each polymerase protein during
vir~l ~ synthesis (5,57,64,77,78). J ~
. t' In UV crosslinking experiments, viral cores were incubated,
respectively, - with (ALHV) RNA 4 containins 32p in it$ cap l structure in
...., (a) "t~e absence of ribonuclepside triphosphates (concli1;ions where on,ly
cleà~age of (ALMV) RNA 4 should take place), (b) with unlabeled capped RNA l"~
in the presence of [a-~2PJ-GTP (conditions where ( 32 p}GTP should be linked
,to the primer fragment gener..ated), and (c) in the presence of aIl .4 -rHTPs • .
The reacttons were then ultraviolet irradiated to 11nk proteins to adjacent
structure. Analysi~ of the rèactiop' pr~~~ts showed that in (~) PB2 vas
crosslinked to capped 11-15 nucleotide,AUMV-derived fragment., that in (b) , 1
PBl\was cr~sslinkèd .to la~l~d guanosine ,.si~ues while in (c) PA vas
associated with the growing ~ chains (77). 'Ba~ed on th!s, PB2 most
likely recognizes and endonucleolytically cleaves cappad maNA ~l-IS
-nucleotides from the cap. PBI la the màlt likeii candida'te to catalyze the ' , .
" initiatJon, of transcription vith the incorporation of a G residue. The , .
Q-
role of PA is still obscare. It. presence may he required to fDrII a'_ ~
'.(,. .. ' complex w!th the other two prateina. l'Mt i., ft lIUIy functJon in linkin,
1 .,.. \
PBl and PB2 in the enzyme complexe Alternatively,"it may function in chain 'r,
elongation. .. tflr. .. "",
l'
(
23
The photoaffinity iabel {,-,2Pl-y{4-(benzoyl-phenyl) met~l amido]-]
me~hyla~~~o.ine SI-triphosphate (or in short. BP~H'GTP) a cap analoaue and
a photo~eaetive'deri~ative of M'GTP wa~ used in similar crossli~king.
exp,eriments de'scrlbed ,above. 'The afOnity label w~s found to be . . ~cros.link.d to PB2 thus' suppotting previous. reports that PB2 rèeognized the
capped ,tr.ueture· of eapped mRNAs (42,57) .• , , . Bxpèriments with pyridoxal 5'- phosphate identified PBI as containing
the' nucleotiele. binding site (64)~ Pyridoxal 51 ·pho-sphate (PLP.) was shoWJ) .. "to r';ver;st"bly inhtbit th~ t in vitro tr~nscription of influenza fow1 ~lague
virus (FPV)~ ~~ever. ~n the presence ~f b~rohydrj.d~, the i~on bec&m8' irreversib1e. This Ls' because PLP forms a' sWiff basè ..,ith a
-
specifie lysine residue in the nueieotide binding site. ,On reduction with-1 t
l'H)-borohydride, the ~nhibition beoame Irreversible. and the PBI protein !O '.
was found to be labeled.
linally. temperature sensltive mùtants.have contributed to the
-elueidation of the functions of the polymeras~-proteins (78). -Temperature , '
.sensitive mutants, temper.aC;ure s~nsitive witQ res~ect t9 the ,
endonueleolYtic funetion. were used to assoeiate the PB2 protein with this " 1 1 •
funetion. Where wild type virus exhibited' simtl'ar ~ates' of endom~eleolytlc , . . .
cleavaae and overa.ll !!! vitro transertption at ,39;5°C (the nonpermiss1ve
t8llperature) and at 33°C (permIssive temperature), tht;:tt8Jl)pe,ature .
•• n.ftive mutant that was known to have a defect: in the genome RNA ,segment ~
ooding for the PB2 prote in exhibited on1y 15% of wlld type' endonueleolytie - 1
-- ~ :"'1
aetivity at 39.5°C. The mutation in PB2 .pparently affected ont y the
end2ele01Ytfe activity sinee subsequent steps:appeared to be temperature 1
"
";;<$:4 -'0
o
o
--' l '
... , .... The data summariz.d above allovs fo~lation of a mechanism for ,
influenza virus transcription ~hat embraces the raIe of .açh polymera ••
, protein and ,its activit tes during transcription. Such a postulated model
is depict~ in Figure 4A and 4B.
Current models of influenza virus traAscription ,visualize the~
polymerase proteins to be associated in a complex during capped RNA-primed
viral mRNA synth~sis (7,37,38,42,59). The acidic polymerase prote in PA, i. "
presumed to be positioned betveen PBI and PB2, vith PBI ,located at the ,
leading end of the complex, and PB2 at the trailing part (7). See Figure "
__ .~ At the start of transcription, the polyme~ase cQIDplex ls located at ,
the 3' -end ~f the' genome RNA templatè (64,77). The PB2 protein .,recognhes
the fully methyla,ted capped structure of a host ceU mRNA. The host èell ,~ • 1 ".
mRNA is bound near t~ the endon~clease site located on PB2. Using its
end'Onucleolytic activity, PB2 cIeaves the capped mRNA approximately 10-13
nucleotides downstream from the capped structure. The cleavage Oecurs only 1
at purine residues, but ,A residues are preferred (42,59,68). ' The capped .
,primer fragment does not appeBr to hydrogen ~nd ~o th. genome'RNA (vRNA) . template, vith the possible excep~ion of the terminal A residue on the
primer fr~gment vhich becomes aligned vith the 3' terminal a residue of the
genome RNA (7,42). A specific interaction is presumed t9 occur betveen tha , ~ .
capped RNA primer fragment and one or more proteins in the transcripta.e (\ 1 1 1
'complex (42,59). Consequently, initiation of transcriptidn appear. to-
occur as PBI adds a G residue ta the ~end of the primer fi.amant a.
directed by' the 31-penult~te C res~due of the vRNA (Diagram c in 'ilure
4). The PBl ~rotein subsequently mayes to the 3' end of the Irovina miNA ,
, chaib vhere it can catalyze addition of .ub.eqUe~t nucleotida.. Initially, \ the PB2 ramains attached to the cap structura, but after 11-1·5 add1i:lonal
, . •
"
..
. ,
t:
l\,
.. ~ ('
l' -)
, '
t'
, "
.'
• 1iaure 4A: Proposed mechanism for the prlming of influenza virus RNA
transcription by capped cellular maNAs. See text for )
explanation. (Taken from Reference 41.)
,
Fiaure 48:" The role, interactions ~d movements of the three
polymerase proteins dudng influenza virus RNA transcript-ioh. \, ,1'
See "text for explanation.: (Taken ~;:om Reference 7.)
, "
, '
".
'. .- . ~
• , . ,
- . - ,
o
\ .
, ,
o
1·· 1
~ o
o
, ,
o
A
'l
. B
""V't'1IUU!.~1iUÇ4~>" '" •
, ,
~
1. CLEAVAGE'OF PRIMER \ l > .12 .. di.teeS
" ..JI\ • Gpppx Y •••••••••••• A pM
, 1ft • GpppX y............. eN·
lO - II " nucleotld ••
2. IN,IT IATION ON' PR IMER OF "RNA SVNTHES IS
'.
P81 .~d i.ted
,u~pGpuPuPuPupCp ••••••• 'Ift...'~ , • GpppX l' •••••••••••• A . ... '.
pP~ ~. .
3. ELONGATION OF CH~IN
. YPÇ'1Pyprpr~~p, •••••• " ·:.· ••• 1 •••••
Il GpppX"'y ••••••••••.•••• APGpCpAP pp •••••••
• (a)
" '
• Ir
(b)
(c)
(a)
,
"
(
<
27
nucleotides Mve baen added to the capped fragment it dissociates from the , .
cap ('7). This is aepicted in Figure 4B (c and dl. If appears that the
polymerase proteins move down the ,growing viral mRNA chain in a complexe
\ ' :
8. bplicat1011\ of the Viral G8n~ t
It was meritioned ~arli~r t&ât transcription is the initial event in
infection bi influenza ~irus as weIl as by other negative strand RNA J •
viruses. Influenza virus RNA replication (synthesis of new minus strand \
RNAs) i5 known to be mediated by newly synthesized virus-specified
proteins. Cycloheximide t an inhibitor of protein synthesis f inhibits RNA
replication but'- does not affect RNA transcription. The polymerase proteins
have aIso> been implicated in virus RNA replication.
The fint step in RNA replication i5 the synthesis of copies of ful1-
lengtD uncapped (+) sense anti8enome copies (cRNAs) using the vRNA as
template. Such an~igenome RNA synthesis i5 primer independent and the
(+)c~ segments are c9mplete transcripts of the vRNA segments and are not
"'polyadenylated. The (+ )cRNAs làck a 5 t -methylated cap structure and
contain pppA at theSr 5 t -termfni tlt pOsitions complementary to the 3'-Us of
the aenome RNA 'segments. It is presumed that virus encoded gene-products
modify RNA ,transcription such tha~ it becomes primer-indpendent and ~bIe to
bypa.s'the polyadenylation signaIs on the genome RNA templates to yield ,
full-length cRNA transcripts (41,45). The antiaenome [(+)cRNA] is then '-
ued •• templatl for prp;eny' vRNA r (- )cRNA) synthesi~. Little ls kD<Swn to
, date of thfs proces •• .
Certain .s~cts of influenza virus RNA synthesis'may be'exploi~ed in
future development of antiviral druas. 10~ instance, uncapped ribopol~rs
(eo1y{AG]) are knoV,Q to potent!y inhibit the cap recognizing viral .. endonuclea.e. Thu. th!. P,bl,.era.e is a potential taraet toward. whi9h
anU-influenz. virus drug. cao be 'directed. '
. .,
o
, ,
o
o
28
.. Although Iess weIl studied t~an that of influ.nza A vi rua , the " ,
transcription process of influenza- B virus appears to he .tmilar. ' Th.
replication of the virus is stfnsitive.!!:!!.!.!2 to a-aman,iUn Q3), the1!!
vitro transc~iptase complex consists of the analogou8 proteins (NP, .PA,,~Bl • • , 1
and PB2) (27), and cloned ~DNAs corresponding to influenza B virus·mRNAs
cont4in heterologous presumably host-cell mRNA-derived sequences at their
appropriatë 5-ends (8,7,10,11,67,68).
In addition, the RNA-dependent RNA polymerase activity of influ~~za
virus has been shown to be inhibited by pyrophosphat~ analogues (e.g.
:phosphonôacetate, phosphonof~rmate, and subst}tuted methylene
• j
~\ dipho~phonates) (15). Infl~nza virus RNA po1ymerase differs süfficiently
from aIl host ~èll RNA polymerases that pyrophosphate analogues can inhiblt - 1 ~ "...
influenza viru:~ RNA polymerase ùnder conditions where host cell RNA
'.\ polymerase ls unaffected. . Pyrophos,Phate analogues are believed to exert
._ .. their'inhibitory effect by complexing with essential zinc ions st the' \ '
active sites of the enzyme, thus preventing tbe binding of nucleoside-
,triphosphates. As in the first case, anti-virus drugs di~~cted aJ&inst th.
viral polymerases may eventu.aÙ,. be more effeëtive than present-day
,vaccines in ameliorating di~ease caused ~. ,th~ Vi~S sinc~ sQch drug
thèrapy would not be iRfluenced.by antifebic variation of influenza virus
surface proteins.
DNA sequencing of cDNA clones der1ve~ from influenza virus-.pecific
RNAs has provided nucleotide sequence information and corresponding deduced - j
amino acid sequences of polypeptide products from multiple oupie. of each ~ ,
,
of the eight genome RNAs of influenza A virus. Dedudad lIIIino .cid
structures f rom multiple èxupI.. of inf lue.nza A vi rus PA (23), PBI < 4 , 71) . ' '
and PB2 (33, 63) proteins have shown a hi,h de,ree of homololY in polymer •••
prote in structure amans diff.rant influenza A virua .train ••
ln
o
" . r '
29
Nuçlaot~ds s~quenc. in~ormation bas also besn obtainsd for ths'five
sho·rt.~t' ga~me RNA segments of influenza B virUs (8.9,10,11,67), but no -
complete sequence has yet baen obtained for any of the influenza B virus 1
RNA segments which encode the polymerase proteins.
'~ Comp.ris9n of nucleotide sequenmces between equivalent genome RNA
"s~aments ~nd mRNAs of influen~a A an~ B viruses has shown variable amounts
of conservation of' open protein-coding frames, miNA structures and primary
-prote~n structure. The open reading frames for the NS 2 and H2 polypeptides
a~a co"ssrved between t~e t~o typ~s of virus (8,9), while the open reading
frame for. the NB polypeptide is unique to influenza B virus (67). Direct
amino acid hom~logy between correspond~ng influenza,A and B virus proteins
has ranged,from le~s than 10% for the NS l polypeptide to 37% for the NP • '1
'polypeptide (11).
In order to continue s!milar comparisons between influenza A and B
virus genes and gene products, and in order to further understand the 1
st~cture and' function of influenza virus polymerase protelns, a library of ; )
!CDNA clones, corresponding to influenza B viru~ RNA segments h.s been , l,\ _
obtained. A single cDNA clone cQntaining a full'represe~tation of tba
- influenza B virus genome RNA segment correspond!ng to the PBl protein has ,
bean Identifled and •• qu~nced •
•
..
o
o
....
o
, . 30
II
1. Grovt.h of Influenu B Viru and Purificatioa of Virua-SI!!Ciflc IlIA
Influenza B/Lee/40 virus was grown in the allantoic caviti •• of 10-day
old hen eggs (obtained from Simetin Enterprises). The elg .urface. ver. ""1
sterilized with ethanol at a position one-third down from the apex of the
egg where a one millimeter indentation was introduced vith an enaraver.
Influenza B virus stock was diluted to a concentration of lOs plaque
forming units (pfu) per ml in Dulbecco Modified, Ragles Medium (DHEH).
Approximately 10~ pfu of virus (0.1 ml) was injected into the chorioallan-
toic space of each egg using a sterile syringe and needle. The ho1e was
then sealed vith hot wax and the eggs placed in an egg incubator at 39°C
for forty-eight hours. At the end of this incubation period, they were
cooled at 4°C for twenty-four hours and then alantoie flui4 vas harvested.
Using forceps, the egg she1ls were cracked open at their air space
ends. A 10 ml pipette was used to co1lect the virus-containins a11antoic
f1uid, which was centrifuged at 5000 r.p.m. for 5 minutes to sediment
,cellular débris. The supernatants were centrifuged at 10,000 r.p.m. for
two hours in ord!,r to pellet the vir~.
The pelleted virus was resuspended in NTE buffer (0.1 H NaCl, 10 mM
Tris-BCl pB- 7.4, 1 mM KOTA) and disaggregated vith a doun~e homolaniz.r • .
- ç 1 The resulting suspension was,layered onto a continuous sucro •• gradient
(15-60% w/v sucrase in NTE) and centrifuled in a Beckman SW28 rotor at
25,000 r.p.m~ at 4°C for 90 minutes. After centrifugation, the viru. had
,--Jsedt..&nted iuto two bands, an upper virus ~d, and a lower band of \ .
residual virus andodebri.. A needle and syringe was us.d to •• pirate the
virus bands. After dilution at a ratio of at l ... t 1/6 in NTI. the virus
wa. concentrated by centrifu.ation ~t 40,000 r.p ••• in a Beckaan T160 rotor
for 30'minutes. '
,,::,iC •
-
o
31
To obtain purified virion RNA, the purified virus wa. r •• uspended in 2
ml of NT! and sodium dodecyl ~ulfate (SDS) was added to a final
concentration of 0:5% (v/v) in order ,to disrupt the vi~s envelope.
Proteina.e K wa. then added to a final concentration of 0.1 mg/ml and
allowed to incubate one hour at 37°e. At the end of the incuba~ion period,
the .olution was extr~cted with an equal volume of 1: 1 phenol:chloroform. -
The resulting aqueous phase was made 0.2 H in sodium acetate and
precipitated with 3 volumes of 95% ethanol. The RNA precipitate vas
redissolved in 0.4 ml TE (10 mM Tris-HCI pH 7.0, 1 mH KOTA) and its
concentr~tion measured by its ultraviolet absorbance at ~60 nm wavelength.
2. Prepàratloa of InflueDA B Vlrus-Spec,ific HeB!8DBer JINAt (1IDAs)
Influenza B/Lee/40 virus was used to infect monolayers of confluent
HeLa cells at a multiplicity of infection (m.o.i.) of 20 pfu/cell. At 10
'hours post infection (h.p. i.) the cells were harvested and centrifuged at
2,000 x g at 20°C for 3 minutes. The cells were was,hed once in cold
.phosphate buffered saline (PBS), and then, resuspended in cold reticulocyte
standard buffer (RSB) (10 mM NaCI, 10 mM Tris-HCI, 1.~mM MgC1 2 ). After
swelling for 5 min. the cella were disrupted using a dounce homogenizer and
centrifuged at 1,500 x g, 20 0 e for 10 minutes in order to pellet nuclei.
An eq~l volume of proteinase K buffer (100 mM Tris-HCI pH 7.4, 50 mM NaCI,
10 mM IDTA, 0.5% SDS) was added to the resulting supernatant and proteinase . ~ va. added to a final concentration of 0.1 mg/ml. The solution was
. incubated at 37~e for one hour and then extracted vith an equal VOIWDe of
lai phanol:chloroform. The resu1ting aqueous phase vas made 0.2 M in
sodium acetate and precipated vith 2.5 volumes ethanol. The RNA
prec1pitate w •• redi.solved :lb 1 ml TB, 7 ml of dimethylsulfoxide (DtSO)
va. aclded and th. mixture vu heated at 45 oC for 20 minutes. At the, end of
o
o
r
o
." . - ... ~"
'32,
. " 1
the incubation period, the lNA va.·precipitated vith 2.5 volu... ethanol, :
vacuum dried and re.utpended PolyA· RNA Val tben .elected on
olilO-dT cellulo.e ~lumn •• )
3. Ilec$roa ..tcroacOpic L~natiaa of ID.fl ....... Vira
Influenza B/Lee/40 viru.e. vere examin.d bl infectir.a Hela cell. vith
the virus, obtaining thin sections of the cells and ... rchinl for pre.cnce
of virus vithin the thin sections. Alternatively, purified influenza
---.~ "'/
virions were negatively stained and examined under the eleétron micro.cope.
Influenza Virus-Infected HeLa Cells
Influenza B/Lee/40 vi~s was used 'to infect monolayer. of confluent ,.
lIeLa cells at a m.o.1. of 30-50 pfu/cell. At 12 h.p.1. the cella vere, . / fixed for one hour in 3% gluteraldehyde in PBS and then rinsed vith PBS.
The cells were scraped from the culture dishes and centrifuged at 5.000
r.p.m. for 5 minutes. Resulting pellets vere fixed with 1% o.mium 1
:tetroxide in Palade's Buffer, and'then rinsed vith Palade'. Buffer. Th.
: cells vere theQ dehydrated in a graded series of Acetone solution.
(SO%~100%). The cells ~ere then impregnated vith a Iraded .erie. rf y
epon/aceton solution.. Cells vere tmpregn.ted in an .pon/acetone
combin.tion at a ratio of 1:3 for one hour, then tran.ferrad ta a 1:1 " .
epon/acetooe combination for another hour, then ta a 3:1 epo~/.cetone
mixture, and'finally tmpregnated in a 100% epon for 3 hour •• They ver.
then placed in a vacuUII chamber at 20·C ta remove air. bubble., _bedcled in
2 CID boat. or block., and baked for tvo day. at 60·C.
GIa.. knives vere u.ed ta obtain thin .actions of the .pec~.
Inive. vere ~ut fram plate lIa.. (LIB Di.tri~or.) uaina a knife ~r _ / J . -.. chine (Type 7801A, LIB Diltributor.). GIa •• uive. vere, IIOUIIted on the
. 1
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o
33
HT-2 Ultramictotqme (Sorvall) and u.ed to .enerate thin sections.of
.pproximately 60-90 nm thickne •• (stlver reflection). ~i~ sections were
transferred to copper coated ,rid .. ··of 300 mesh Bize CJB EH Service) "and 1
Mtained in 4% uranyl acetate in 50% ethanol, a8 weIl as in Reynolds lead
citrate.
Pre-cleaned grids were coated with 0.5% formvar in
carbonized in a Kinney carbon evaporator to stabilize
Three microliters of a 1.6 x 1010 pfu/ml suspension 0
,~
o
\.
Acetate, and
dropped onto the Jrid and allowed to adsorb for five e The solution
was drawn off with filter paper, leaving only a on the "grid. The
film was allowed to dry before the grid was washed in dis by
quick immel~ions in drops of distilled water.
briefly onto filter paper to draw off exeess water. Afterwards, a drop of
1% phosphotungstic acid was plaeed on the grid, and ÜDDediately drawn off
'" ,vith f!-lter paper.
Blectro~Hicroscopes
The Philips, 300 and the Philips 201 electron microscopes vere used to
analyze the specimens. An electron !ma,e filiD was used with the Philips , . ~oo electron microscope, while a~fine.grain 35'mm film was use4 with the
Philip. 201.
4. PnparatiCIII of -IUI-I.II.,I_ ~ IRA
Gena.e RNA was iodinated by incubating approximately 10 ~g of RNA for
10 minute. at 60·C in 50 ~l of buffer containing 0.04 mM pot.ssium iodide
(1C1), ·0.1 H sodium aeetate pH 4.8, 10 mM th&llic chloride and' 1 mCi nUI.
~
'.
(
, \.
o
0-
--- - ------
" ./
34 '
, The reaction vas stopped by the addition of 2 ,JI Jl-mercaptoethanol. and th.
RNA vas precipitated with 2.5 voI11lD8 ethanoJ. at -20·C ana anaIyzed by .el o ,
electrophoresls (Fii. 10).
s. Hol.ecul.ar _ CloaiDa of Influeau B Viru-Speciflc Db
Tvo cloning strategies vere used to obtain cDMA cÎOQes specific to . ., '.
influenza B virus RNAs. These strategies are presented schamattcal.!y in , "
Figure 7 and are discus,ed at greater length belov.
A. Clon1!yr StratesY l '
(1) Cloning ,- Ribonuclease_ H Hethod . .
Purified influenza B virl:'s genome RNAs (vRNA) vere tailed- at their
J' -ends with pOlyadenyl).c acid using E: f2ll poly A' polymerase (Bethead.
Research Laboratories) (70). The polyadenylation rea~tion mixture
contained 50 mM Tris-HCl pH 7.9, 100 mM roBgnesium chloride (MgC1 2 ), 0.02 tnH
sodium thloride (NaCl), 0.5 mg/ml bovine serum album.!n (BSA), 5 ~g vRNA,
100 JJCi [a- 32PJrATP and 10 units poly.A pol~rase. A two minute reaction
was estimated by gel electrophoresis to add approxima.tely 30-50 adenyHc 1
acid residues to the 3' end of each genome RNA. Réactions were Ftopped by .
extraction vith an equal volume of phenol, then extracted .ga~ vith An
equal volume of chloroform and then precip"itated vith three volumes of
ethanol. "
The resulting polyadenylated influenza B vtrus-specific aenome RNA was
1 -
reverae transcribed into cDN! using rever.e tran.cripta.e and
qligodeox,.-thymidylic acid 12 ' 11 as primer. A typical reaction mixture
contained 50 mM Tri.-HCl pH 8.3, 42 mM Pota.sium chloride (XCI), 8 IIIH
} HaC12~ 0.4 mM dithiothrietol {D'M'), 0.2 mM aach of the four deoxynucleoUde
• 0
'- - -
~ ............ --------~.-------------------------------~---~~~~
c
c
'lJ
f •
Cl
• , ,'},
"
35
triphosphate. (dNTPs). 4 mM so6ium pyrophosphate and 50 units'of reverse~
transcriptase. The mixture vas incubated at J7°C for one hour and
extracted with an. equal volume of phenol and· then an equal volume of
chloroform fofloved 'by ethanol pre~ipttation. An aliquot of each reaction
mixture vas radioactively labeled and reserved for analysis on
polyacrylam,ide gel electrophoresis (PAGE) while t~e major portion vas
incubate~ in the absence of radiolabel. . .
Second s,trand cDNA synthesis was performed using the ribonuclease H .
, ". method of Gobler and Hoffman (25). Approximately 1:0 J,lg of reverse
transcripts still' attached to template RNA were used in a reaetion
cont~ining 20 mM Tris-HCl pH 7.5, 5 mM HgCl 2 • 10 mM ammonium sulphate
[(NHIo)2S0IoJ, 100 mM KCI, 0.15 mM ~-nicot.inamide adeqine dinucleotide
(~-NAD). 50 JJg/ml BSA., 40 ~ each of the four dNTPs, 8.5 units/ml !. coli
RNase H. 230 units/ml -f>NA polymerase 1. 'and 10 units/ml ~. ~ DNA 11gase.
The reaction mixture was incuba~ed at 12°C for one ho ur and then
transferred to 22°C for one hour. The .reaetion W8'S stopped by the ,addition
.of EDTA to a final concentration of 20 mM, then extracted with an equal
volumè of phenol and then ah equal volume of chloroform a~d precip!,tated
vith tpree ~ol~s of ethanol. 'An aliquot of the reaction mixture was
incubated in the presence of 2~ ~Ci [a- J2 P]dCTP and reserved for analysis
by PAGE. The major portion of the reaction was illcubated in the absence of.
radiolabel and the reaction products w~re desalted"on a Sephadex G-50
~olumn to ramave ùoincorporated triphosphates.
The resu~ting double-,tranded cDNAs (ds-cDNAB) were homopol,..r tailed
"
. vith deoxycytidylic acid residues. at their 3'-ends uaing terminal
deoxYnucleotidyl transfera,e CTdT) (47) in ~ 100 ~l reaction containing 0.2
mM dithiothrietol (DTT), 2S mH Tris-cacodylate pH 1.0. 0.1 H potassium .
cacodylate, 1 mM cobalt chloride (CoCl 2 ), 0~2 ma/ml !SA, 2 mM dCTP~ 50 ~Ci . r·
o.
o
o
, .....
36
[~-32PJdcTP and 5 units termin.l transfer.se (Bethe.da R •••• rch
Laboratories). The react!on vas carried out .t 37°C for 3 and 5 minute
time 'tntetvals. The reaction ~as stopped by incubation .t 'O·C for fiv. '
minutes to inactivate the terminal tr4Rsferase. Phenol and chlorofo~ , ' dl
1 l' \<t""" extractions vere then performed .folloved by èthanol precipitation':
Samples from the first and second strand s~thesis and, from-the
dC-tailing 'pf ds-cDNAs were analyzed on 2.8% polyacrylamide gel~ containina .,
8 H urea (Fig: 8). , ;
(ii) Plasmid Insertion and Transformation
DeOX~Cyt~~I-tai,led double, stranded -cONAs vere anne~~ed to Pst
I-linearized dG-tailed pBR322 ONA (Bethesda Research laboratories) in ••
hybridization bufler comprised- of 10 mM Tris pH 7.6, 1 mM EDTA; and 150 mH
NaCl. The reaction was incubated at 70 G C for 15 minutes l transferred
ÜMnediately to a 5loC water bath for two hours and then laft at room f
te~perature for an additiona! two hours. Resultirig recombinant plasmid • .
would "e lexpected to reconstitute new~ Î!! l sites a~ each end of thel
ins~rted cDNA sequences. These recombinant ONA molecules vere subsequently
used to transform the RRl strain of E, coli. , - r-The transformation buffer contained 10' mM Tris-HCI pH 8.1, 10 mM
ca~ ..... fum ch,loride (CaCi l ) and 10 mM magnesitpll chloride (HaCI1)' Recombinant .
\ ONA was resuspended in the transformation buffer .nd mix.d vith competent'
cells of the RRI strain of !. coli. As a control, .f!S I-linearized,
dG-tailed pBR322,was also taxen up in transformation buffer and u.ed to,
tr.nsfora competent RRI cells. The control reaction was u.ed to e.~imat.
the level of background tr.n.formation result!ng from contamlnatina
nonlin .. rlzed p~322 mol.cule.. The reaction. v.re cooled on le. for
thirty minutes and then he.t 'shocked for 90 seconds at 37°C. 'On. ml of
... ~
(
(
~-o,
~-... ~&T'
c'
Luri~ Broth (LB) ~a. t~added and the incubation ~a. continued at 37°C
for ~ne hour. ~e rea~tio~'produc~s vere,plated on LB ag~r containina 10
~g/ml tetra~y~line and i~cubated st ,37°C'for 24-48 hou;s.
Resultin8 colonie. were replica plated on both tett:acycl.ine-containing
LB agar and LB Agar containing 250 ~i/ml ampicillin in order ta select for~ \
, r , tet,raeyél1ne-resis~ant, ampioUlin-sensitive (Tet , Amp ) colonies. Such
colonies wete expected to contain virus-specific insert cDNAs.
'(tH) <Restriction'Analysis
Tetracycline-resistant and ampici~lin-sensitive (Te~r, Amps) colonies
were purified an~ their plasmid ~NA isolated. The plasmids were thep
analyzed for the len~th o~ insert DNA th~y'contained by Pst, 1 digestion . .
A rapid mini-prep technique .as used to i~olate plasmid DNA. Ten ml
ovemight bacterial cultures 8rown in LB containing 20 ~g/ml tetracycline
were pelleted and then resus'p~nded lin 0.8 ml of a Jysis buffer containing
50 mM Tris-HCl pH 8:1, 50 mM KOTA, 8% sucrose (w/v), and 5% Triton X-LOO.
Pifty ~l ,of a freshly prepared 10 mg/ml lysozyme solution was added. ~e
reaction vas left at room 'temperature for 15 miuutes and thèn boiled for 90
~econds at 100°C. They vere th en centrifuged at 14,000 g for 20 minutes , ,
and the supematants decanted into nev microcentrifuge .tubes ta which 1 ml
of isopropanol was added. The tubes ver~ th en cooled on dry ice for 5
minutes and centrifuged at 14,000 g for 10-15 minutes. The résulting
" " Bupematant. were discarde~ and the pellets were vacuum dried and then "
re.U8pended in 480 ~l of TB buffer. Ten ~l of a 1 mg/ml·RNase A solution 1
vas added and the tubes vere incubated at 37°C for 20 minutes. , .
Sub.equently, 10 ~l of a 10 mg/ml proteinase K solution was added, and the
tube. were incubated at 65°C for ten minutes. ~traction with phenol and .
th.n chloroform were ~hen perfor.med and the resulting DNA was precipitated
vith ethanol.
-0
o '. '
o
. '
. ,
. .. 38
. ' Restriction endonuclease analysis wa. carried out uing, the enzyme l!!
1. A typical reaction in a 100 ~l reaction volUme contained: 50 mM NaCI, .
10 mM HgC1 2 , 1 mM DTT, 100 ~g/ml BSA, '25 mM Tris-HCI (pH 7.8), • approximately 2 ~g DRA 'and 8 units E!! I. The,reactions wer~ incubatad at
1
37°C for two hours, the DN! was extracted wit~ phenol and then chloroform ,',
and ethanol precipitated. The pellet was resuspended in agarose gel . -'1
loading ~uffer.(30% glycerol, 0.25% bromophenol blue,.0.25%-xylene cyanol)
and analyzed on 1% agarose gels (Fig. 9).
B. Clonina Straten II \ - .
(i) Hyb~idization of cDNAs 'Separately Derived from Virus-
Specifie Genome RNA and mRNA to Yield ds-cDNAs ' ~
Influenza B~virus genome RNA was purifièd, polyadenylated and reverse i" _ ,
transcribed into cDNA as previously described for Cloning Strategy 1 (see
above). After reverse transcription, ho~ever~ the RNA template was
hydrolyzed .in 1.6 N sodium hydroxide (NaOH) and 0.5 H &DTA at 70°C for 30 1 •
/
minutes. -After neQtralization by addition of an equal volume of 1.6 H 7
- sodium acetate (pH 5.0), the cDN! was phenol extracted and separated from
unincorpo~ated dNTPs (desalted) on a Sephadex G-50 column. Hessenger RNA
(mRNA) from influ~za virus infected Hela cells was similarly reverse
transcribed, template-hydrolized, phenol ext~acted and desalted. cDMAs
derived from gertome RNA and cDMAs derived trom miNA wer. tailed at thelr ... ..tl J •
JI-end with deoxycytidylic acid residues using TdT in separa te reactions. \ .
The raaction conditions were similar to the terminal tranafera.e r.action
performed for 'Clonin'g Strategy l as described above. The full-lenath, . - ,
deoxycytidylic acid-t'aUed (+) and (- )-stranded cDNA molecule.
corresponding to genome RNA segments l, 2 and 3 were selected by
,/'
(
, \
o
," 39
Q
.lectrophor.si.4n 4% polyacrylamide sels containin~ ~ H urea, eluted'from ,
th. S_l., and hybrtdiJ:ed one to another. to form double-stranded cDNAs " '\
, "
(U) Plasmid In.artion pnd Transformation (. 1
1 1
'The ds-c~ vere annealed to Pst I-cut, , , \
deoxyg~nidylic acid-tailed . \ .
pBR322 DNA J. BetheBda Research Laborat~ries)., The resultins recombinant 1
mol.cul.s vere u.ed to transform,g. E!!!. strain RRI. The annealins and " ""!,,.
transf4rmatlon conditions are the s&me as described àbove for Clonins
..stratelY I.
(iU) . Restriction Analysis
B f t i ti d 1 1 i ç d T' t r Amps e ore res r c on en onuC ease ana ys s vas per~orme on è ,
colonifils, colony "cracking tl was used ta si~e the plasmid DNA ta find out
whether or not colonieS contained inserts. !bat is, whole colonies were
disrupted and analy'zed by gel el-tctrophoresis. The "cracking" bll:ffer
contained 50 mH NaOH, d.5J SDS, 5 mM ROTA and 0.025% bromocresol green.
Sterile toothpicks vere used ta Bcrape bacterial colonies from àgar ~ ,"
plates and transfer them t~ mi;rocentrifuse tubes containing 75 ~l of
craëking buffer (50). The colonies were ~cerated asainst the wall of the
tube usina the toothpiek. The tubes vere immediately'vortexed, for five . '.
minut .. , l.ft at room temperature fôr 30 minutes, centrifuged at !4,OOO x g
for 5 minut •• , and 50 ~l of the resulting supernatants were analyzed on a
1% a,arose ,el. >
. R •• triction .ndonuel .... analy.t. vas perfo~d with a vari.ty of
. enayme.. The diaestion conditions ver. tho •• recommended by the
aanufacturer ••
. "
r .
o
. '
o
·0
.1
40
.h
Purification of Plasmid DNA: Kaxiprep . .. T~ ml of an overnight broth culture containing the bacterial clone of
", interest vas inoculated into one liter of LB containing 20 ~l/ml-
tetraéy~line and plaèed in 'a 37°C sha~ing incubator. A spactronic 20
spectrophotometer (Bausch and Lamb) ~as used_~o monitor,the optlcal ,
absorbance. of the culture at 660 nm wâvelength (OD"o)" When the cultura
attained an OD reading of-0.4 to 0.5, 100 ~g/ml chlàramp~enicol was added
for plasmid amplification and the culture was incubated overnight.
The r~sultinB bacteria were pelleted by centrifugation at 5000 rpm ~~r
ten mlnutes. The cells from \ liter of broth culture were resuspended in
20 ml: of a splutiolil of 25% sUfrose (w/,v) in 50 mM Tris-Hel pH 8.0 and' 4 ml , '
. ~f freshly prepared 10 mg/ml lysozyme was added. The ,cells were le..f,t on
ice for 5 minutes. Four ml., of 0.5 H EDTA were a~ded and the mixtur-e was
left on lce for 5 minutes. Subsequently, 32 ml of Triton lysis mixture (50 . ,
mM Tris-HCI pH 8.0,62.5 mM EDTA, 0.4% Triton X-100)'wete added and the
, mixtur~ was incubated on ice for .ten minu~ Bacterial cell walls were , •. "1
pelleted, in. a Bec1c:mfn Ti6~ rot0.,r 'for one hour at 40,000 x g. ' The ' . \ ,. . .:"
supernatant was saved and weighed'. Polyethylene glycol (PlG) 8000 and 5 H
sodium chloride were ea~h added to the supernatant in an amount.eG\U&l' t~
10% of the ~ss of the sup~rnitant.' . Th~ mixture was shaken to diss\lve the
PEG and sodium chloride and then le ft at 4°C overnight.
The re~ltina PEG pre~ipitate was centrifuged at 5,000 x g for 15
minutes and the supernatant discarded. Twenty-eight ml of IX SSC (150 mM 1
NaCI, 15 mM sodium citrate) was added to dissolve the pellet ~nd then 26
grams.of cesium chlôrid~ was added. The mixture wa. centrifused at 71 for
15 minutes. Tha. ftlpematant was mixed vith 2 ml of a 2 mg/ml' athidt um
bromide .olution ~d the re.ult!n. material vas ~an~rifuled at 40,000 rpa
in a Backman T14Q rotor for 48 hours until i.opycnic equilibrium was J>
reached. .
, "
- 1
1 •
(
41
Banded pl~ .. id DNA va. collected by puncturing the tubas with a
n •• dle: Bthidiua bromide vas' removed by extraction with isoamyl alcohol
and the DNA preclpitated with ethanol. This cesium chloride gradient
centrifusation ~as repeated a second time after which the DNA was dialyzed
overnight' to remove residuB:l, cesium chloride.
(v) Purification of Pl.smid Insert DNA by Agarose Gel:
Electrophoresis
The purified plasmid DNA vas digested vith 1!! 1 and electrophoresed
on a 1% agarose gel in-IX TBE buffer (100 mM Tris-borate, 2 mM KOTA). ~ \
Aftèr ethidium bfomide staining, a gel sliee containing the insert pNA vas 'Il
visualized under UV illumination. cut out o'f the gel and placed into,
dialysis t~bin8 containins a small amount of IX TBE vith 5 ~g/ml BSA. The
tubins-vas clamped at both ends and immersed in a shallov iayer of IX ,TBE.
Blectric current vas passed through the buffer for one hour at ,100 voles ~
during which timethe DNA migrated out of the sel fragment and onto the . . ' wall of the dialysis tubing. The polarity of the 'cu~rent was then reversed
. for 2.minutes to releas~ the DNA from the~wall of the tubing into the
buffer. The buffer containins the DNA was subjected to extraction first
with phenol and then with a 24:1 (v/v) mixture of chloroform:isoamyl
alcohol. The DNA vas ethanol precipitated and further p~rified using
Blutip ion ~xchange chromatosraphy columns"(Schlei~h8r and Schuell).
(vi) End-Labeli"1 and Strand Separation of Insert DNA
Th. pur!fied in.ert DNA w •• dise.ted with appropriate.restriction ,
*Dz,.. •• Prior'to end-labeling. S'-te~ln.l phosphate groups were removed
fra.'tbe DNA by bact~ri.l alkal!ne pho.phat •• e (BAP) in a reaction
containins 0.05 fi Tris (pH 8 •. 1)~ :0.01 li HgC1 2 and 200 units MP. The
l o
o
o
\ , kf 42
" ;.,'
reaetion vas ineubated at 37·C for half an hour, transferred to 6S·C for ; 1 ~;:~,."
one hour, extraeted.vith phenol and ehloroform and ethanol precipitated.
The dephosp~orylated DNA vas 5' end-labeled in a kinasinl re~ction . ~
containinl 50 mM Tris-HCl pH 9.~, 10 mM MgCI 2 , 5 mM DTT, 0.1 mM spermidine-
-BCI, 0.1 mM !OTA, 200 ~Ci Ilamma(y)- J2 P]-ATP, 10 units T4 polynucleotide
kinàse, and" 1-10 ~g DNA. The reactlon was incubated at 37°C for one hour
after-vhich the DNA vas pnenol extraeted and ethanol precipit~ted. The
labeled DNA vas analyzed on 4% polyaerylamide gels. Gel slices containin,
desired end-labeled restriction fragments were cut out of the leI and the
,DNA was eluted from the gel by crushing and overnight 1umersion in "cru.h
and soak buffer" (500 mM ammonium aeetate, 10 mM mAgnesium Acetate, 1 mM
KOTA, 0.1% lw/v] SDS). After ~~ntrifugation, the acrylamide gel piece.
vere pelleted. DNA-containing supernatants were collected and ethanol \ .
precipitated. Th&DNA pellets were resuspended in denaturation buffer (1
mM EDTA, 50 mM NaOH, -7% sucrose, 0.05% [v/vJ bromophenol blue, 0.05% [w/v)
xylene cyanol) and heated at BO°C for ten minutes ta denature the double
stranded DNA. The DNA was immediately ~ooled on ice ana lo,ded on a 5%
TBE-buffered nondenatuz:ing aerylamide gel that had baen pre-chille'd
overnight at 4°C. Eleetrophoresis vas carried out at 300 volts overntaht 1. , \> ,
"to separate s!nkle-stranded DNA species vhich wer~ detected by ,
autoradiography and eluted from the' ,els as described previously.~
(vii) DNA Sequence Determination
DNA sequeneing was performed by the èhemical el .. va,e method of Max ..
and Gilbert (51). A series of ba •• -specifie ehemical cle.va, •• of DNA i.
central to thi. method. Dimetpyl .ulfate, formic .eid, alkali, and 1
hydra.ine Ar. uaed to modify purine and pyrimidine ~ ••• re.pectively at ,
....... --j
1,
, " L 1. ~.
o
43
~ specifie points of the DNA. Piperidine vas subsequently used ta remove the , ,
modlfied ba.,s trom the ribo-phoaphate baekbone and cause ~-elimin4tion of :; '\'
l' phosphate. from sugars causing ~NA strands ta break .t those points.
Pive base~speeific reaction. vere performed. The reaction, for
modification of guanine (G) residues eontained 200 ~l dimethyl, s~lfate -
(DHS) buffêr (50 mM sodium cacodylate pH 8.0, 19 mM KgC1 2• -1 mM EDTA), S'pl
[ '2PJDNA, and 1 pl DHS. The reaction ~as incubated at 20°C for ~ minutes t
and .topped vith 50 pl DHs stop bufler (1.5 K sodium aeetate pH i.o, 1.0 K
mereaptoothanol, 100 ~Blmi tRNA). Dimethyl sulfate modifies guanine
residues by ~thylating the ]-ni.trogen of guanine. Methylation of the . . nUrogen. disrupts the electronie structure around the guanine "and on , ,
addition of piperidi·ne, the 7-methylguan~ne iso-pened. lormic acid was
used ta westen Adenine and guanine glycosidic bonds by protonation of
purine ri~g nitrogens in a G + A reaction containing 10 pl water, 10 pl
[J2PJDNA and 50 pl 88% formic acid (51): The reaction wa~ !ncubated at t
-SC for ~O minutes and stopped by addition of 250 ~ of stop buffer (0.3 K
a ,cetate pH 7.0, 0.1 K !OTA, 25 ~8/ml t~ carrier). In ~ one-step base- _
sp ifie ohemiea! cleavage .. alkal! was used ta open Adenine and cytosine
ring- in an 'A + C reacUon. The reacUon /as composed of 5 pl [3 2PJ DNA
• and 100 pl AC butter (1.2 N NaOH, 1 mM EDT~", A,7-minute incubation was
earried out at 90·C and the reactton vas terminated vith 150 'pl of 1 N
Ilaeia1 acetic acid. Hydraz~ne vas used in a T + C and in a C reaction ta
open the" thymine and cytosine ring_ respectively.. The C reacUon was .'
carried out in th. pre.ence of .0dium·chloride.(NaCI) ta suppress the~,
ruetion so that only C would ruct appreciably. The ~ "+ C reaetion ' o
eontarned 10 ~l'vater, 10' pl [ i2plDNA and 30 pl hydrazine. Incubation wa. 1
e: for tan .1nute.:at .20·C and va_ te~nat.d by hydrazine stôp buffer (O.~ K
, _odi~ aeetate pH 7.0, 0.1 mM EDTA. 25 PJ/ml tRNA). The C re3ction
contained similar components but contained 5 H NaCl· instead of water.
o
j
, ,
o ·
44
lollowins the base modification r .. ctions de.cribed above, thr ••
volume. of cold ethanol vere added to chilI and preeipitat. the DNA. A
's~cond ethanC?l prect,htation freed the »NA frOli re.idual bas.-lIOdificaUon
reasents and a final ~tbanol rin.e of the pellet removed exc ••• sodium
Acetate. The pellets vere vacuUII clried and re.uspended in 100 J.ll of 1 M
piperidine in order to'displace aIl rinl-opened bases from th.ir luaars and
catalyze the.~-elimination of phosphates from the empty susars, thus ,
cleavins the DNA (51). The piperidine-containing tubes vere heated at 90·C
for half an hour and then successive lyophilizations were performed to
remove the piperidlne. Thus, the original end-labeled DNA was cleaved in
separate reactions as follovs: at guanines (G), atMboth guanine and 1
adenine residues (G+A), at both adenine and cytosine residues (A+C). at
both pytosines. and thymines (C+T), and at cytosine resid~es (C) •. The 0'
nested set of cleavage products senerated in this manner vere taken up in
sequencing gel loadins buffer (80% [v/v] de-ionized formamide, IX'TBE [pH
8.3], 1 mM !DTA, 0.1% lw/v] xylene cyanol, 0.1% [v/v] bromophenol blue),
heat-denatured and analyze~ on etther 8% or 20% polyacrylamide sequencing
sels containing 8 M urea. 1
(vUi) Computer-Assisted Analysis . The DNA and protein sequence analysis progr~ of -J)evereux !1 al (22)
1
and Postell (International Biotechn~logies Inc.) vere used, re.pectively,
on the V~ comp~ter and the IBM-PC computer. Relative hydrophobicity va.
calculated by the prosram of Kyte and ~olittle (43).
-
... ' , ~
-
III. USUL'lS
1. Cloafec Stratgy 1
" ,
4S '?
•
Two strategie. vere used to' perfor.m molecular cloning of tofluenza B
virus-specifie RNA (39). 80th of these strategies are outlined in Figure , " .
In Cloning Strategy 1. !. S2!! poly A polymerase was used to add 30-50
adenylic acid res!dues to the 3 ' -ends of the influenza B virus genome RNA
s.amants as described in Materials and Hethods. This polyadenylated g800me
-RNA va. reverse transcribed into cDNA using reverse transcriptase and 1
oli,o~dT12'1' a. primer. (a32Pl dCTP vas used to radiolabel an aliquot of
the first strand synthes!s reaction. Incorporation of radioactive
deoxycytidine residues allowed uniform internaI labeling of the cDNA such
that vhen analyzed on a denaturin, gel. cDN!s corresponding tu the RNA
segments could be visualized by autoradiography. cDN!s visualized in this
manner are sean in lanes 1 and 3 of Figure 8.
Second strand synthe,sis vas performed by the RNase H method of Gubler
and Boffun (25). The enzymes ribonuclease H (RNase H). the Xlenow
fraJm8At of DN! polymerase land 1. ~ DN! lisase were used together to
synth.d"z. the second strand cDNA. RNase H introduces nicks in, the RNA
t8lll~late of th. geD0mt. RNA: cDNA hybrids resulUng fram first s-trand ,
.ynth •• i. and thus create. multiple RNA primers (see Fig. 7). These
primer. are elon,ated by DN! polymerase. fillin, the gaps created by RNase
H. Liaa •••• als remaining nick. and the result is complete double-stranded
cDNA. An aliquot of th •• econd strand reaction mixture containing
nonradioactive firat strand cDNA va. radioactively labeled vith [œ- J2 P)dCTP
and analyzed along.ide first strand cDNA on a denaturing 2.8%
polyacrylamide gel. This is shown in Figure 8t lane 2.
r 46 -,
..
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;',j~:,
-: .c..4 . , • . ,
.. , o ,
•
• Figure S: Electron lIiC'roaragb' ot-Lnfluenza vi~ particls. HeLa eelb ,.-'
...
;.... , ,/ , ~;t' ~ ...... ".
vetre infected vith influenza virus and hàrve.ted at 12 houri
post infection (h.p.i.). Magnifieation 416,666X. RNP,
ribonucleoprotein; L, lipid bilayer; S, spike projections; PM, \
plasma membrane; ER, endoplaSllic reticulum •
~.
•
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.; ,
1'· r- i' f.
;;....-- #
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L~ v ...... .J r ~ ) 1
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48
"
, FiÎl!re 6:
. 1 .'
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,
Infl~nza virions, negatrvely staineo vith 1% pot •• ~~
phosphotungstiê acid ('P.!'A). These virus partieles vare , ,
calculated to have, a 'èHamet~r, ranging fram 119 nm to 130 nm,. ., .. ~ ...
vhieh ls similar to the 80-,120,nm diameter of influenza virus ....
as determined by o~hers [45,49,66). Hagnificati~n 156,800X. St
spike projections; L, lipid bilayer.· ....
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~III,
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Figure 7: ,~cheme "'?f the two cl~ning strategies used to obtain cP~ clones
specifie to influenza B virus RNAs. Both Clon~ng Strategy land
Cloning "Strategy II are desc'ribed in the Materials and Hethoda •
section.
, -
\ .
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..
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• ~.
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CLON'N0 2
"yAMA 3' ~ 'Pol A .
• PoI~~'" ,'MAA _,s'
. 1 OIlgo dT12-" A • t A• verN "anacriptaa;e
cONA S'TTTT 1 ITflrm'n.'
. ,T,lIn.f.r •••
$'TTTT __ ----CCCC y
; 1 1 .' IIi ·IT.rmlnel tTraNt.ra ••
'L-...... GOGO III • 1 GGGa
TT-~ .-cONA TT eccc
CCCC~ 1 • 1 1 ':1-TTT
. ,.'~ ~CCCC TT1Tt
1T.'~lnel - 1)ansf.r ...
CONA:t .~"V , Tm 5'
+OIIaO. d1,2-18 1 R.T •
-' 51
) , 1
, \
CLONING 1
,"---_ s' <.,aHA·
)'AAAA ____ s,
,Qflgo f'12-18 • R.T.
-, TTTT' • 3' cONA YAAA.A_ •• _,_ 6' VRtM.Hybr'd
If AN ••• H ~ ...DNA "'"
E.coll Uva •• '
S'TTTT 3' YMA"] 1 , 1 , 5' d,-cONA
\ . f.T.rmlna •
tTranat.,. ..
ecce 3' 1 1 1 1 .5' ~,
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o
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52
Approximately 15-20 deoxycytidy1yl residues ,were then added at the,
3 1 -ends,of the double stranded cDNAs using terminal ~eoxynucl.oti~yl
transferase (TdT) in 2, 3 and 5 minutes tailing reactions. Radioactiv. . ' ~
aliquots of these reactions are also ~hown\in Figure.S. , \
Deoxycytidylic acid-tailed double stranded cDNAs were then .~nealed to
pBR32~ DNA previously digested with the restriction enzyme P.t 1 and tailed ! '
with deoxyguanosine residues. The resulting recombinant DNA molecules vere
used ta transform the RRI strain of~. coli. Since th!. clon!ng strategy 1
employe3 purified genome ~ from virions in the presume~ absence of Any
host.-derived RNA spedes" Any resulting tetracycline-~esistant ampicillin;'
sensitive colonies should have contained virus-specifie cloned cDN!
species. Tvo hundred and fifty tetracycline-resistant, ampic!llin-
gensitive colonies vere initially obtained. Plasmid DNA from these. clanes
wâs ananlyzed for length of insert DNA_J?Y E!1 '1 digestion and gel
'electrophor~sis (Fig~ 9). Some of the clones could be seen to contain DNA
inserts ranging in size from 300 to approximately 1900 base pairs. These ,
.clones are p!esently being analyzed for p01ylIM!ra$e gene--specific inserts.
2. Clonina Stra$eu II
" '
Si'nce the genome RNA segments encoding the influenza B virus
polymerase ~roteins have been estimated to be 2.3'to ~.5 lb in lenBt~,
Cloning Str.t~gy I_did not provide full-length cDNA clones of thes. ,.n.s.
Menee, an alternate cloning strategy was employed involving gel selection
for full-length cDNA copies of polymerase gen •••
Influenza B virus genome RNA waS again polyadenylated and rev.r ••
transcribed into cDNA usina reverse transcripta •• and olilod.oxyth~idylic
acid 12 0 11 as a primer. 'Total p01y A-containing RNA from influenza B virus-
l
53
, .
/.
\,
'.lIure 8-. Clo~ing StratelY 1. - Influenza B/LeeL'O yi nlS genome RNA was
polyadsnYlatsd and reverse transcri~d into çDNA. The resulting
eDNA-RNA hybrids were ~ed for s~cond strand Synthes~ by the
-'
RN .. s B _thod of GubIer and Hoffman (25). L4lIe 1 and 3: first 1 ~ \ "'.
strand cDNA. Lape 2: second strand cDNA. Deoxycytidylic actd
re.idues were subsequen~,iy ad~éd -ta the 3' ends of double
stranded cDNA i~ a 37minute (J') and 5-minute (S') tailing
reaction using TdT. Lanes Rsa 1 and ~ 1 are marker lanes
~ cont.inins DNA segments of the lengths notèd in n~cleotide .. base
pairs. Kolecular species were analy~ed on a 2.8% polyacrylamide
sel containing 6 H urea.
"
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.'.
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0 ...
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1
.... 1 1 2 3 HInf .. 1 • 1 t t ,
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de -1'tIII!!I
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54
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Ftaure 9: l!1 1 digest'ions of soma ?f the recombinant plasmid DNAs
, "
re8ulting from Cloning Strategy' l. Lanes'Rsa l, Pst I and Hinf - - -. • lIJ ..
1 conta~ size markers of pBR322 DRA digested with these , reat't-iction endonucleases' to give DNA fragments of the sizes - ,
noted in nucleotide base pairs. ~ 1 digestion linearizes the
pBR322 D~ into a single specles of DNA 4362 base pairs in
length. Lanes 1-10 represent DNA from different bacterial
clones. DNAs were analyzed on a 1~ TBI-buffered agarose gel •
...
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56
., "
"." ~ . ...
..
...
IIIWI"'!,II!".III!': "'3 --~--"""'II'!4,_'"'!'t-."II!*"';o"'Jj!'l!1!"')'''II;;!".!Ié''''",1II'!4.'''': '!'!9 .. ;;>W4M!!III. ~.~:"I!:._~t 4t!'ll!,.;o"~."~?;:r, ~~tll"l.<"'IQI"""-'r.II"!.~I,"'-';1r!'p;:-r,,\T.'"'· ":"I\~~:r:;--,;:r;7'!",~'"""':.,,., .. ~r:.~~--;, ~-,-- ~ .. ---;--;- \.. ..
1
c'
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57
i1~fected cells vas similarly transcribed into cDNA. RNA templates were.
r~ve~ by alkaline hYdrOlY~iS and'then appro~mately 15-20 deoxycytidytic
acia residues vere added t~the 3'-ends of both the (+) and (-) sense cDNA
copies in separate reactions using TdT. The results of this are shown in 1
ligure 10. The ~af1es designated "Ait represent internally labeled reverse
transcripts derived separately from, influenza B virus senomé RNA segments
and influenza B virus-specUic mRNA. The lanes marked "B': represent the
products of deoxycytidylic acid (dC)-tailing of such reverse transcripts. \ . ,
lull length dC-tailed cDNAs corresponding, respectively, to 8enome RNA and
.. to mRNA specifie to 8enome RNA segments 1, 2 and 3 were selected by
electrophoresis on 4% polyacrylamide gels containing 6H urea, eluted from
the gels, and then hybridized to form aouble-stranded dC-tailed cDNAs
(d~.-cDNA) •
These ds-cDNAs were hybr~dized to Pst I-cut deoxyguanidylic
acid-tailed pBR322 DNA (Bethesda ~esearch Laboratories) and the resulting .;;:1,.:
recombinant molecules were used to transform 1. ~ strain RRl. Because
of the cloning strategy employed (synthesi% of one strand of the ds-cDNA
from purified virus genome RNA which was essentially free of contaminating "
nonviral species), aIl, tetracycline-resistant ampicillin-sensitive colonies
were ex~ecte~;' be inf~uenza B virus-specifie. Nine such colonies were
obtained. The colonies were iniUally analyzed by colony "cracking" to
roughly estimate insert length. The result is shown in Figure Il. Two
bacterial colonies appeared to carry plasmids with large ins~rts. These '1 '
~ vere designated pBP4 and pBP7. Plasmid'DNA was digested with ~ l to
excise the insert DNA sequences from pBR322 sequences. The lengths of the
insart DNA. wera analyzed by electrophoresis on a 1% agarose gel (lig. 12).
Influenza B virus pol)zmarase genes have previously baen estimated to he
2.2-2.5 lb in 1anath. Dis •• tion of pBP4 with Pst l generated two insert-
~
l·
"
58 '
l'.
. , ,
o
Pigure 10: 'Cloning Strategy II. Ana1ysis' of cDNAs synthèsized u8ing
. ') -
, . - 0 '
gen~me RNA (vRNA) and messenger RNA (mRNA) s$parate1y as ,
. templates' and .... deoxycytidylic acid-tailing of the resultin.
reverse transcripts. Lanes A: internally, labeled cDNA. Lanas
B: Sing1e-stranded cDNAs tai1ed for 2 minutes with
deoxycytidy1ic acid restdues using TdT and radiolabeled on1y in
the hom9polymer taila. [ 12511 vRNA: purified genoms RNA
segments internally~labeled at cytosine residue~ (8) and used
as molecular weight marke,rs. Holecular species were analyzed "
on a 2.8% polyacry~amide gel containing 6\" ure •• ~ . ,
, , ,
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• " '.
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59
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60
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-Pigure Il: CloninB StrateBY II. Analysis of,recombinant coloni •• b,'
colony t'dràeking" to estimate in.ert length. Pl ... Did. pr •• umed
..
9
- ' to conta in long inserts are present in,lane 4 (pBP4) and lan.
10 (pBP7). Plasmid DNAs vere analyzed on a 1% TBI-buff.red
agaros. gel.
, J • .:......
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P,st. 1 digesiion of pBP4 'and pBP7 DNA4 ,Lanes fUrked Âv. .nd Alu ..-- -1
, "
!l'he higl't molecular weight speciès (>1746 base , .
~'pairs) in i~es 4"~nd ? represent linearized pBR322 DNA. The;:
.' , ,
" '
" , t , r
two 'DNA fraaments resulting from Pst 1 di8~stion of 'the pBP4 '" • 1 f .. ~ ~ • , ,
1-
insert DNA have beeit ~~signated 4A and 45.
analyzed on '4' 1,% ~E-ag~rose' gel. '
. , , ,
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655 521 0
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64
derived bands, corresponding to a total insert length of approximately 2300
base pairs with ,an internaI Pst 1 site. The insert DNA of p8P7 app~ared to
be sli~htly shorte~. Plasmid,p8P4 was selacted for further analysi ••
Preliminary restriction enzyme analysi. of pBP4 DNA wa. perfo~d with . ,
a number of restriction endonucleases. Enzyme. which diga.te~ the in.ert ,
DNA into fraamants of moderate si~e (300-600 base pairs) ware .elected for
sequencing. Figure 13 shows DNA fragments generated by the restriction '-
enzymes Dde 1 and H!n! l, while an overall sequencing strategy, includina
~ the experimentally-derived partial endonuc1ease cleavale map of the p8P4
insert DNA is shown in Figure 14.
pBP4 DNA was purified by cesium chloride gradient centrifugation and
digested with Pst 1 to excise the Insert DNA. The resulting E!l 1
restriction fragments 4A and 4B (Figure 12) were separated from pBR322 DNA
by agarose gel electrophoresis and eluted from gel slices. The resultina
insert DNA fragments were redigested with the enzymes outlined ,in Figure
14, and then 5' end-Iabeled us!ng T4 polynucleotide kinase and [y-,S2P1ATP.
The resulting DNA fragments were radiolabeled at both S' ends. These
5' -ends were 'segregated by alkaline denaturation and electrophoretic
separation of the resu~ing slngle-stranded species on non-denaturing 5%
polyacrylamide gels. A typical result is shown in Figure 15. In thi.
figure, insert DNA Pst 1 restriction fragments 4A and 4B have been - ,
redigested with restriction endonuclease ~ 1. The largast DNA specie.
generated when fragment 4A is cut with ~ 1 (dedgnated Al in Fig. 15) ha.
partially separated into two sirtgle-stranded DNA special (dasignated AIA
and AIB). However, the denat~ration and relulting •• paration into .lna1e
stranded ( •• ) species was not usually complete. ~ome faster-mlsratina
double-.tranded (d.) specie. (de.isnatad Ale for the Al 'traament) c.an be
seen. It la likely that the single-.tranded species partially renatured
, 65 \
, .
,Piaure 13: Restriction en~onuclease analysi. of pBP4 pla_id rand insert
»NA. Lane. Al and BS contain marker DNA segment. of the
len,ths noted in nucleotide ba.e pairs.' Lane A2: total pBP4
»NA dilest~d vith Hinf 1. Lane A3: purified f!S l digestion,
fraamant 4B of the pBP4 DNA (Fig. 12) redigested vith Hinf 1.
Lane'A4: purified E!! l fragment 4A redisested with ~ l.
Lane BI: Pst l fragment 4A redigested with Dde l. Lana B2:
~ l fragme~t 4B redisested vith Dde 1.' Lane B3: total pBP4
, DNA 'disestad vith Dde 1. Land B4: total pBP4 DNA digested
vith.~ l and Pst 1. Pragments were analyzed on 4%
polyacrylamide gels.
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,
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1652
~~. ~~8
- '.
66
67'
( ,
Figure 14: Partial "Heavage map and sequencing strategy i pBP4 D~. Experimentally derived restriction endonucleas cleavage sites
are repr~~ented as small upright arrows along the length of the
cloned insert DNA. The direction of mRNA transcription ts from
left to right. Nucleotide l corresponds ta .the 3' -terminal
nucleotide of the virion genome RNA segment. "The zigzag at
each end represents G-C linker regions. Short upright bar~
bracketing horizontal arrows delineate restriction fragments
used for sequencing. The foot of each arrow represents the
t~striction site labeled with [y-S2plATP and polynucleotide
kinase. The length of each arrow represe'nts the amount of
sequence information'obtained from each restriction fragment •
•
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0 %
--- ----ëJ .. !x:E~ 'z
.'
1 1 1 1 oz:Ea.
---
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--
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Pi,ure 15: Panel A: Blectrophoresis of alkaline-denatured pBP4 DNA Pst
I-derived after redigestion with Hinf I. AlA~and AIB represent
the separated strands of the larBest fragment (Al) r~sulting
from Hinf l digestion of the ~arBer fragment (A) resulting from
Pst 1 digestion of the pBP4 insert DNA. Ale represents
renatured Al. Other pBP4 Pst 1, Hinf I-derived fragments are
labeled accordinBly. Strands were sepa~ated on a
, non-denaturing 5% polyacrylamide gel. ..
1
Panels Band C: , Nucleotide sequence analysis of strands A2A and A2B from panel
A. Sequence l'adders for each strand were analyzed in separate \
loadings on a 20% sequencing gel, an 8% gel run for
approximately 4500 volt hours (short run), and lm 8% Bel run .
for approximately 13,000 volt hours (iong run). Str~nd A2B
represent~ one terminus of the insert DNA as evidenced by the
,pr •• ence çf a poly-G tract.
... ~,
0
0,
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r 70
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o " :;:tIr
.... '." ot.
-....... -...... - :a-,-,
L -... - F-
i> .... - ... 4
'. ~: Ik· • ,,"
. '. .. .-
.. .. .~ "'~
7 ..
.,
0'
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71
durin. elect~ophoresls. Such 'renaturation occured to varying degr~es with 1
.. ch DNA .pecies. The single-stranded species were eluted fro~ the
polyacrylamide gel. ,
More than 95% of each strand of the pBP4 insert DNA wa~ sequenced by
the chemical cleava.e method of Kaxam and Gilbert (51). AlI restriction
.ite. used for end-Iabelin. vere overlapped by sequence analysis of DNA 1
fraamants eut with different restttction enzymes (Fig. 14). The short
regiows of sequen~~ which were ~etermined on only one strand were sequenced
on at least two different occasions and no ambiguities were seen in the
sequencins gels representing these regions. The internaI Pst l site within
the ,insert DNA was overlapped by sequence analysis of a restriction
fragment derived by Kbo II digestion of total pBP4 plasmid DNA which had
not ~en digested with E!! I.
Autoradiograms of typical sequencina gels are shown in Figure IS. A
single 36 cm sequencing gel run cannot resolve more than approximately 100
bases of a sequencing ladder. Three gel runs are therefore employed: a
20% gel run capable of resolving the first 50 nucleotides, Jan 8% gel run
that has been run for approximately 4500 Volt' hours, and flnallY another 8% , 1
gel run for approximately 13,000 volt hours. The top of the readable
sequence ladder of each gel overlaps the bot tom of the next such that for a
'aivan DN4 fragment (such as A2) sequence info~tion of approximately
300-400 nucieotide. can be read from the S'-labeled end for each DNA strand
( •• a. A2A and AlB). A. previously mentioned, both strands were sequençed,
so that the sequence ~btained for one .trand cou Id partially or completely 1
coapl...nt the •• quence obtained for the other strand • •
, Initial .equence an.lysis of the pBP4 insert DN! revealed end
sequenc.. that matched known 3' - and S' -end .equence. of influenza B virus
genome RNA segments as determined by direct RNA sequenci~j (21). Thi. wa.
•
o
•
72
a direct confirmation that the pBP4 DNA vas bath influenza B viru.-specific
and a complete representation of a genome RNA segment. The complete
nucleotide sequence of the pBP4 insert DNA is shown in ligure 16. Thirteen
additional nucleotides vere found betveen the nucle~tide correspondinl ta
the 3'-end of the virion RNA segment and the G-C linker region. Thase are
presumably nonviral sequences derived from the S'-end of an influenza ~
vhich had acquired them from a cellular miNA during the transcription
initiation process of virus mRNA synthesis (42).
The genome RNA segment from vhich the pBP4 insert DNA was derived'i.
2368 nucleotides long. It has a 5'-noncoding region of 20 nucleotides
followed by a single long open protein-coding reading frame. This open
reading frame extends from the first possible initiation codon for protein
, synthesi. at nucleotides 21-23 ta a termination codon at nucleotides
2277-2279. This region i5 capable of coding for a protein of 752 amino
acids including the initiating methionine with a calculated molecular ,
weight of 84,407 daltons. A tract of 6 Adenine residues found at
nucleotides 2347-2352 by analogy to influenza A viruses (71) probably
represents the polyadenylation site of the mRNA.
The size of the genome RNA segment and protein corresponding ta the
pBP4 insert DNA were compatible with that of an influenza B virus
polymerase gene and protein (61). The amine acid sequence deduced ftam the
long open reading frame was compared ta the known influenza A virus
pol)'JD8rase genes (4,23,33,71) using a best-fit algorithm (22). Little
homology (less than 7%) was found with the PB2 and PA protein. of influenza
A virus.' However, comparison of the amino acid sequence of the pBP4 in.ert
DNA with that of the influen~a A/WSN/33 virus PBl protein revealed -t-
extensive homology between the influenza A and B vind PBI proteins. The
amino acid homology between th. PB1 prote in. of. the two viruse. 1. shawn in
73
c.
, ,
..
~. L
, t · l
lipr. 161 Nucleotide sequence of the J!BP4 DNA corresponding to the <..
influenza B/Lee/40 viru. genome ~ segment encoding the PBl ,
proteine The deduced &mino acid sequence of the PBl protein is,
.hown. Nucleotide numbering i~ shown above the nucleotide
.equenC1t. Nucleotide 1 corresponda to the J'-ter.minal
nucleotide of the'genome RNA segment. It is preceded by.13
non-viral nucleotides (see text for éxplanation). !mina acid
numbering is shown'in parentheses at the end of each line. The .
tinderlined amino acid residues are those which are homologous . . -vith residues vi~~in the influenza A/WSN/33 virus PBl protein
sequence (71) assuming that misalignments hàve occurred.
respectively. in the regions between amino acid resi4ues 394
and 400 and between residues 422 and 425 of the influenza B
virus sequence. These regions are designated by arrows. Sea
text for furthar explanation.
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74
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1
ligur~ 11. ,Assuaina that minor misalignments had occurred between the PBl .. , .
prot~ins of influenza A and B viruses wit~ the introduction of a sIngle
amino acid deletion in the reaian qetween amino acid residues 394 and 400
(the, first set of arrows in Figurè 16) 'and an insertion of two amine acids
betwee~ r§sidues 422 and 425 (second set of arrows in ligur~ 16), it
beC0m8. p.Dssi~le to a11an the PBI proteins of the two viruses to :.,--~--
demonstrat~ 61% ove~all amino acid homology. This is the highest degree of
\ .-amino ac~d ~~~jet to be fo~d between equi~alent proteins of
; influenza A and B viroses (see Table 1-). For this reason ft was concluded
that the pBP4 insert DNA corresponds'to the influenza B virus genome RNA
segment encodina the influenza B virus PBl protein.
~e distribution of charged amino acid residues present in the
influenza B vi~s PBl protein was analyzed in Figure 18. Basic amine acid o
residues have:been highllghted ln blue and acidic residues in red. The q.
influenza B virus PBl protein, like the influenza A protein (4,23,71), is a ,
hiah1y basic protein. I~ one assumes a charge of +1 for each arginine and
lysine, +0.5 for each histidine, and :1 for each gl~tamic and aspartic acid
" ' "'l ~e. idue, the influenza B virU$ PBl protein can be calculated ta have a net
~harge of +,~O st pH 7: O.' ~ makes i t '5 lightly 'less bas ic 'than the PBl
protein of inf~uenza A/WSN~j'whiCb has a net charge of +27. As can be
seen in liaure 18, c~r8ed residues have a relatively even dist~ibution
throughout the influenza A and B virus PBI prot~jns. Of the 108 basic
"lno acid re.idue. in the inf~uenza B virus protein (12 histidines, 57
,., ly.ine. and 39 ar .. irn\n~s) * basic residues are conserved at 81 èO,fresponding 1
posttion5 in th. infl~enz. A!WSN/33 PBI protein. Slmila~ly, 42 of the 82 . ~
acidic reaidu •• (42 ,lutamic acid and 40 aspartic acid) of the influenza B
PBI proteln al'. conserved .s acidic r.aidue. at th •• ame positions withln '1.
'the influenza A virus protein.
t .'
• ''''l
.. ...et!
. " -_/ ~) , "
.~76 /' II ( 1'--, . 1
1
, ,' .. , A
.. ... .. '
, "
, , .
Figure 17: . qomparison of the deduced amino"acid sequences of the PBI '.
proteins of the B/Lee/40 and A/WSN/33 (71) strains of influen.a
virus. Allowing for a one residue deletion between amino .cid
residues 394 and 400, and a two residue addition between
.residues 422 and 425 of the inf~penza B virus sequence, the ;.; U'
sequences can be aligned such that the boxed residues are
conserved between the strains •
.\
" .
1
- ....... -i'1I! :? ,
.)
o
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c
rLi.J.AII'-' 1 y-A h- Asp - Ly 1 Rlth 1 yrr.uh.l- p r-oJTliiJCys - G 1 ft tri'Pllle- r le-Asprr.;:lLau-Asprt7ilPrO!&1 u-HUI1I.- Ph.U!.\t Thr- A 1.- A.n-G 1 Il - S. rlil.ûAr9l!r..uJ l1a-A,p~Leu l)'slAWva l- I\tt-G 1 UUs.d"tt-ASIIf..I..:&.UGI \I_~! y-~It_ GI u-Il.-
Ph.4lli1 Val- Lyl-"sn - /l.rr;ï1.lys -liS-l.u- P .. o-A h-lys - As n -Argl'lYsl '" 1 y- Pht-lluo/l.-lys e~illlt-PrO-IIItti:YslY.l -Thr~ Hf s-Ph.- G 1 ft - AroI.!.i.ù:Arg-Arg- V.l-A"9 - "IP-Asft 0 lit t - Thrl.l..X.U lys-Mtt- Val - Th,-'l" Thr-II.- '" 1 y~Arll-
rLYil"IPIA'ril r 1.- Th .. -A 'g- VI I-'! unYrll1t-LY. \l..UtG 1 "~L'u- A,n - Ln -Ar,-Sul.!.ut btu- r 1. \OiO;oA..-..:.....;..; ...
l.u-Ly. -Ar-,-Arg-A 11- lle-Ah- Thr- A lIJiYl[ la L'II-b .-A .. -Ar -Ah-l1a-AlI-Thr- Pr0ti.!.tJ".t .... __ ..:..; __ = .... ..-.... __ :;... .... Oof
T hr-G! U-Ar'1f n.-ft'-: r-iA'P-S,rfP"Njf Il.Hrp- Phl-Ar.A, p - Phi-CyS E 11'-" 1. - P ,-ft VII- L. u-w ... ..;;.::.:.-.;.:...;,;,:~ 1 h- hr-ryr- ___ 'i __ r- ]_ A'"-Gln~GluilrR-rht-ArtAs"- V.I-llu ---=J'-A!'- Pr_ Il,-MI t-
Pro- hp ~ Pht-Asn fT'I"ël. P rorr;;;J G 1 u-Ar9- T yrJ'l'ï"nlI, 1 u- La u hr- r9 A lal'tYST LI u- Lys JtyiJL a u-l/S ~ Phe - Phe -A s nG 1 u- "1t~A la-Slri.!.l.!tAIP~ lys-Tyr- Phl~AIP- Ser Thr-Ar Lysl!.uJ 11_-G 1 u\UlJ !l--Ar91!.!::.2J' Llu- -leu-
77
(19 )
(46 )
(73 )
(100 )
(121)
(154)
(181)
(208/
( 235)
(262)
(289 )
(J16 )
(Hl)
(J10)
( 397)
G 1 II-G 1 u 1 y- Thr-A 11- St r -liu-St r- P ro-GI y- Me t - lIet-"et -G 1 Y -M.t-Phe -A 1 n-Mtt-Ltu -Str- Thr- VII - Ltu-G 1'1- Va 1 - (4lZ) 1I.-Asp &1 -TlIr-Ala-Se"-l.u-S.r-Pro-Sl -Met-IItt-Mtt-G! -Mlt-Phe-AS"-Mtt-llu-Ser·Th,-V.l- -1 - S.r-Ile-
A ro· ASp"'l y- Ltu-leu- va Il" lIjAIP' '" 1 y-Gly·P,o- Asn·Ltu- Ty C-","Jltu Ly' -Ah~l l- LU-L,y- U!. S.r _AlR- G J x-Gl y-rrR-Aln-LIU' Tr"-M~ IL' ...... "';";O __ .:;10 ... ;.;.;. ... ____ .;.;r.-;.;;,;.
T y~."'ft-ll.I;:ni~ Pro-IO 1 ul'T'1rllYIJGly-A,Î - L. ulL,u- H Il tp;';\Gl" T rp·Glu-lt" • G 1 u-"PUUl61n~Iy"Ar!-L.v_ Cys-Alnlttajl.u'-'l. ............ _"""...r.II ..... Il.·lYI - 61 ,,41TilAIPmt'l Th,(Pro-1 h- Hh·G'T y - p,.0lll.rrm Lysrr.t1A. Plfyr'~JP-1 1a. O"lS' .. -G 1)' hr - H S - Ir' rp y .'-AU.AIllt!lUV., UlIJ"tt Pro· A 1.-Hh-Glx- P"o A 11.tJ..uJ As"L!!!.UG 1 u Tlr.~!p-" II-V.I "1. - Thr Thr - K 1 s - Slr- Tr
Arg- Thr Il.-'ro .. ~ __ .. ____ .. ______ ~~~~~~
(449)
(416)
(503)
(S3() )
( 557)
(584 )
(611 )
(6J8)
(665)
( 692 )
:an-t:u- ,K.- GY ïtA 1.- Cy.IJ!iiJAs"I!'ëriA hi!t ... Tyr.Xrij LYII~,o.O.I.: 1 ylG 1 n·HI S Slr·MIt LIu "u-A 1.· Il. t A ll-H t S _ ( 719) _'''' •• M- ''''- ~ lit lys· , ".l.!!It ho ÙI.l:f Se,~~, r- Tyr-AralAr, • 'ro- VI I-~ lx! Il.·S... r·M~ v.l li 1 u-A la - "et V Il-~tr-
~~~~rrra~rtll:Î:p-:' t-efHI'l utr.'a Ty't'l U· Sar-Ci y.lrtt".t- S"ILYI-G fijASPr;ïi'iJGl u-lys - A hliittlA 1. - H h.L.u- ( 146) La.tâ tAtat 'L.- 1'- ~ ·t.~.uPh._~_y-~'r-~lr·Ar .. /1.-LyS_b~'·Gl!dGlul2.UTh~-Glu-f"l!!UILy.-IIt-Cy,-Gly-Glu(TT;l"y.Tyr,"tt P521 (·TERMINUS Str-Thrt..!.!.:tClu-Glu.Clu-Ar9-Ar9-titn_(ys (757) C-TER"IIIUS
.ç<
fi
•
-:---__ &II" .... ------a.~···~'::.~,·"'9i·- -".'
Figure 18: Distribution of char8~d amino acids in influenza A and B :PBl ---. ..
proteins. Basic amino acids are highlighted in blue, acldic
residues in red. The PBl prote in !sI a highly basic protein
with a ,,)let charge of +20 at pH 7.0 for the influenza B/Lee/4Q
virus prote!n and +27 for the influenza A/WSN/33 virus pr-otein ,
(71) •
, ,
•
)
o •
... ~ .. _.,.-
c
o
.. " ....... ; . ,.
• u ...... ,.~t •. , Il 'l •• i·.,J)~ tt.~ " ' •• ~11 •• ,,1 ;."II,IIT':I'a-lt.::1 J:' a.,- (It) 1 .t. JI '" tlO'ft .... '1" ...,' ......... ~.h ".I.&&..:.:aI\h __ le! _''''''ü.LaI''.~_.t !.' !;:,
... ... ... '. " a .. 1 .... '." " f,
, .. ' '" "001
nUI
f'U'
(1"1
UOIJ
f' 1\ ~
,1., ,
f ".1 .. It,
, .. " f ,rt. lUI)
'U" ,UU
(., .. rHIt
U ...
"'" ,-, "'" t ... ,
'MU
, .. , '"'' INlt
~_l!IU
J
79
f'
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o
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-'( T
1 1 80
An intriguing aspect of the structure of the PB1 protein. of influenza
A and B viruses exists in the regio~ between amino acids 399-427, where a
conserved hydrophobie region thirty amino acids in length is found (Figs.
17, 18). The hydrophobie nature of this region of the protein is more
readily visualized in the hydropathy plot shown in Figure 19. When the
pro~ein folds to assume its native confinDation, it is possible that it
folds 'in a manner sueh that this hydrophobie region is tucked away in the
interior, away from the acqueous milieu. This conserved hyd~ophobic region
may, alternately, piay a role in the interaction of this protein with the
other protein or RNA components of the ribonucleoprotein (RNP) and/or the
transcriptase complex. This conserved ~ydrophobic region also eontains the
longest stretch of exact amine aeid homology (comprised of 23 amine acids)
found between the influenza A and B virus PBl protei~s. This ean be seen
in Figure 17, residues 400-422 of the influenza B virus PBI sequence.
Praline residues are highly conserved in the PB1 protelns of influenza
A and B viruses. Of the 30 proline residues found in the influe9za A virus
protein, 27 are conserved in the same relative positions within the
influenza B virus molecule. Chou and Fasman analysis of the influenza >
A/WSN/33 virus PBl prote in (71) has predicted that 33% of the molecule
would be a-helical. This finding becomes interesting when one eonsiders
that 90% of the proline residues found in the influenza A ~irus PBI protein
are eonserved in the influenza B virus protein. Proiine reaidues exhibit
great mobility in proteins, including puekered flip-flop conversions of
their pyrrolidine ring and rotation of the puckered pyrrolidine ring about
the Ca-earboxyl bond (60). Horeover, the bulky proline residua cannot ha
accomodated in a straight a-helix, and disrupts it, allovina bands and
change of direction. Therefore, the hilh level of proline con.ervation
implies severe constraint in the organization of a-helie.l relions and
reverse turns in the influenza virus PBl proteins.
/
81
,
Pigure 19, Rydropathy plot o~ PBl proteins of the B/Lee/40 and A/WSN/33
strains of, influenza virus. Relative hydrophobieity and 1
hydrophilieity of the proteins along their amino acid sequences r
vere calculated by the metbod of Kyte and Doolittle (43) using
" a seapent length of 9 amino acids. The consecutive scores are
plotted fram the amino to the carboxy termini of the proteins,
and the midpoint 1ine corresponds to the grand average of the ./
. hydropa~~ of the amino aeid composition ~f most proteins whose
l sequences are known (43). Relatively hydrophobie regions of . the molecules are shown above the midpoint line and relatively
hydrophilic regions are below.
..
._'~
o
o
,/ 82
J'
o •
o i .. ~~~~~ ,., 1 : ~.,""" -
---
l '
•
_.p" -, ---------~- '-~~i'-
f
o
83
. A total of 17 cy.tein. re.iduu vere found in, the influenza B virus \
PBI protein. The influenza A!WSN/33 virus PB. prote1n contains la eyst.ine
re.idua., 6 of vhicb are consarved in th~ same positions in the influenza B
viru. molecule (ligure 17).
'.
, '
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84
" IV.
Influenza viruses are negative strand RNA viruaes that cause
respiratory disesse in humans (53). They have ~ segmented aenome which
accounts for the high fraquency of recombination seen in these viruae ••
Genetic recombination via genome RNA segment rea.sortment ia comman and
important in these viruses. This phenomenon appears to be directly ""-
responsible for the variabili~y in viral surface antiaenicity and expIa in.
the ability of influenza viruses to continue ,ta severely affact vorld
health (3,16,32,80). For this and other reasons influenza viruse. have
attracted immense interest and have been intensively studied. H~ltip1e.
examples of each of the influénza A virus genome RNA segments have heen
cloned and sequenced and nov six of the eight influenza B genome RNA
segments have also been sequenced (revieved in 45).
X-ray chrystallography bas revealed the three-dimensional structure of
the hemagglutinin and functional domains and antigeni~ reaions have baen
identified on this molecule (41,80). Huch additional infonDation has heen
obtained by expressing various of the viral genes usina eucaryotic and
procaryotic expression vectors (6,19;46,79,83).
Inflùenza viruses have several extremely interestina a.pects ta t~ir
life cycles such as their transcription proca •• wherein capped ho.t-cell
mRNAs are used as prLmers (42,57,59,68,17,78). This aspect ~f viral RNA
synthesis appears to be perfo~d by the viral polymerase protein.. Now
that the work of Krus and ,othera CS, 7 , 37,42,57,59,7n (.; be~ to
elucidate the individual roles of th. influenza viru. ~ymera •• prot.ina
in transcription, another approach to aainin, .ore in.i,ht into .pecifié
functional domain. of th... protein. i. to ca.pare the prt.ary .tructur ..
of the corre.pond!na influenza A and B virus polymera.e protein ••
.j
j
c
()
85
Con.ervation o~rÛDary structure betveen these definitaly,but distantly
related viru.es i. likely to identify prote in domains under severe
functional constraint.
A variety ofl.pproaChes have heen used ta synthesize double-$tranded
cDNA (ds-cDNA) froID viral and eukaryotic RNA templates. Most methods hegin
vith poly(A)" mRNA vhich ia transcribed to ds-cDNA. A comman procedure
employs reverse transcriptase (an RNA-dependant DNA polymerase) to convert
the mRNA to first strand cDNA (47,50). The RNA template is subsequently
removed by altaline hydrolysis to yield a single-stranded cDN! which is
allowed to fo~ a hairpin-like structure at its J'-end. This hairpin is
then used to prime second strand synthesis mediated by,g. coli DNA
polymerase or by reverse transcriptase. The resulting first-strand cDN! is
covalently connected to the second strand cDNA. 51 nuclease must then be
used to digest and open up the single-stranded loop to produce double
stranded cDN!. The major disadvantage of this method results from this 51
nuclease step vhich leads to loss of viral sequences correspond!ng to the
5'~terminal ~egions of the miNAs.
The tvo clon!ng methods described in this manuscript each has its
adv~tages. In Cloning Strategy l, genome RNA was used as a template to ,
synthesize first strand cDN! which served as template for second strand
synthesi. mediated'by Ribonuclease H. This approach can be contrasted with
that in Cloning Strategy II where the first strand cDN! derived from mRNA
va. hybridized to the first strand cON! derived from polyadenylated genome
RNA. In Cloning,Strategy II, the region corresponding to the size range
expected for the polymera.e-specific cDNAs (representing copies of mRNA and
aenOile RNA) vere cut out of gels and hybridized dne to another to form the
d8-cDNA. By doina thi., it v .. po •• ible to avoid second strand synth .. is
vhich i. u.ually much le •• efficient tban first .trand cDNA .ynthe.i ••
1
1 )
o
o 1
l
86
Horeove~. the level of background coloni •• resultina from undesired ( ••••
host) contaminants was essentially el~inated as campared to method. usina
only total mRNA from infected cells as tamplate.
The aenome RNA segment encodina the PBl prote!n of influenza B virus
was found to be 2368 nucleotides 10n8 (Figure 16). It has a sin.le Ion.
open reading frame capable of codina for a prote in of 752 amino acids
includina the initiating methionine vith a calculated molecular wei.ht of
84,407 daltons. The predicted primary structure of the PBI protein of
'influenza B virus showed extensive homology with its influenza A virus
counterpart. Table 1 summarizes what is so far known of protein homolo.y
betveen equivalent proteins of influenza A and B viruses. Althou.h protein
homology,ranges from 9.7% for the nonstructural prote!ns (NSl) to 61% for
the PBI proteins of both v!ruses, by far the greatest homology exists for'
• the PBI polymerase protein. The hydropathy profile in Figure 19 shows that
the influenza A and B virus PBI prote!ns hfve a similar hydropathic
profile. Extensive homology (Fig. 17) exists throughout the length of the
influenza A and B virus PBI proteins. lt is noteworthy .that even amanl th.
conserved amino acids on1y 30% are encoded by exactly·the s&me nucleotid.
triplets. These are predominantly amin~ acids for vhich the genette cod.
is not degenerate. Thus it appears that protein structure is con.erv.d to
a far greater extent than is nucleotide structure. Such conservation of
prote in structure over and above the nucleotide structure arlues for a hi.h
degree of functional constraint on the protein structure.
lt se8mS that the role of the polymer ••• pro teins in RNA synthe.t.
puts them under gre.ter functional and structural constr.int than is
apparent for other viral proteins. Such constra}nt probably relates to the
enzymatic activity of the polymera •• prot.ins in the ribonucl.oproteln
(RNP) complex dur!n. transcription (7,37,38,42,59). A •• ociation of
1 !
87
TABLE 1
p
RNA Sesment Protain Straios Compared References % Romology·
1,2,3 Polymerase
4
s
6
7
8
(PBI) B/Lee/40 vs. A/WSN/33 (70) 61%
H8IIID&g,lutinin (HAl) B/Lee/40 vs. A/PR/8/34
B/Lee/40 vs. A/PR(8/34
Nucleoprotein (NP) B/Lee/40 vs. A/PR/8/34
Neur&lDinidase (NA) B/~e/40 vs. A/Udorn/72
l/Lee/40 v~. A/Udorn/72
B/Lee/40 vs. A/Udorn/72
Nonstructural (HS l ) B/Lee/40 vs. A/Udorn/72
B/Lee/40 vs. A/Udorn/72
(10)
(66)
(8)
(8)
24%
39%
37%
25%
14%
9.7%
~6.2%
• . Romolo8Y calculated for best alianment, disregarding additions, \
insertions and delations. , ,
b Re,ion betweert &lDino acid residues 116 and 363 only of the influenza B
virus protein sequence. ~ 1
Exact % homolo,y of the remaining regions of th.
mole~ulea dependa on choice of MUltiple possible alignments.
\'
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88
polymarase proteins with the RNP complez .. y requir. an opttmum
con~Jrmation durin. transcription. The conserved hydrophobie r.lion
(Plgure 17) may play a role in the interaction of the PBI protein with the 1
proteins or RNAs of the polymerase complex or it may function in producina
.the required protein conformation of the RNP. In the conformational chana. 1
from a random coil to the native structure thi. region, which is laekir~ in
eharged residues, may beeome buried in the interior to allow the prot.in to
assume a specifie conformation.
In addition, the conservation of proline and cysteine re.idues
de~erves mention. Prolines are incompatible with a-helices and have earnad
the name of "helix-breakers ll• This is due to the bulky pyrrolidine rinl of
proline residues, which are not e8sily accomodated in an a-helix. In
addition, the ring undergoes puckered fJip-flop conversions and rotations
about, the Ca-carboxyl carbon bond whieh has the net effect. of disruptlng
a-heliees (20,60). The fact that prolines are highly eonserved between the
influenza A and B virus PBI proteins suggests that the organization of
a-helieal regions is under severe funetional constraint in this protein.
Cystei~e r~sidues are also relatively conserved between the PBl protein. of
both ·viruses. This is not ~~rprising sinee eysteine residues play a ~.ry
important role in the folding of proteins. They can be reduced to form
disulfide bridges and are crucial in providing polypeptide chain
erosslinking as weIl as providing reaetive sulfhydryl group ••
Th~s, a large degr8e of functi~nal eonstraint .ppear. to ha a central
theme in the evolution of the PBI prote in. of influenza A and B viru. •••
The high overall amino acid conservation, and of prolin •• , cy.tein •• and
the internaI hydrophobie reaion in particular may ha required for the
prot.in to attain a .peeifié conformation whieh "Yoanabl. it to int.ract
) \
--~ -"
,.
( »
19
favorably withOthe othor prat. in. of th. vl~ transcripta.e coaplex ••
they ex.cute th. very important function. of RNA replic.tion and
transcription . .
_.
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\
o
._.
c.
()
1.
\ \
--
/,
V. IIILIOGRAPIIY
,
90 -
. ,
.. At.ond. J.W. and R.D. Barry. 1979. Genetic recoabination between tvo
• strains of fovl plague virus: Construction of senetic .. ps. VlrololY
nf 407-415. .... ---- ~ -----
2. Almond, J.W. and V. lelsenreieh. 1982. Phosphorylation of the ""
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3. Berton. H.T •• o
C.W. Naeve and R.G. Webster. 1984. Antisenie st~ctur. ~
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6. Bos. T.J •• A.R. Davis and D.P. Nayak. 1984. NH2-te~inal hydrophobie
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--"
..
.r
, '
-0
o
""'t; , ,~
- ,
91 r
• 9. Briadis, D.J., R.A. Lamb and P.W. Choppin. 1982. Sequence of RNA ...
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t
14.
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(
t 18. Davis, A.R., A.L. Riti and D.P. Nayak.) 1980. ,rnfluanza dafactive
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o 1
o
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6
. ~.
'ô ,.'
--
c
(
, . j
94 ...
37: ~vù .. i, 1., 1. Hizumoto and A. lshi}laJDa.. 1983. (
RNA, polymera.e,of
influenza vin.. IV. Catalytic propertie. of the capped RNA 1
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38. X.wakami, 1., Je. Hizumoto, A. I.hihama, ~. Shinozaki-Yamaguchi and Je •. ' '\
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,
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