Proc. Nati. Acad. Sci. USAVol. 88, pp. 5187-5191, June 1991Biochemistry

Negative charge at the casein kinase II phosphorylation site isimportant for transformation but not for Rb protein bindingby the E7 protein of human papillomavirus type 16

(in vitro mutagenesis/ras/protein structure)

JULIANE M. FIRZLAFF*, BERNHARD LUSCHER*, AND ROBERT N. EISENMANDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98104

Communicated by Edwin G. Krebs, March 25, 1991 (receivedfor review August 24, 1990)

ABSTRACT The human papillomavirus E7 protein isphosphorylated at the two serines in positions 31/32, which arepart of a consensus sequence for casein kinase II (CKII). In thisstudy, we have investigated the effect of CKII phosphorylationsite mutations, all of which lead to unphosphorylated E7proteins. The replacement of the two serines by unchargedalanine residues drastically reduced the ability ofE7 to cotrans-form primary cells with ras, whereas negatively charged as-partic acid at the same positions produced only a slight effect.This difference was not reflected in the plO5Rb binding or theE2 promoter transactivation capability of these two mutants.Mutations that changed the CKII consensus without alteringthe serine residues also resulted in a loss ofphosphorylation andtransformation. This indicated that negative charge at posi-tions 31/32 provided either by phosphorylation or by a neg-atively charged amino acid is necessary for efficient transfor-mation without significantly affecting plO5Rb binding or trans-activation.

Human papillomavirus type 16 (HPV16) is believed to beinvolved in the etiology of human cervical cancer (1). Intumors, the viral DNA is frequently integrated (2) and onlythe early reading frames E6 and E7 are expressed (3). Recentstudies have demonstrated that the expression of E6 togetherwith E7 is necessary for the transformation of primary humancells (4-6). In addition, it was shown that the expressionof the E7 open reading frame (ORF) in the absence of E6is capable of transforming rodent fibroblast cell lines(7-10).The E7 protein shares several functional properties with

the ElA protein of adenovirus type 5 in that E7 is able tocooperate with an activated ras gene product in the trans-formation of primary rodent cells (11-13) and it can transac-tivate the adenovirus E2 promoter (12). In addition, it wasrecently shown that the E7 protein, like ElA, participates ina complex with the retinoblastoma (Rb) gene product plO5Rb(Rb; refs. 14-16).The E7 ORF of HPV16 encodes a nuclear, zinc-binding

phosphoprotein with a calculated molecular mass of 11 kDa(17-19). The E7 protein is phosphorylated in vivo at only onesite, Ser-31/32 (16, 20). In this region, which lies just Cterminal to the Rb binding region (residues 17-26), a consen-sus sequence for phosphorylation by casein kinase II (CKII;Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu; residues 30-37) can befound. In this study, we have explored the effects of site-specific mutations within the CKII phosphorylation region oncotransformation, transactivation, and in vivo interactionwith Rb.

MATERIALS AND METHODSConstruction of Mutants. A 4.4-kilobase (kb) HindIII/

EcoRI fragment of plasmid p1059 (nucleotides 79-4468 ofHPV16; ref. 12) was cloned into pBS+ (Stratagene). For thesite-directed mutagenesis, a kit from Amersham was usedaccording to the manufacturer's recommendations with aminor modification: double-stranded recombinant vectorDNA was digested with Nco I (at site 863 in HPV16) andHindIII (at site 932 in pBS+) followed by the isolation andheat denaturation of the large fragment. After the Exo IIIdigestion step, this denatured DNA was added to the muta-genesis reaction mixture to serve as primer for the second-strand synthesis. The following oligonucleotides were usedfor the mutagenesis [changes compared to wild type (wt) areunderlined]: 5'-ATC CTC CTC CTC TGC AGC GTC ATTTAA TTG CTC AT-3' (alanine), 5'-TC ATC CTC CTC CTCGC GTC GTC ATT TAA TTG CTC AT-3' (aspartic acid),5'-GC TGG ACC ATC TAT TTG CTG CTG CTQ CTG TGAGCT CTG ATT TAA TTG CTC ATA ACA G-3' (glutamine).A HindIII/Kpn I fragment, encompassing the promoterlessE6 and E7 ORFs (nucleotides 79-880 of HPV16) from the wtand mutant clones, was introduced into the mammalianexpression vector pEQ176P2. The parent vector pEQ176 (tobe described elsewhere; A. Geballe, personal communica-tion) was derived from pON249 (21) and contained thecytomegalovirus immediate early promoter (a 1.1-kb PstI/Sst I fragment) followed by a polylinker and driving theexpression of the bacterial 8-galactosidase gene. InpEQ176P2, the majority of the ,B-galactosidase gene wasremoved. The HPV16 E6-E7 wt and mutant fragments wereinserted into the polylinker of pEQ176P2. Simian virus 40sequences provided the signal for poly(A) addition and aeukaryotic origin of replication. Double-stranded sequencingof the entire E7 sequence in the recombinant plasmids usedfor the transfections confirmed the wt and predicted mutantalanine, aspartic acid, and glutamine sequences, respec-tively.The mutant E7 ORF from plasmid ADLYC (kindly pro-

vided by P. Howley, National Cancer Institute), whichcontains a deletion in the Rb binding site and does notassociate with Rb in vitro (15), was excised and cloned intopEQ176P2.

Transfections. COS-7 cells were transfected by the methoddescribed by Chen and Okayama (22) with 20 ,ug of E7 wt ormutant plasmid DNA per 100-mm plate. After 48 hr, the cellswere labeled with [35S]methionine or 32Pi and the radioim-

Abbreviations: CAT, chloramphenicol acetyltransferase; CKII,casein kinase II; HPV16, human papillomavirus type 16; ORF, openreading frame; Rb, retinoblastoma susceptibility p105 protein; wt,wild type.*Present address: Institute for Molecular Biology, MedizinischeHochschule Hanover, 3000 Hanover 61, Federal Republic of Ger-many.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991)

munoprecipitations followed by SDS/15% PAGE were per-formed as described (20). The anti-HPV16E7 antibody wasraised against a bacterial trpE-E7 fusion protein (20).The preparation of primary baby rat kidney (BRK) cells

and the transfections were carried out as described (23) using1 ug of E7 (mutant) plasmid DNA, 1 Lg -of pT24 (24)expressing an activated ras gene, and 8 ,ug of salmon spermDNA per 60-mm plate. After 3-4 weeks, the plates werescored for morphologic transformants.For the chloramphenicol acetyltransferase (CAT) assays,

NIH 3T3 cells were transfected in duplicate as describedabove with 15 Ag of E7 (or E7 mutant) plasmid DNA, 3 ;Lg ofpEC plasmid (25) containing the E2 promoter of adenovirusdriving the CAT gene, and 2 ixg of pEQ176 plasmid per100-mm plate. Forty-eight hours after transfection, the CATassays were performed essentially as described by Gorman etal. (26). f-Galactosidase expression was measured in acolorimetric assay using 4-methylumbelliferyl j-D-galacto-sidase (27), and the amount of input cell lysate was adjustedfor transfection efficiency using the P3-galactosidase expres-sion levels for normalization. The extent of acetylation wasassessed by densitometry using the bioimage system (Bio-Image, Ann Arbor, MI).

All cells were maintained in Dulbecco's modified Eagle'smedium with 10o fetal bovine serum.In Vivo Binding of E7 to Rb. Two days after transfection,

the COS-7 cells were labeled for 1 hr with 0.5 mCi of[35S]methionine (1 Ci = 37 GBq) as described above. Thecells were then Iysed in L buffer (250 mM NaCl/50 mMHepes, pH '7.5/5 mM EDTA/0.5 mM dithiothreitol/0.1%Nonidet P-40/0.2 mM phenylmethylsulfonyl fluoride/0.5%aprotinin/5 mM NaF/10 mM /3-glycerophosphate) and cen-trifuged, and the supernatant was incubated with C36 anti-Rbmonoclonal antibody (a gift from P. Whyte; see ref. 28) and2 ul of rabbit anti-mouse IgG antiserum. Alternatively, apolyclonal rabbit antiserum raised against a synthetic peptideencompassing amino acids 896-913 of Rb (from P. Whyte)was used. The incubation was carried out in the presence ofprotein A-Sepharose CL-4B beads (Sigma) for 1 hr at 4°C.The beads were washed three times with L buffer and theproteins were separated by SDS/15% PAGE. An aliquot ofeach cell lysate was incubated with 10 ,ul of rabbit polyclonalanti-HPV16E7 antiserum and protein A-Sepharose CL-4Bbeads in the presence of 0.5% each of Nonidet P-40, deoxy-cholate, and SDS. After incubation for 1 hr at 4°C, the-beadswere washed three times in RIPA buffer (20) and analyzed asdescribed above.

RESULTSGeneration and Expression of Mutant E7 Proteins. The E7

protein of HPV16 is phosphorylated in vivo at Ser-31/32within the CKII consensus sequence (16, 20). To study theeffect ofalterations in the CKII site on the transformation andtransactivation functions ofE7, three mutations (Fig. 1) weregenerated by site-directed mutagenesis. The two serines inthe CKII site were replaced by two nonpolar (alanine) andtwo acidic (aspartic acid) amino acids. For the third mutation(glutamine), the six acidic residues of the CKII site weresubstituted by glutamines. The wt and mutant E7 sequencestogether with E6 were cloned into the mammalian expressionvector pEQ176P2 under the control of a cytomegalovirusimmediate early promoter. To monitor the expression of theE7 proteins, COS-7 cells were transiently transfected withthe E7-expressing vectors, labeled with V35S]methionine, andimmunoprecipitated with a previously described anti-HPV16E7 antibody (20). As shown in Fig. 2A, the immuneserum precipitated a protein of =19 kDa (lanes 2-4) in thecells transfected with the E7 wt, alanine, and aspartic acidexpression plasmids, respectively, whereas no such protein

7II I 1i 1leI/

FIG. 1. Structure of the wt and mutated E7 ORF of HPV16.(Upper) Diagram represents the E7 ORF. The first methionine, theposition ofthe sequences necessary for Rb binding (15), and the CKIIsite (20) are indicated. (Lower) The wt and mutant DNA sequencesat the CKII site are 'shown, with altered nucleotides in italics. Thecorresponding amino acid sequence is depicted underneath with thesubstituted amino acids highlighted.

could be detected in the cells transfected with the vectorpEQ176P2 (lane 1). The E7 protein does not migrate accord-ing to its calculated molecular mass of 11 kDa in SDS/PAGEand the 19-kDa protein is of the size expected from previousstudies (3, 20). The glutamine mutant protein had a slightly

A vector wt Ala Asp Gin

- 200

97

- 68

- 43

-- 25

- 18

B

- 18

2 3 4 5

FIG. 2. Expression of E7 wt and mutant proteins in COS-7 cells.COS-7 cells were transfected with the vector alone; plasmids ex-pressing the wt E7 protein; or the alanine, aspartic acid, andglutamine mutant proteins as indicated. The cells were labeled with[35S]methionine (A) or [32P]phosphate (B) and immunoprecipitatedwith anti-HPV16E7 antiserum. The positions of molecular massmarkers are indicated (in kDa) on the right.

5188 Biochemistry: Firz1aff et al.

I-,".."I--,

Proc. Natl. Acad. Sci. USA 88 (1991) 5189

slower electrophoretic mobility compared to wt. In cellsexpressing the E7 aspartic acid mutant, an additional band ofsmaller molecular mass was detected. In pulse-chase exper-iments, no precursor product relationship between these twoforms was observed. The half-lives of all the E7 wt andmutant proteins were the same (data not shown).To assess whether the mutations had the predicted effect

on the ability of the proteins to be phosphorylated in vivo, a

second set of COS-7 cells transfected in parallel with thedifferent E7 constructs was labeled with [32P]phosphate andimmunoprecipitated with anti-E7 antiserum. As shown inFig. 2B, phosphate could only be incorporated into the wt E7protein, whereas no phosphorylation was detected in theproteins in which the acceptor serines were mutated toalanine or aspartic acid or in which the consensus sequencearound the acceptor serines was altered (glutamine mutant).

Cotransformation of BRK Cells. The E7 protein of HPV16is capable of transforming primary BRK cells in cooperationwith an activated ras oncogene product (12, 13). We thereforeexamined the transforming potential of the mutant E7 pro-teins in this assay. Primary cultures of kidney cells from6-day-old Fischer rats were cotransfected with pT24, ex-

pressing the activated T24 Ha-rasl gene, and the differentE7-encoding plasmids. After 3-4 weeks, the cells werestained and foci of transformed cells were counted (Fig. 3).The transformation of the BRK cells was not greatly affectedby the aspartic acid mutation relative to wt, whereas thealanine and glutamine mutations severely impaired the coop-eration of the mutant E7 protein with the ras oncoprotein.Two types of transformed BRK cells transfected with the wtand aspartic acid mutant constructs were observed, one offibroblastoid and the other of epithelioid morphology (datanot shown). From both cell types, immortalized cell linescould be established and expression of either wt or asparticacid mutant E7 protein was detected (data not shown). Incontrast, none of the alanine, glutamine, or vector trans-fected cells gave rise to immortalized cell lines.

In Vivo Binding of wt and Mutant E7 to Rb. We nextassessed whether the mutations at the CKII consensus siteaffected the ability of the different E7 proteins to bind to Rb.Since Rb is constitutively expressed in COS-7 cells (data notshown; ref. 29), we introduced wt and mutant E7 proteinsinto these cells and determined whether these proteinsformed a complex with Rb. Two days after transfecting thedifferent constructs into COS-7 cells, the cells were lysedunder mild conditions to preserve potential protein-proteininteractions. The E7 wt (lane 2) as well as the three mutantE7 proteins (lanes 3-5) were found to be coprecipitated with

(O 30A,0

0

-0

U)20

°

n

0)

w t Ala Asp Gln vector

FIG. 3. Transformation of BRK cells with ras and E7 wt ormutant proteins. BRK cells were transfected with the different E7protein-expressing plasmids and examined for transformed foci after3-4 weeks. Results from five different experiments are presented andthe standard deviation is indicated. Three experiments in which theoverall number of foci was reduced were not included but gave thesame results.

an anti-Rb monoclonal antibody C36 (Fig. 4A) consistent withprevious work, demonstrating association of Rb with wt E7(14-16). The total amount ofE7 proteins present in the lysatewas determined by precipitation with an anti-E7 antiserum(lanes 7-10). The amount of E7 proteins coprecipitated withRb in three independent experiments was compared to thetotal amount of E7 protein by densitometric scanning. Be-tween 1% and 10% of the wt, alanine, and aspartic acidmutant proteins were bound but no consistent, statisticallysignificant difference in the binding between these threeproteins was found. However, we cannot rule out the pos-sibility of minor differences in the binding affinities. Incontrast, we consistently detected only between 0.3% and0.5% of the glutamine mutant protein present in the Rbcomplex, indicating that the rather extensive mutations in theglutamine protein had a more general effect on the structureof the protein and prevented efficient binding.To demonstrate the specificity of E7 binding to Rb in our

assay, we also transiently expressed in COS-7 cells a mutantE7 protein that has been previously shown to be unable tobind plO5Rb in vitro (15). The construct was derived fromplasmid ADLYC, containing a deletion offour amino acids inthe Rb binding domain of E7 (15). After immunoprecipitationunder mild conditions, using a rabbit polyclonal antiserumagainst an Rb peptide (Fig. 4B) or the monoclonal antibodyC36 (data not shown), only the wt E7 protein was coprecip-itated (lane 3), whereas ADLYC mutant E7 protein was not(lane 2), even though approximately equal amounts of the twoproteins were present in the transfected cells (lanes 4 and 5,respectively). In addition, E7 was not immunoprecipitatedunder mild conditions with a monoclonal anti-myc antibodyor with a polyclonal rabbit anti-mouse IgG antiserum (datanot shown). Furthermore, no coprecipitation of other tran-siently expressed proteins such as c-myc and erbA, which arenot known to associate with Rb, was observed with the C36anti-Rb antibody (data not shown). Taken together, weconclude that phosphorylation of E7 is not required for Rbbinding.

Transactivation of the Adenovirus E2 Promoter by E7Mutant Proteins. The E7 protein is also functionally similar tothe ElA protein of adenovirus in its ability to transactivatethe E2 promoter of adenovirus (12). To test the mutant E7proteins for this function, we used plasmid pEC (25), whichexpresses the CAT gene under the control of the adenovirusE2 promoter. As an internal control for variations in trans-fection efficiency, the plasmid pEQ176, which encodes j3-ga-lactosidase, was used. These plasmids were cotransfected induplicate into NIH 3T3 cells with the different E7-expressingplasmids or the vector pEQ176P2. A CAT assay was per-formed from the cell lysates (Fig. 5A) and the percentage ofacetylation was measured by densitometry. Fig. SB repre-sents the combined results of four independent experimentsdone in duplicate. The transactivation potential of the E7protein did not appear to be grossly affected by the alanine(lanes 3 and 4) or aspartic acid (lanes 5 and 6) mutation in theCKII site with 44.5% and 35.6% acetylation, respectively,compared to 38.5% for the wt protein (lanes 1 and 2). Thetransactivation of the glutamine mutant (lanes 9 and 10) wasreduced to 14.5% acetylation, which was still well above thevalue for the vector (1.2%), which represents the basal levelof the E2 promoter (lanes 7, 8, 11, and 12).

DISCUSSIONIn this study, we have determined that the phosphorylationof the E7 protein of HPV16 at Ser-31/32 is important for itstransformation function. The presence of negatively chargedamino acids such as aspartic acid can substitute for the twoserines at this position, whereas uncharged alanine residuesare unable to do so. In addition, altering the consensus

Biochemistry: Firz1aff et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

vector wt Ala Asp Gin vector wt Ala Asp Ginm= --'

2uu

97

68

43

-AL

-:v. *- ..-

E7^w2. 3 1 0

B vectOc A wI~ A vci vector

I:25

18

m

I;-a. - 200

- 97

- 68

FIG. 4. Binding of E7proteins to p1O5Rb. (A)COS-7 cells were transfectedwith the different E7-ex-pressing constructs as indi-cated (see also Fig. 2), la-

25 beled with [35S]methionine,and immunoprecipitatedwith C36 anti-Rb antibody(lanes 1-5) or with anti-E7antiserum (lanes 6-10). (B)COS-7 cells were transfectedwith constructs expressingthe wt E7 or the ADLYC E7mutant protein as indicated,labeled with [35S]methio-

18 nine, and immunoprecipi-P tated with a rabbit anti-Rb

peptide antiserum. The posi-tions of molecular massmarkers are indicated (in

AS3 4 > - kDa) on the right.

sequence for CKII phosphorylation around Ser-31/32 pre-vented phosphorylation and severely impaired transforma-tion by E7. The binding of wt and the mutant E7 proteins toRb was retained but a reduction in the binding affinity of theE7 glutamine mutant protein was observed. The transacti-

AWt Ala Asp vector Gin vector

* ,. * A, * t

* S * :* * a 4

1 2 3 4 5 6 7 8 9 10 - 2

B Wt Ala Asp Gin vector

% aostylation 38.5 44.5 35.6 14.5 1.2

standard deviation 3.17 0.89 6.77 4.92 0.88

* average of 4 independent expenments done in duplicate

FIG. 5. Transactivation of the E2 promoter of adenovirus by thedifferent E7 proteins. NIH 3T3 cells were transfected in duplicatewith the vector; E7 wt; and mutant alanine, aspartic acid, andglutamine protein-expressing plasmids as indicated, together withplasmids pEC and pEQ176. (A) Cell extracts were normalized fortheir ,3-galactosidase expression and assayed for CAT activity. (B)The average percentage acetylation from four independent experi-ments done in duplicate is shown and the standard deviation isindicated.

vation potential of the E7 protein was not the determiningfactor for the transformation ability.

It is not known at this point whether both Ser-31/32 arephosphorylated simultaneously or only one at a time with apossible preference of one over the other. After mutagenesisof either serine in trpE-E7 fusion proteins, the remaining onecan still be phosphorylated by CKII in vitro (16), suggestingthat both serines can be CKII targets. Similar conclusionshave been drawn for the Myc oncoprotein, which can bephosphorylated by CKII at multiple adjacent sites (30). In E7,upon substitution of the two serines at positions 31/32 by twoalanines, this region is only slightly altered compared to havingunphosphorylated serine at this position (data not shown)when analyzed for protein conformation and hydropathy (31,32). Therefore, the alanine mutant can be considered as a

constitutively unphosphorylated E7 protein. To mimic a fullyphosphorylated state, two aspartic acids were introduced inplace ofthe two serines. This raises the negative charge ofthisregion by 2 but not as much as phosphorylation of the twoserines, which would add the equivalent of 3 negative charges.

In our tests of the above-mentioned mutants for cotrans-formation of BRK cells in conjunction with an activated ras

gene product, it was evident that the aspartic acid mutanttransformed almost as efficiently as the wt E7 protein,whereas the alanine mutant transformed 10 times less well(Fig. 3). Therefore, it can be concluded that the introductionof negative charge at positions 31/32 is necessary for efficienttransformation by E7 and it seems likely that CKII phos-phorylation may act to positively regulate this function of theE7 protein. Evidence is accumulating that a phosphorylatedserine or threonine sometimes can be functionally replacedby a negatively charged amino acid (33-35).

In the glutamine mutant protein, the acceptor serines werein place but the 6 acidic amino acids around them thatconstitute the CKII consensus site were changed into glu-

A

Rbt

I

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Proc. Natl. Acad. Sci. USA 88 (1991) 5191

tamines. As predicted from studies with synthetic peptides(36, 37), no in vivo phosphorylation was detectable at the sitewith the altered consensus sequence. This result strengthensthe argument that such sites are targets for CKII in vivo.While these studies were under way, it was reported (38,

39) that CKII site mutations have only a limited effect ontransformation. Since these were single amino acid changesthat are likely to interfere only partially with phosphoryla-tion, the small effect on transformation is not surprising. Incontrast, in two other reports mutations of the two serines atpositions 31/32 to Arg/Pro (16) or only arginine (40) reducethe ability of the E7 protein to induce transformation butthese changes introduce amino acids that are structurallyvery different from serine. Nonetheless, taken together withour data they provide strong support for the importance ofphosphorylation at this site.Although CKII phosphorylation can alter the transforma-

tion potential of E7, it is clear from recent studies on both E7(16, 38) and ElA (41) that the binding of Rb is another factorpossibly involved in the transforming potential of these twoproteins. Since the CKII site is adjacent to the Rb binding site(see Fig. 1), it was suggested that CKII might regulateassociation with Rb (20). However, in this study no consis-tent difference between wt and alanine and aspartic acidmutant E7 proteins in their in vivo binding to Rb was found.Only the rather extensive glutamine mutant protein showeddecreased binding efficiency. This result is in agreement withreports on other E7 mutants showing that mutations in andaround the CKII site do not abolish in vitro Rb binding (16).The possibility that binding of Rb protein is not sufficient fortransformation is also supported by the fact that mutations inthe N- and C-terminal regions ofthe E7 protein, outside oftheRb binding site and CKII recognition sequence, can affect thetransformation capacity of E7 (38).Comparison of the wt with the alanine and aspartic acid

mutants indicates that no gross alteration in the transactiva-tion was detected, indicating that phosphorylation may not beimportant for the regulation of this activity. As the alaninemutant as well as a mutant described by Edmonds andVousden (p35/36 Asp/His; ref. 38), whose transactivationpotential was highly impaired, are both able to transform, itcan be concluded that transformation is not mediated throughtransactivation by the E7 protein. Up to now, no cellulartargets for transactivation by E7 have been reported.Our data show that there is an additional function of the E7

protein separate from binding to Rb but critical for transfor-mation, which is mediated by a negative charge at the CKIIsite. Therefore, it would be ofinterest to know what the levelsof CKII expression are in cells that are targets for HPVtransformation and to what extent environmental signalsmight influence both CKII activity and E7 function.

We are grateful to Drs. A. Geballe, P. Howley, and J. Nevins forplasmids and P. Whyte for anti-Rb antibodies. We thank J. Cooper,P. Kaur, and P. Whyte for critically reading the manuscript; E.Tolentino for oligonucleotide synthesis; and P. Goodwin and S.Spyropoulos of the Image Analysis Laboratory for densitometry.This work was supported by Grant PO1CA28151 to R.N.E.

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