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Multiple Isoelectric

Forms

of

Poliovirus

RNA-Dependent RNA

Polymerase: Evidence for

Phosphorylation

LYNN J. RANSONEt AND ASIMDASGUPTA*

Department of Microbiology and Immunology andJonsson Comprehensive CancerCenter, University of California,

Los

Angeles,

School

of

Medicine,

Los

Angeles, California

90024-1747

Received 5April1989/Accepted 16 July 1989

Poliovirus-specific RNA-dependent RNApolymerase(3D""')waspurifiedto apparenthomogeneity. A single

polypeptide ofanapparentmolecular weight of 63,000catalyzes thesynthesis ofdimeric and monomeric RNA

products in response to the poliovirion RNA template. Analysis of purified 3D""' by two-dimensional

electrophoresis showed multiple forms of3DP"', suggesting posttranslational modification of the protein in

virus-infected cells. The two major forms of3D1"' appear to have approximate pI values of 7.1 and 7.4.

Incubation of purified3DP"' with calfintestinalphosphatase resulted in almost complete disappearance of the

pl7.1 form andaconcomitant increase in the intensity of the pI 7.4 form of3DP"°. Addition of32P-labeled

P,

during infection of HeLacells with poliovirusresulted in specific labeling of3D1"" and3CD, a viral protein

which contains the entire3D"'" sequence. Both3DP"" and3CDappeartobephosphorylatedatserine residues.

Ribosomalsalt washes prepared fromboth mock- andpoliovirus-infectedcells contain phosphatases capable of

dephosphorylating quantitatively the phosphorylated form (pI7.1)of3DP"'. The positive-polarity, single-stranded RNA genome of

picornaviruses is replicated by a virus-encoded, RNA-de-pendent RNA polymerase found in the cytoplasms of

in-fected cells(2). The first RNA-dependent RNA polymerase wasdetected in cells infected withmengovirus,amember of

thepicornavirus family (3). The most studied

RNA-depen-dent RNApolymerase, however, is that of poliovirus. The poliovirus RNA-dependent RNA polymerase (3DPOJ) (25)

results from proteolytic cleavage of a polyprotein synthe-sized from poliovirus RNA incells infected with the virus (26). This enzymeis responsible for replication of the viral genomeinthecytoplasmsof infected cells(5, 10, 12, 13, 19, 29,30). The input viral RNA (plus strand) is first copied by

the RNA polymerase to generate a cRNA (minus strand) which serves asthe template for synthesis ofprogenyvirion

RNA (2). In vitro synthesis of minus-strand RNA from a virion RNA template requires 3DPo1 anda host cell protein

called host factor (4, 9, 11). A synthetic RNA primer, oligo(U), can replace host factor in the in vitro reaction, suggesting a role of host factor in the initiation of minus-strand RNAsynthesis (9).

We have purified 3DPo1 to apparent homogeneity. We presentevidence suggesting that 3DPo1 isphosphorylatedin

vivo at serine residues. We also show that virus-specific protein, 3CD, which containsthe entire 3DP01 molecule, is

also phosphorylated at serine residues. The purified 3DPo1

catalyzes oligo(U)-primed synthesis of covalently linked dimeric RNA inadditiontomonomeric RNAproducts. We discuss the possibleroleofphosphorylation of 3D-"" in the

replicationof thepoliovirusRNAgenome. MATERIALS AND METHODS

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Phosphocellu-lose was obtained from Whatman, Inc., (Clifton, N.J.).

Unlabeled nucleotides wereobtained from Calbiochem-Be-*Correspondingauthor.

t Present address: Molecular BiologyandVirology Laboratory, The SalkInstitute, San Diego, CA 92138.

hring (LaJolla, Calif.). Poly(U)-Sepharose 4Bwasobtained from Pharmacia FineChemicals (Piscataway, N.J.). Radio-isotopes werepurchased from Amersham Corp. (Arlington Heights, Ill.) and ICN Pharmaceuticals Inc. (Irvine, Calif.). Cells and virus. HeLa cellswere growninSpinner cultures

with Joklik modified essential medium supplemented with

6% newborn calfserum and infected withpoliovirus type 1 (Mahoney strain) aspreviously described (10).

Labeling proteinswith[355]methionine. Tolabelpoliovirus proteins with [35S]methionine, cells were suspended, after virus adsorption, in Earle saline supplemented with 5% dialyzed fetal calf serum and all the amino acids except methionine. [35S]methionine Trans-label (specific activity, 1,000Ci/mmol;1mCi/4x 108cells)wasaddedtothe culture after 2.5 h of infection, at which time host cell protein synthesiswasinhibited sothat onlypoliovirus-specific

pro-teins were labeled. Cells were harvested by centrifugation

5.5 h after infection, and the pellets were washed with phosphate-buffered saline andstored at -70°C.

Labelingof infectedcellswith

32Pi.

Theprotocolfor infec-tionwas the same as thatdescribed above except that the cell culture medium was phosphate free (24). At 2.5 h

postinfection, 32p;(2mCi/4 x 108 HeLacells)wasadded to the culture. Cells were harvested at 5.5 hpostinfection as

described above. For quantitation ofphosphorylation, the

polypeptidebandswereexcised from sodiumdodecylsulfate

(SDS)-polyacrylamide gels and soakedinliquid scintillation fluid andradioactivitywasmeasured inaliquid scintillation counter.

Purification ofpoliovirus polymerase. Poliovirus polymer-asewaspurified by phosphocellulose chromatography

(frac-tion II)asdescribedpreviously (10). FractionIIpolymerase was further purified by fast protein liquid chromatography on a 1-ml mono S column (Pharmacia). The enzyme was

eluted witha15-ml,50-to-500mM lineargradientin bufferA

(50 mM Tris hydrochloride [pH 8.0], 10% glycerol, 0.1% Nonidet P-40, 5 mM 2-mercaptoethanol). Peak fractions

containingpoly(U) polymerase activitywere dialyzed over-nightin buffer B (bufferA[20% glycerol] and 50 mM

KCI).

Furtherpurification of the fraction IIIpolymerase involved

4563 0022-538X/89/114563-06$02.00/0

CopyrightC 1989, AmericanSociety for Microbiology

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passage through poly(U) Sepharose 4B,followedby elution of the enzyme with a 50-to-400mMKClgradient. Fractions containing poly(U) polymerase activity were pooled, con-centrated, and dialyzed against buffer B. Fraction IV poly-merase was then passed through a second poly(U) Sepha-rose 4B column and eluted as previously described. The enzyme (fraction V) was stored at -70°C in small aliquots.

Poliovirion RNA was prepared as previously described (28). Invitro RNA synthesis with polymerase and methylmercu-richydroxide gel electrophoresis.Thestandard reaction mix-ture of oligo(U)-dependent RNA synthesis contained the

following in 50 ,ul: 50 mMHEPES (N-2-hydroxyethylpiper-azine-N'-2-ethanesulfonic acid) (pH 8.0); 5mM magnesium acetate; 4 mMdithiothreitol; 10 ,ug of dactinomycin per ml; 30 mMKCl; 20 ,uM zinc chloride;0.2mMeachATP, GTP, andCTP; 1 ,uCi of

[ox-32P]UTP;

5,uMunlabeled UTP; 1 ,ug of poliovirus template RNA; and 0.1 jig of fraction V

polymerase. Reactions contained 100 ng of oligo(U) perml.

Incubation was for 30 min at 37°C. For analysis ofthe in vitro product, the phenol-extracted, ethanol-precipitated la-beled RNAwas suspended indiethylpyrocarbonate-treated water, denatured with 15 mM methylmercuric hydroxide, and analyzed on a1%agarosegelcontaining 10 mM meth-ylmercuric hydroxide as described by Bailey and Davidson

(1).

Phosphoamino acid analysis. 3DPo1 was located on SDS-polyacrylamide gels bydirectautoradiography,and the band was excised from multiple lanes and electro eluted as de-scribed elsewhere (27). The isolated

3DPo1

was then acid hydrolyzedin the presence of bovineserum albumin,

oval-bumin, lysozyme,and amixture ofphosphoserine, phospho-tyrosine, and phosphothreonine. The phosphoamino acids

were then separated by high-voltage paper electrophoresis

as described elsewhere (27). The paperwas dried,

autora-diographed, and then stained with ninhydrin in acetone to

visualize the phosphoamino acid markers.

Dephosphorylation reactions. Polymerase in buffer B was

added to reaction mixtures containing 30mM HEPES (pH

8.0), calf intestinalphosphatase (CIP),boiled CIP, or

mock-orpoliovirus-infected ribosomalsalt wash(RSW)(24)atthe

indicated concentrations. Each reaction mixture also con-tained 5

jig

of bovine serum albumin per ml as described

elsewhere (18). One unit of CIP activity was defined asthe amount which hydrolyzed 1.0

jiM

p-nitrophenyl phosphate

top-nitrophenol and

Pi

permin at 25°C. CIP was boiled for 15 min prior to its addition to control reactions. The reac-tions were prepared on ice, initiated by incubation for the

appropriateamounts of time at 30°C, and then prepared for

two-dimensional gelanalysis.

Two-dimensional gel electrophoresis. Two-dimensional gel

analysiswas performed as described by O'Farrell (22). The

first dimension was an isoelectric-focusing gel in the pH range of3 to 10 or 6 to 8 as indicated previously (22). The second dimension was a 12.5% SDS-polyacrylamide gel. After separation of the proteins in two dimensions, the gel was fixed, dried, and autoradiographed with X-AR film (Eastman Kodak Co., Rochester, N.Y.) and a Cronex inten-sifier (Du Pont Co., Wilmington, Del.).

RESULTS

Purification of

3DP''.

In a previous study, we reported

purificationof

3DPo1

from the soluble phase of infected HeLa cellstotwopolypeptides of approximate molecular weights of63,000 and 35,000 (17). The 63,000-Mr polypeptide was

3DP01,

whereasthe35,000Mrpolypeptidewasof host origin

l 2 3 4

94-68 - -..

-3D

43-3 0

-FIG. 1. Purification ofpoliovirus polymerase. Poliovirus

poly-merase waspurified from 2 x 109 HeLa cells infected with

poliovi-rus asdescribed in Materials and Methods. Fraction II, III, and V polymerase preparations were analyzed by SDS-PAGE followedby

silverstaining of the gel. Lanes: 1, molecular weight markerproteins (numbersonthe leftaremolecularweights inthousands); 2through

4, pooled and concentrated fraction II, III, and V polymerases, respectively. 3D, 3DPoI.

(17). This highly purified preparation of 3DPo1 catalyzed the synthesis ofcovalently linked dimeric RNA products from poliovirion RNA templates in the presence of an oligo(U) primer. Experiments utilizing 5'-32P-labeled oligo(U)primer

and all four unlabeled nucleotide triphosphates suggested that the viral RNApolymerase elongates the oligo(U)primer copying the virion RNA template (plus strand) and that the newly synthesized minus strand snaps back on itself to

generate a template-primer structure. This structure is then elongated by the polymerase to form covalently linked dimeric RNA products (16, 17). To examine the possibility that the

35,000-Mr

polypeptide may play a role in the oligo(U)-primed synthesis of dimeric RNA products, at-tempts weremade to furtherpurify the viralpolymerase. We developed a modified purification scheme for 3DPo1 which

involved salt gradient elution through phosphocellulose (fraction II)and fastprotein liquid-mono Schromatography

(fraction III), followed by salt gradient elutionthroughtwo consecutive poly(U)-Sepharose columns (fractions IV and V). Figure1 showspolypeptide profilesofdifferent

prepara-tions of 3DPo1 as analyzed by SDS-polyacrylamide gel elec-trophoresis (PAGE)followedbysilverstaining.Thefraction

II polymerase contained several polypeptides (Fig. 1, lane 2). Subsequent purification of fractionII enzymeby monoS

(Fig. 1, lane 3) and poly(U) Sepharose chromatography resulted in several-thousandfold purification of 3DPo1 (data

notshown). Infact,onlyonepolypeptide ofanapproximate Mr of63,000 could be detected in the final

poly(U)-Sepha-rose-purified enzyme preparation (Fig. 1, lane 4). Further analysis of fraction V 3DPo1 by two-dimensional gel

electro-phoresisfollowedby silverstainingshowedonly one major

spot at an Mr of 63,000 witha pl of 7.4 (Fig. 2B). Another faint spot ofapparentlythesameMrwhichmigratedto apl of 7.1wasalsodetected.Comparisonofsilver-stainedspots in the fraction V enzyme preparation (Fig. 2B) with a

partially purified 35S-labeled 3DPo1 preparation (Fig. 2A) showed that the silver-stained major (pI 7.4) and minor (pI 7.1) spots comigratedwith two35S-labeled 63,000-Mr spots

at pIs 7.4 and 7.1, respectively. These results suggest that

highlypurifiedpreparations of the enzyme might contain at least two forms of 3DPo1 with the same Mr but different isoelectric points.

The

63,000-Mr

3DPo'synthesizesdimericRNAproducts. To examine the nature of RNA products synthesized by the

purified 3DPol enzyme, fractions II, III, and V were incu-batedseparatelywithpoliovirionRNAtemplate and allfour

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A.

3CD-

-3D- _

B

3D-- IEF

I$I

LU

0

U,)

71 74

FIG. 2. Two-dimensionalgel analysis of3DI3o. (A) Analysis of

partially purified [35S]methionine-labeled viral proteins by two-dimensionalgel electrophoresisfollowedby fluorography. (B)

Anal-ysis of fraction V polymerase by two-dimensional electrophoresis

followed by silver staining of the gel. Arrowheads indicate the

positionsof3DI)1. IEF,Isoelectricfocusing. E, Extractcontaining [35S]methionine-labeledviralproteins.

ribonucleoside triphosphates inthe presence or absence of

oligo(U). Labeled RNA products were denatured with 15 mMmethylmercuric hydroxideandanalyzedonagarosegels containing10mMmethylmercury.AllfractionII, III,and V

enzyme preparations were dependent on oligo(U) (Fig. 3). No labeled products could be detected in the absence of

oligo(U)withfraction III and Vpolymerases (Fig. 3,lanes 3 and 5). These results contrast those in a recent study in

1 2 3 4 5 6

2X_>

Ix- 4I -35S

OLIGO'UU -t-

-FIG. 3. Invitro RNA synthesis of dimeric RNA by poliovirus polymerase. Fraction II, III, and V polymerases were used for synthesis of labeled RNA from the poliovirus RNA template in the

presence ofoligo(U) asdescribed in Materials and Methods.

La-beledRNAs synthesized in vitroweredenaturedby 15 mM methyl

mercuryandanalyzed byagarosegel electrophoresisin thepresence

of10 mM methyl mercury. Lanes: 1 and 2, 20 ,ug of fraction II

polymerase; 3 and 4,1,ug of fractionIIIpolymerase;5and6,0.1,ug

offractionVpolymerase; +,witholigo(U); -,withoutoligo(U). 1X

and 2X, Monomeric and dimeric RNAs, respectively. The arrow

indicates theposition ofanRNAspecieswhichmigratesfaster than

2X RNA and is notdetected in the reactioncontaining fractionV

polymerase.

which Plotchetal. observed oligo(U)-independent synthesis of dimeric RNA molecules inresponse topoliovirion RNA by the viral polymerase expressed in Escherichia coli (23). The majority of the products synthesized bythefraction II

andfraction III 3DP°1 consisted of labeled35S RNA;

how-ever, someproducts of higher molecular weightswere also synthesized. These higher-molecular-weight RNAs con-sisted of two bands, one migrating at approximately twice (2x) the size of single-stranded RNA and one migrating faster than the 2x RNA. Incontrast, the fraction Venzyme

synthesized mainly the 2x product and some monomeric RNA. The product (Fig. 3, arrow), which migrated a little faster than the 2x RNA, was not detected in the reaction containing fractionVenzyme. Atpresent, wedonotknow

howthis product is synthesized by 3DPo1. However, since synthesis of this product is dependent on oligo(U), we

suspect that it results from the snap-back of the newly synthesized minus-strand RNA that is notquite fulllength.

In other words, while copying the plus-strand template

during synthesis, the labeled minus strand can snap backon itselfprior to reaching the 5' end of the template and prime itself for synthesis of a complementary (plus) strand. Indeed,

ourpreliminary experiments indicate that the product does not contain sequences complementary to the 5' end of template RNA. These results suggest that highly purified

3DP01,

which consists of one silver-stainable polypeptide, is capable of catalyzing synthesis of covalently linked dimeric RNAmolecules in response to oligo(U)-primed poliovirion

RNA.

3DP'" is phosphorylated. Analysis of the most-purified 3DPo1 by two-dimensional gel electrophoresis showed that there were multiple forms of the molecule which differed only by theirnetcharges (Fig. 2). To investigate the possi-bilitythat 3DPo'undergoes posttranslational modification in

infected HeLa cells, virus-specific proteins were labeled with[35S]methionine. Labeled polymerase was then purified from the cytoplasms of infected cells as described above.

[35S]methionine-labeled fractionVpolymerase was analyzed by two-dimensional gel electrophoresis (Fig. 4). Twoforms

(pls 7.1 and 7.4) of labeled3DPo' weredetected again (Fig. 4A). In thisparticular preparation, however, the pI 7.1 form of 3DPOI was much more abundant than the pl 7.4 form, unlike in the preparation shown in Fig. 2. Treatment of

labeledpolymerasewith 0.01 U(Fig.4B)and 0.1 U(Fig.4C) of CIP prior to two-dimensional gel analysis resulted in

almost complete disappearance of the pl 7.1 form and a

concomitant increase in theintensity of the p1 7.4form. A

similar resultwasobtained by usinga different preparation of 3DPo1 (Fig. 4D and E). However, when CIP was

inacti-vatedby heating at 100°C priortobeing mixed with 3DPo1, theintensitiesof spots1(pl 7.1) and 2(p17.4)wererelatively

unchanged (Fig. 4F) compared with their intensities in the

control(Fig. 4D). Mixing equalamountsof eachsample(Fig. 4DtoF)providedfurtherproofthat thedisappearanceof the pI 7.1 form (spot 1) is accompanied by an increase in the

intensity ofthepl7.4form(spot2)(Fig. 4G).Theseresults

suggestthat thetwodifferent formsof 3DPo1 could represent differentphosphorylation states ofthe molecule.

If3DPo1 is phosphorylatedinvivo,it should bepossibleto

label3DPo1 by addition of

32Pi

during infection of cells with

poliovirus. Experiments were performed in which parallel

cultures of infected cells were labeledwith [35S]methionine

or

32Pi

and 3DPo1 was then purified from 32P;- and

[35S]methionine-labeled infected cells. Figure 5A shows

analysis by SDS-PAGE of 3DPo1 purified by phosphocellu-lose (lanes 1 and 4), mono S (lanes 2 and 5), and poly(U)

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35 1 2

B

35S

F.

32p

35S 3

14

5

61

7

-M

C.

32p

F*

-

* -3CD

E.P F S+ P

~ 3D...1 i.

[image:4.612.344.531.76.322.2]

.w mo 3 D

FIG. 4. Two-dimensional gel analysis of buffer- orCIP-treated

[35S]methionine-labeled fraction V polymerase. [35S]methionine-labeled 3DPOl was treated with buffer B, CIP, or boiled CIP as

indicated in Materials and Methods. (A) Buffer B;(B)0.01 U ofCIP; (C) 0.1 U of CIP; (D) buffer B; (E)buffer Bplus0.1 U ofCIP; (F) boiledCIP; (G) mixture of samples from panels D, E,and F.Spots: 1,pl 7.1; 2, pl 7.4.

Sepharose (lanes 3 and 6) column chromatographies. A 32P-labeled band (lane 6) comigrated with

[35S]methionine-labeled 3DPo1 (lane 3). Comparison ofmono S-purified

35S-and 32P-labeled proteins (Fig. 5A, lanes 2 and 5, respec-tively) showed that aviral proteincomigratingwith 3CD, a putativeprecursorto3DPo1, was also labeled by32p;.

Anal-ysisof35S-and35P-labeledproteinsbytwo-dimensionalgel electrophoresis showed that the 32P-labeled bands ofMrsof

63,000 and72,000 comigratedexactly with [35S]methionine-labeled 3DP01 and 3CD, respectively (Fig. 5B andC).

Both3D'1" and 3CDarephosphorylatedatserineresidues. To determine which amino acids in 3DPo1 and 3CD are phosphorylated, 32P-labeled proteins were excised froman

SDSgel, electroeluted,andhydrolyzed with6 MHCl. The

resultingphosphoaminoacidswereanalyzedbyhigh-voltage

paper electrophoresis at pH 3.0. Acid hydrolysis of both 3CD and 3DPo1 yielded spots which comigrated with phos-phoserine molecules (Fig. 6).

Dephosphorylation of3DP°I in cell extracts. To determine

whetheruninfected HeLa cellscontainanactivity capable of phosphorylating 3DP01, crude cell extracts were prepared from mock- and poliovirus-infected cells (RSW). These

extractswerethenmixedwith[35S]methionine-labeled 3DPo'

and unlabeled ATP. In these experiments, a shallower pH gradientwas usedtofacilitate better separation of the two

forms of3DPO'. Incubation of purified3DPo'with ATP alone

didnotchange thepatternofmigration of the twoforms of

3DPo',

suggesting that fraction Vpolymerase didnotcontain akinase capable ofphosphorylating the polymerase

mole-cule(Fig. 7,comparepanelA withpanelB). Incubationofan

RSWpreparedfrom mock-infected cellswith3DPo1 and ATP resulted in complete disappearance of the acidic form (pl

FIG. 5. One- and two-dimensional gel analysis of [35S]methio-nine- and 32P-labeled polymerase. (A) SDS-PAGEanalysis. Lanes: 1, [35S]methionine-labeled fraction II polymerase; 2, [35S]meth-ionine-labeled fraction III polymerase; 3, [35S]methionine-labeled fraction V polymerase; 4, 32P-labeled fraction II polymerase; 5,

32P-labeledfraction III polymerase; 6, 32P-labeled fraction V

poly-merase;7,sameaslane 1 with 20 times theamountof protein.(B to F) Two-dimensional gel electrophoresis. (B) [35S]methionine-la-beledfraction IIIpolymerase;(C) 32P-labeled fraction III

polymer-ase;(D)[35S]methionine-labeled fraction V polymerase; (E)sameas panel Dexceptpolymerase is 32plabeled; (F) mixtureofsamples frompanels DandE.

7.1) and a concomitant increase in the intensity of the relatively basic form (pl 7.4) of 3DPo1 (Fig. 7C). Similar contrasting results were observed with an extract derived from poliovirus-infected cells (Fig. 7D). These results

sug-gest that crude cell extracts contain one or more phos-phatases capableofdephosphorylating3DPo1 and thatunder

the conditions of theassay,phosphatase activitiesprobably far exceed those of the kinases.

1 2 3 4

. .

-Ser

p

-Th

r-P

-Tyrr

P

- Origin

FIG. 6. Phosphoamino acidanalysisof 3Dpol and3CDby high-voltagepaperelectrophoresisofhydrolyzed3DP01 and 3CD. Lanes:

1, hydrolyzed 32P-labeled 3CD; 2,sameas lane 1 with one-halfthe amountofsample; 3,hydrolyzed32P-labeled

3DP'l;

4,same aslane

3 withone-halftheamountofsample.Anautoradiogramis shown.

The positions ofthe marker phosphoamino acids as detected by ninhydrinstainingareindicatedontheright.

A.

D.

2

A

B.

1 2

E.

2

3CD -

-3D-

-1 2

C.

2

i2

G.

1 2

-3D

D

35S

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A

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D

FIG. 7. Dephosphorylationof 3DPo1 by cellextracts. [35S]methio-nine-labeledfraction V polymerase was incubated with buffer B,

ATP,orRSWs prepared from mock-orpoliovirus-infectedcells and

analyzed by two-dimensional gel electrophoresis with apH 6 to8

gradient asdescribed in Materials and Methods. (A)Buffer without

ATP; (B) buffer with 1 mM ATP; (C) mock RSW and ATP; (D)

poliovirus RSW and ATP.

DISCUSSION

We havepurified 3DPo1 to apparent homogeneityfrom the soluble phase of HeLa cells infected with poliovirus. The

purified enzyme, which contains a single polypeptide ofan

apparent Mr-63,000, synthesizes both monomeric and

di-meric RNA products in response to virion RNA template with anoligo(U) primer.

Two linesofevidence presented heresuggest thatatleast

a fraction of 3DPo1 is phosphorylated in vivo. First, the purified protein exhibitsatleasttwoformshaving isoelectric pointsof7.1and 7.4 whenanalyzed bytwo-dimensional gel electrophoresis. The acidic form can be partially or

com-pletely converted to the relatively basic form by treatment withCIPorphosphatasespresentin crudecellextracts(Fig.

4 and 7). Second, 3DPo1 can be labeled with

32pi

when the

labelisaddedduring infection of HeLacells with poliovirus.

3DPo1 labeled in vivo with

32Pi

comigrated with

[35S]methio-nine-labeled 3DPo1 when analyzed by one- and two-dimen-sional electrophoresis. In addition, we found that 3CD, a virus-specific precursor protein which contains the entire

3DPo'

sequence, is also phosphorylated invivo.

The extent of

3DPo'

phosphorylation was approximately

0.2mol ofphosphatepermolof 3DPo1 (Fig. 5).Conclusions

fromthisresult, however,should beconsidered tentative, as

the amount of phosphate

(32pi)

per mol of 3DPo1 varied considerably fromoneexperiment toanother. Likewise, the

ratioofthepI 7.1 formtothe pl 7.4 form of

[35S]methionine-labeled polymerase in differentpreparations varied greatly.

For example, in Fig. 2A, approximately 10% of total 3DPo1 was found to have a pl of 7.1 (phosphorylated form),

whereas in Fig. 7A, almost 50% of thetotal 3DPOI hadaplof

7.1.We donothaveapreciseexplanationforthis;however, it could be due to copurification of various amounts of phosphatasesindifferentpreparations of the enzyme.

We were unable to immunoprecipitate 32P-labeled 3DPo1 from virus-infected cells, although [35S]methionine-labeled

3DPo1 was readily immunoprecipitated. It is possible that antibody prepared against a fusion protein (Trp-3DPoI)

ex-pressed in E. coli does not recognize the phosphorylated formof

3DPo'.

Alternatively, it may be difficult to detect only 10 to 20% of phosphorylated 3DPOI in virus-infected cell extracts simplybecause of competition by a vast excess (80 to90%)ofunphosphorylated3DPo1 for immune immunoglob-ulin G.

Theactivities of a number of proteins have been shown to beaffected by their phosphorylation states (for a review, see reference 8). This tendency was initially observed in studies of enzymes involved in respiration and metabolism and has since been seen in RNA polymerase 11 (7), simian virus 40 large T antigen (18), vesicular stomatitis virus phosphopro-tein NS (6), and interferon-induced, double-stranded-RNA-activated protein kinase (15). Phosphorylation also plays a major role in the regulation of protein synthesis. Initiation factors, ribosomal proteins, messenger ribonucleoprotein

particles, and amino acyl-tRNA synthetases have been shown tobemodified by phosphorylation (for a review, see reference 21). Whether phosphorylation or dephosphoryla-tion of

3DPo1

plays any role in theinitiation of RNA replica-tion is not yet known. Our preliminary results suggest that

dephosphorylationof

3DPo1

by CIP enhances its RNA chain

elongationactivity. A recent publication from the laboratory of E. Wimmer suggested that3DPo1 may have adirect rolein the initiation of RNA synthesis, most probably via its involvementin uridylylation of VPg (29). In light of this, it is interesting to speculate as to how the phosphorylation state of

3DPo1

mayaffectis ability to replicate the poliovirion RNA templates in vivo. Gillis-DeWalt and Semler have described a poliovirus mutant which is defective in the protease responsible for processing precursor proteins (14). Conse-quently, the mutant virus is unable to produce 3DPo1 in sufficient quantities. However, despite this defect, the over-all RNAsynthesisby this mutant virus is similar to that seen with the wild-type virus. It is conceivable that a small fraction of the total pool of

3DPo1

in the infected cells may represent the active form of the polymerase. This active

form could be one ofthe two isoelectric forms observed in thestudies presented here.

Previous results from this laboratory demonstrated that

hydrolysisof ATP was required for in vitro RNA synthesis

of minus-strand RNA from a poliovirion template in a reaction catalyzed by

3DPo1

and host factor (20). Morrow et al. also demonstrated that host factor could be a 67,000-Mr

protein with a possible protein kinase activity (19). The

obviousquestionis whether thekinase present in host factor

preparations is capable ofphosphorylating

3DPo1.

Because we purified host factor from the RSW of HeLa cells, we asked whether RSWs could phosphorylate

3DP01

in vitro. Such crude preparations as those seen in Fig. 7 contain

phosphatases and cannot be used to address this question. Experiments in the near future will determine whether highly

purifiedhostfactor is capable ofphosphorylating

3DP"'.

ACKNOWLEDGMENTS

Thisworkwassupportedby Public HealthServicegrantAI-18272

from the National Institutes ofHealthtoA.D. A.D.isamember of theMolecular BiologyInstituteat theUniversity ofCalifornia,Los

Angeles, and issupportedby an American Cancer

SocFety

Faculty Research Award.

We thank members ofthe A. Dasgupta laboratory for helpful discussions during the course ofthe work. We also ttmnk l0 0.

Wettstein for assistance with thephosphoamino acidanalysis. LITERATURE CITED

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Figure

FIG.1.rus aspolymerase(numbers4,meraserespectively.silver pooled Purification of poliovirus polymerase
FIG.2.followedpartiallypositionsdimensionalysis[35S]methionine-labeled Two-dimensional gel analysis of 3DI3o
FIG. 4.boiledlabeled(C)indicated[35S]methionine-labeled1, pl Two-dimensional gel analysis of buffer- or CIP-treated fraction V polymerase.[35S]methionine- 3DPOlwas treated with buffer B, CIP, or boiled CIP as in Materials and Methods
FIG. 7.gradientATP;ATP,analyzednine-labeledpoliovirus Dephosphorylation of 3DPo1 by cell extracts

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