0022-538X182/030767-14$02.00/0
Expression of
the
PRC II Avian Sarcoma Virus Genome
BECKY
ADKINS,1'
2tTONYHUNTER,1
AND KARENBEEMON't*TumorVirology Laboratory, TheSalk Institute, San Diego,California 92138,1 and Department ofBiology,
Universityof California atSanDiego, LaJolla, California920932 Received 8 September 1981/Accepted 14 October 1981
We found that the genomic RNAofthe replication-defective avian sarcoma
virus PRC II was 4.0 kilobases long. ANorthern blot analysis of the viral RNAs present in PRC II-transformed cells showed that the PRC II genome was expressed as a single 4.0 kilobase mRNA species. In vitro translation of poly-adenylic acid-containing 70S virion RNA yielded two highly related proteins of 110,000 and 105,000 daltons (P110 and P105), which were synthesized from messenger activity that sedimented as expected for the 4.0 kilobase PRC II genome (at 25 to 27S). P110 and P105 were identified as in vitro translation products of the PRC II genome by immunoprecipitation and tryptic peptide mapping and were the only PRC II-specific polypeptides detected by in vitro synthesis. Inaddition, we found that immune complexesprepared from PRC II 70Svirion RNA in vitro translation products contained atyrosine-specific protein kinase activity. A comparison of the in vitro- and in vivo-synthesized proteins revealed that PRC11-transformedcellsalsocontained 110,000- and105,000-dalton proteins, whichwereindistinguishablefrom invitro-synthesized P110 and P105by electrophoretic mobility andtryptic peptide analysis. Both P110 and P105 were
present in producercells and in seven individual nonproducer clones. A pulse-chaseanalysisshowed that P105 was theprimary translation productof thePRC IIgenome and that P110 was derived from P105 bypost-translational modifica-tion.Under conditions of long-term labeling with[3
S]methionine,
P110 and P105 werepresent in a molar ratio of approximately 1:1. These results indicated that the transformation-specificproductof the PRC II genome, previously referred to as a single component(P105), actually consists of twopolypeptides related by post-translational modification.Recently,threeclassesof aviansarcomavirus have been defined on the basis ofbiochemical and immunological differences among their transforming proteins (5,25). PRC II and Fuji-nami sarcoma virus (FSV), members of one avian
sarcoma virus class, are replication-defective
transforming viruses which induce sarcomasin vivo and transformation of avian fibroblasts in vitro. The FSV genome has been shown to containgenetic informationwhichwasprobably obtained by recombination with cellular
se-quences.Thesehost-derivedsequences, termed fps,arelocated in the middle of the FSV genome andare flanked by helper virus sequences (19, 22). The RNA genomes of FSV and PRC II
appear to beclosely related, as determined by nucleic acidhybridization, and may be the
prod-ucts of two independent recombinations
be-tween an avian leukosis virus genome and the
samecellulargene (35).
Chicken embryo fibroblasts transformed by
t Present address: Institut furVirusforschung, Im Neuen-heimer Feld 280, 6900 Heidelberg, WestGermany.
tPresent address:DepartmentofBiology,TheJohns Hop-kinsUniversity,Baltimore,MD 21218.
PRCIIcontain aphosphoprotein, P105,which is similar to FSV-encodedP140 in that it is
com-posed of helper virus-derived gag
protein
se-quences which arecontiguous with transforma-tion-specific sequences from thefps region (5, 24). Since P105 is the only viral gene product which has been detected in transformed,
non-producercells byimmunoprecipitation, it is
cur-rently the onlyidentifiedtransformation-specific product of the PRC II genome (8). Thisprotein
canbeimmunoprecipitatedwithantiseraagainst p19and p27, twovirioninternal structural pro-teins which are coded for by the 5'-proximal portionof the gag gene (5, 24). TBR serafrom rabbits bearing tumors induced by Rous
sarco-ma virus (RSV), a member of a second avian sarcoma virus class, also precipitate P105 by recognition of the gag determinants (5, 26). Immune complexes prepared with anti-p19 or
TBR serumfrom PRC II-transformedcells
con-tain a tyrosine-specific phosphotransferase ac-tivitywhichphosphorylates eitherP105aloneor
bothP105 and theheavy chain of immunoglob-ulin, respectively (5, 26). Because of this protein kinase activity observed in immunoprecipitates
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which contain P105 and the elevated levels of
phosphotyrosine in proteinobservedin PRC
II-transformedcells, P105 has beenimplicatedas a
tyrosine-specific protein kinase in vivo(5).
Although PRC IIand FSVareaviansarcoma viruses, it hasbeen demonstratedrecently that theSnyder-Theilen strain of felinesarcomavirus (ST-FeSV) and the Gardner-Arnstein strain of FeSV code for proteins which are related to
FSV P140 and PRC II P105 as determined by
immunological criteria and tryptic peptide map-ping (4, 5). In addition, hybridization studies have shown that FeSV virion RNA is partially homologoustothefps region of the FSVgenome (35). Because the FSV and PRC II genomes have extensive homology in the fps region, it seemslikelythat the PRC II and FeSV transfor-mation-specific regions are also partially ho-mologous.
It was of interest to determine whether the PRC II genome expressed
transformation-spe-cific proteins other than P105. We approached
this problem in two ways. First, we examined
PRCII-specific intracellular RNAsinanattempt to identify species which could serve as mRNA's forproteins otherthan P105. Second, weexamined the in vitrotranslation products of
PRC II 70S virion RNA and compared these
with the viral proteins synthesized in PRC II-transformed cells. In this report, we present
evidence that a single PRC II-specific RNA species gives riseto twohighly related formsof
P105 both in vivo and in vitro. These two
proteins, designatedP110andP105,appeartobe
related inaprecursor-product fashion by
post-translationalmodification. Inaddition, weshow that the PRC II proteins synthesized in vitro
display an associated protein kinase activity in immunecomplexes.
MATERIALSAND METHODS
Cells and viruses. PRC II virus stockswereprepared
fromPRCII-infected chickencellsoriginally provided byP.K.Vogt,UniversityofSouthernCalifornia,Los
Angeles. Rous-associated virustype 2 (RAV-2) was
also obtained from P. K. Vogt. FSV was obtained fromH.Temin(University of Wisconsin, Madison),as
describedbyLeeetal.(22)andBeemon(5),and from H.Hanafusa(The Rockefeller University, New York, N.Y.),asdescribed by Feldmanetal.(17)and
Hana-fusa et al. (19). All virus stocks were grown on gs-chf- chicken embryo fibroblasts(CEF)thatwere prepared fromeggssupplied by SPAFAS, Inc.,
Nor-wich, Conn., asdescribedpreviously (1, 7).
Virus and viral RNApreparation. Virus,total virion
RNA,and70Svirion RNAwerepreparedasdescribed previously (1, 7).
Cellular RNA preparation. Whole-cell RNA was prepared from infected and uninfected CEF, as de-scribedpreviously (1, 20). Forpolysomal RNA,PRC
II-infectedCEFwerewashedandlysed,anda
postnu-clear supernatant was prepared essentially as
de-scribed by Lee et al. (21), except that the lysis buffer contained 0.01 M vanadyl-ribonucleoside complex (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) as an RNase inhibitor. The postnuclear superna-tantcontained 0.1 M KC1, 0.005 M MgCl2, 0.025 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesul-fonic acid) (pH 7.5), 0.01 M vanadyl-ribonucleoside complex,1%Nonidet P-40 (NP-40), and0.5% sodium deoxycholate. The polysomes were then pelleted through a layer of 1.0 M sucrose and a layer of 2.0 M sucrose in the same buffer lacking detergent and vanadyl-ribonucleoside complex in a Beckman SW41 rotorat30,000 rpmfor 12 h. The RNA wasextracted from the polysomal pellet by the method of Lee et al. (21).
RNAhandling.The conditions used for the
prepara-tion ofpolyadenylic acid [poly (A)]-containing RNA, thedenaturation of RNA by dimethyl sulfoxide treat-ment, and sucrosegradient sedimentation have been described previously (1, 7).
Forgel electrophoresis, RNAs were treated at 50°C for 1 h with a solution containing 0.008 M sodium acetate (pH 5.2), 50%o (vol/vol) dimethyl sulfoxide,
14%(vol/vol)glyoxal (AldrichChemical Co.,
Milwau-kee, Wis.), and 400 ,ugofcarriertRNA(Boehringer
Mannheim Corp., Indianapolis, Ind.) per ml. RNAs were then fractionated on a 1.2% agarose gel in a buffer containing 0.036 M Tris base, 0.03 M NaH2PO4, 0.001 M EDTA, and 0.5% sodium dodecyl sulfate
(SDS)(pH7.8)for 720 V-h.Preparation of the gelfor
transfer to diazobenzyloxymethyl paper and prepara-tion of the diazobenzyloxymethyl paper were per-formedessentially as described byAlwine etal. (2), except that 0.2 Msodiumacetate (pH4.0) wasused instead of borate buffer.
Hybridization to RNA coupled to
diazobenzyloxy-methyl paper.After transfer of the RNA from thegel,
thediazobenzyloxymethylpaper wastreated with
pre-hybridization buffer(50% formamide [BDH], 0.75 M
NaCl, 0.05 M HEPES, pH 7.5,0.2% SDS, 0.005 M
EDTA, 1% [wt/vol] glycine, 200 ,ug of sonicated, denatured calf thymus DNA per ml, Sx concentrated Denhardtsolution [13]) for 4 h at 41°C.Hybridizations wereperformed with1 x 10'to5 x 107cpmof probe in 12 ml of hybridization buffer (the same as
pre-hybridizationbuffer except with 1xconcentrated
Den-hardt solution) plus 3 ml of50%dextransulfate for the times and at the temperatures indicated in thelegend toFig.2. Thepaper wasthen washedextensively with 2x,1 x,0.5x, and0.1xSSCcontaining0.2% SDS and exposedtoKodak X-OmatRfilm withanintensifying
screen(1x SSC is 0.15MNaClplus 0.015Msodium citrate).
Probepreparations. 32P-labeled cDNA,,p (contain-ing DNA sequences complementary to the entire PRC IIandhelper virus genomes)waspreparedby incubat-ing heat-denatured PRC II 70S RNA in a mixture
containing0.05 MTris-hydrochloride(pH8.0),0.01 M
MgCl2, 0.03 M 3-mercaptoethanol, 0.12 M KCI, 0.5
mMdATP, 0.5 mM dCTP, 0.5 mMdGTP, 2 mg of calf thymusprimer per ml,4,uM[cK-32P]TTP(293Ci/mmol; New England Nuclear Corp., Boston, Mass.), and avianmyeloblastosis virus reversetranscriptasefrom J. W. Beard (Life Sciences, St. Petersburg, Fla.) at 37°Cfor60min. TheDNA wasextracted with phenol-chloroform andseparated fromunincorporated materi-alby passageoveraSephadex G-75 column inabuffer
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containing 0.15 M NaCl, 0.01 M Tris-hydrochloride (pH 7.4), 0.001 MEDTA, and0.1% SDS.
To prepare a32P-labelednick-translatedprobe, we
used arecombinantlambda clonecontainingan inser-tionof the ST-FeSV genome, which was kindly pro-vided by C. J. Sherr, National Institutes of Health, Bethesda, Md. (34). Nick-translationwas performed on the entire recombinant clone essentially as de-scribed by Rigby et al. (29), except that the reaction
buffercontained 0.01 MTris-hydrochloride (pH7.5),
0.01 M MgCl2, and 0.001 M dithiothreitol. The
reac-tion was stopped and treated asdescribed above for
thepreparationof cDNAprobes.
Probepreparations weretreatedwith 0.3 N NaOH for 10 minatroomtemperature and neutralized with HCI before they were added to the hybridization
buffer. a
In vitro translation.An mRNA-dependent
reticulo-cyte lysatewas usedfor in vitrotranslation,as
previ-ously described (1, 6). Completed reactions were
incubatedfor 20 min at37°Cwith 50 gLgof RNase A perml in the presenceof 0.01 M EDTA beforeanalysis
on12.5%SDS-polyacrylamide slabgels (6, 7).
Trypticpeptide
mapping.
Two-dimensional tryptic peptide analysis of [ S]methionine-labeled proteins wasperformed as previously described (1, 7).Radiolabeling and immunoprecipitation. The proce-dures used for labeling cells with [35S]methionine (Amersham Corp., Arlington Heights, Ill.) and
32P,
(ICN, Irvine,Calif.) and forimmunoprecipitation have been described previously (32). TBR serumwas ob-tainedby injecting the Schmidt-Ruppinstrain of RSV subgroup D into newborn rabbits (9). Rabbit antiserum against avian myeloblastosis virusp19 was provided by D. P. Bolognesi, Duke University Medical Center,
Durham, N.C.
Proteinkinaseassay.Our assay of theproteinkinase
activitypresentinimmunecomplexeswasbasicallyas
describedbyCollettandErikson(12), except thatthe
immunoprecipitation buffer contained NP-40 as the
only detergent. Immunoprecipitates adsorbed onto fixedStaphylococcusaureuswerewashed with 0.15 M
NaCl4.01 M sodium phosphate (pH 7.2) and then
incubated for 10 min at 30°C in 20 ,ul of a buffer
containing0.02 M PIPES
[piperazine-N,N'-bis(2-eth-anesulfonic acid)] (pH 7.0), 0.01 MMnCl2and1 to5 ,uCiof
[.y-32P]ATP
(specific activity, >2,500 Ci/mmol; NewEngland Nuclear Corp.). Samples wereassayedby SDS-polyacrylamide gel electrophoresis,
autoradi-ography, and scintillation counting of radioactive bands.
Analysis by partial proteolysis.
[35S]methionine-la-beledproteinsingelslices werepartially digestedwith S.aureusprotease V8andanalyzed usingthe methods described byCleveland etal.(11), with the modifica-tions of Eckhart et al. (15).
RESULTS
Comparisonof PRCIIvirionRNA andPRC
II-specificintracellularRNA.The expression of the PRC II genome has been studied by utilizing antiserumwhichrecognizes viral structural pro-teindeterminantsorantiserum which recognizes undefined determinants coded for by the fps region (4, 5, 8, 24). In this way, a single polypep-tide, P105, has been identified as a translation
product of the PRC II transforming virus
genome. However, it is possiblethat additional
polypeptides which cannot be detected with
these antisera are necessaryfor transformation
by PRC II. For this reason, we wanted to
determine the size and, therefore, the coding capacity of the PRC II genome. This was accom-plished by preparing 32P-labeled PRC II 70S vinon RNA, denaturing this RNA by glyoxal
treatment, and fractionating the RNA on a neu-tral agarose gel. This analysis (Fig. 1, lane 3)
demonstrated that the PRC II 70S virion RNA
complex contained two major discrete RNA species. The larger RNA was approximately 7.5 kilobases (kb) long, which was consistent with the sizes of the RNAs previously described for avian leukosis viral genomes (19, 20, 22, 36). Therefore, it seemed likely that the larger RNA species corresponded to the helper virus genome. The smaller RNA was approximately 4.0 kblong and presumably represented thePRC
7.5kb- ia
-4.8kb-
JIM
4.0
kb-
.4*-1 2 3
FIG. 1. Comparisonof32P-labeledPRC II and FSV
virion RNAs. PRC II- and FSV-transformed CEF werelabeled with
32Pi
for 6 h inphosphate-freemedi-um. Thesupernatantwascentrifugedat2,000 rpm for 10 min to remove the cells and then centrifuged at
45,000 rpm for 30 min in a Beckman SW50.1rotor to
pelletthe virus. Virion RNAwasprepared by phenol
extraction, ethanol precipitation, and glyoxal
treat-ment, as described in the text. The samples were
resolved on a 1.0%o neutral agarose gel. Thearrows
indicate the positions of chick 28 and 18S rRNA markers run on the samegel. The FSVpreparation in
lane 2 contained a small amount of contaminating rRNA.Lane1, FSV virionRNA(virus obtained from
H. Temin); lane 2, FSV virionRNA(virus obtained fromH. Hanafusa); lane 3, PRC II virionRNA.
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[image:3.491.286.409.300.536.2]II genome. Ifthis entire moleculewere translat-ed,an RNAof this size would yielda 130,000-dalton (130K)to140Kpolypeptide; this ismore
than the observed molecular weight of105,000
for the PRC II transformation-specific protein. However, it is unlikely that the entire RNA
molecule is utilizedfor translation because the avian leukosis virus sequences at both ends of the genome probably include large untranslated regions.SinceFSVcodes for a somewhat larger butrelated protein, we were interested in direct-ly comparing the PRC II and FSVgenomes by the same method. Figure 1 shows the results of side-by-side electrophoresis of glyoxal-treated PRC IIvirion RNA andtwopreparations ofFSV virion RNA. Although the helper virus RNAs appeared to be the same size in all cases, the FSV-specificRNA was 4.8kb long, or 800 bases larger than PRC II RNA.
To explore the expression of the PRC II genome, we undertook an analysis of the PRC II-specific intracellular mRNAs. Since many
avian and murine retroviruses express internal
1 2 3
7.5 kb
-4.0 kb- * 2.9k6 - _ .A
A
codingsequencesinthe form ofspliced,
subgen-omic-sizedmRNA's(3, 10, 14, 16, 18, 20, 23,30, 31, 33, 36), we were particularly interested in whether PRC II-specific mRNA's smaller than 4.0 kb couldbe detected. Totestthispossibility, total cellularpoly(A)-containingRNA and poly-somal poly(A)-containing RNA were prepared from PRC TI-transformed CEF. These RNAs weredenatured with glyoxal andfractionated ona1.2% neutralagarosegelinparallelwithRNAs prepared in a similar fashion from RAV-2 avian leukosis virus-infected CEF and uninfected CEF. The RNAs were thentransferred to diazo-tizedpaper andhybridized as described above. Figure 2A shows the
hybridization
pattern ob-servedwhenweuseda2P-labeled
cDNAprobe prepared fromPRC IT 70S virion RNA. A com-parison between the virus-specific RNAs in RAV-2-infected CEF and the virus-specific RNAs in PRCII-infectedCEFshowedthatboth contained 7.5- and 2.9-kb RNA species thatwere capable of hybridizing with this probe. However, an additional band at 4.0 kb was
1 2 3 4 5
4.0kb
-B
FIG. 2. Identification of PRC II helper-related and PRC IItransformation-specificintracellular RNAs. Total cellularpoly(A)-containing RNAs from PRC II-transformedCEF,RAV-2-infected CEF,anduninfected CEF
andpoly(A)-containingpolysomal RNA from PRC II-transformed CEFwerepreparedasdescribed in thetext.
The RNAsweredenatured withglyoxal, fractionatedon a1.2% neutral agarosegel,and transferredtodiazotized paperasdescribed in thetext.Theblotswerehybridized witharepresentative32P-labeledcDNAprobeprepared from PRCII70S virionRNA(A) anda32P-labelednick-translatedprobepreparedfromaAST-FeSVDNAclone (B).Hybridizationswereperformed inabuffercontaining10odextran sulfateat41°Cfor4h(A)or at37°Cfor 23 h(B). The blots werewashedatthehybridizationtemperatureas described in thetextandwereexposedto Kodak X-Omat R film withanintensifyingscreenfor 1.5 h(A) and 2 weeks(B).Sizeswereextrapolatedfrom RNAof theSchmidt-Ruppinstrain of RSVsubgroupDrun onthesamegels,assumingsizes of9.5,4.8,and2.8 kb forgenomic, env, andsrcmRNA's, respectively(D.Schwartz, personalcommunication). (A)Lane1,2.5 ,ugof PRC II whole-cellRNA; lane 2,2.5p.gof RAV-2 whole-cellRNA;lane3,1.0,ugofuninfected whole-cell RNA.(B) Lane 1,0.1 jgof PRCII70S virionRNA; lane2, 2.0 ,ug of PRC IIpolysomal RNA;lane3,2.5 ,ugof PRC II whole-cellRNA; lane 4, 1.5 ,ug of RAV-2 whole-cellRNA;lane5,1.0,gof uninfected whole-cell RNA.
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observed exclusively in theRNAfrom PRC II-transformed CEF.
A similar blot was hybridized with a
32p_
labeled probe prepared by nick-translating a recombinant DNAclonecontaininganinsertion of the entireST-FeSVgenome.SincethePRCII and FeSV genomes share some commontrans-formation-specificsequencesbutlackhomology in otherregionsofthegenome(Beemon, unpub-lisheddata), we expected that thisprobe would recognize onlythose mRNA'swhich were spe-cific to thePRC II genome. AsFig. 2Bshows, the FeSV probe hybridized to only the 4.0-kb RNAinpolysomal and totalcellularRNA prepa-rationsfrom PRC II-transformedCEF. The 4.0-kb RNA in preparations from PRC II-trans-formedCEFwasidenticalin size to the smaller RNA in denaturedPRC II 70S virion RNA. No PRC II-specific subgenomic mRNA's were de-tected with either the PRCII probe, the FeSV probe,orwith aprobe prepared froma recombi-nant plasmid (p 53) containing a925-bp insert consisting ofthe5'- and3'-terminalsequencesof the Prague-B RSV genome (data notshown).On the basis of this analysis, we concluded that the 7.5- and 2.9-kb RNAs observed in both RAV-2-and PRC
Il-infected
CEF represented thehelper leukosis virus genomic size and env mRNA's, respectively. In addition, we tentatively con-cluded that the PRC II genomewasexpressedin infectedCEF as one major RNAspecies 4.0 kb long.PRC II transforming protein synthesized in
vivo and in vitro consists of two highly related components. We wanted to determine whether the PRC II genome contained coding informa-tion for polypeptides other than P105. To do this, we utilized the method of invitro transla-tion ofvirionRNA, which has been useful for identifying potential transforming proteins of RSV (7, 27, 28). PRC II 70S virion RNA was heat denatured, and the poly(A)-containing RNAwas selected forfractionation on neutral sucrose gradients. Each gradient fraction was translated in vitro in a micrococcal nuclease-treated rabbit reticulocyte lysate, and the pro-tein products were resolved by SDS-polyacryl-amide gel electrophoresis. Figure 3 shows that the poly(A)-containing virion RNA contained twopredominant messengeractivities, one cod-ingforaheterogeneous band of approximately 76K andasecondcoding for a similarly hetero-geneous bandwhichmigrated in the 105K range. The76Kpolypeptide was synthesized primarily from RNA which sedimented at 35S, whereas the 105Kprotein was madefrom 25 to 27S RNA. Tryptic peptide mapping of the
[35S]methionine-labeled 76K polypeptide synthesized in vitro showed apattern similarto thepatterns
which we have observed previously for Pr76919en-coded by the avian leukosis or sarcoma virus genome(1, 6). Under certain conditions of elec-trophoresis,itwaspossibletoseparate the het-erogeneous 76K band intotwocomponents with apparent molecular weights of 80,000 and 76,000. The tryptic peptide pattern of the 80K proteinwasidenticaltothatof the 76K
protein
(data not shown). It seemed reasonable that these two polypeptides represented translationproducts
of different natural helper virusge-nomespresent in thePRC II stock. In support of
thishypothesis, wefound that infection of CEF withlimitingdilutionsof the PRC II virus stock produced one transformed clone which
con-tainedonlyasingle76K polypeptide (see below andFig. 6, lane4).
The heterogeneous band in the 105K range could also be resolved into two major compo-nents with apparent molecular weights of 110,000and 105,000. Thetryptic peptide
diges-tionpattern of the 110Kproteinwasidenticaltothat of the 105K protein (Fig. 4). The tryptic peptide maps of these two in vitro-synthesized proteinsappeared to behighlyrelated to the map of the P105 protein immunoprecipitated from PRC Il-transformed CEF (Fig. 4A). Mixing
ex-periments with P105 synthesized in vivo and either 110K or 105K synthesized in vitro
con-firmed the identity ofthe in vitro and in vivo products (datanotshown).
The observation of a 105K doublet synthe-sized in vitro prompted more carefulscrutinyof theinvivo-synthesized P105. Side-by-side elec-trophoresis of the in vivo- and in vitro-synthe-sized proteins demonstrated that the in vivo product could be resolved into two major com-ponents which comigrated with their in vitro counterparts (Fig. 5). However, one striking difference between the in vivo- and in vitro-generated doublets was the relative labeling in-tensities of the two components. The major product synthesized invitro was the 105K poly-peptide, whereas the relative amounts of the 110K and 105K proteins synthesized in vivo understeady-state labeling conditions appeared
tobeapproximately equal.
In addition to the 76K and 105K doublets, a number of smaller polypeptides were synthe-sizedprimarily fromRNAin the 25 to 27S region of the gradient (for example, the 42K protein doublet synthesized from messenger activity which cosedimented with the 110K and 105K messenger activities). It was possible to assign these proteins to one of two distinct classes, each containing helper virus structural protein tryptic peptides (data not shown). Members of
one class contained only gag-related tryptic peptides andpresumably resulted from
prema-ture termination of translation within the gag portionsof the PRCIIgenome. The second class
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180 K
-105 K
-76 K
42 K
-34 K
-355
18
ins
FIG. 3. In vitro translation of PRC II virion RNA. Thepoly(A)-containingfraction of heat-denatured PRC II
70S virionRNAwasdenatured withdimethylsulfoxideasdescribed in the text, loadedonto a10to30%sucrose
gradient, and spuninaBeckman SW41 rotorat25,000 rpm for 16 hat22°C.The RNA in eachgradientfraction
wasthen translated in amessenger-dependentrabbit reticulocyte lysate in the presence of[35S]methionine,and theproducts wereresolved on a12.5%acrylamide slab gel. Thepositionof35S RNAwasextrapolatedfrom 28 and 18S rRNA's run in aparallel gradient.
ofproteins contained pol peptides in additionto somegag peptides. We did notdetectany fps-specific tryptic peptides in proteins from either
of these classes.
Another polypeptide of approximately 34K wassynthesized fromRNAin the 18Sregionof
the gradient. Tryptic peptide mapping ofthis
protein demonstrated that itwasidenticaltothe 34Kprotein whichhasbeendescribed previous-lyasthetranslationproduct ofacellularmRNA
packaged intothevirions ofmostaviansarcoma
and leukosis viruses(1).
We expected that any internal coding
se-quences within the PRC II genome would be
expressed aspolypeptides translated from frag-mented RNAs with sedimentation values less than 25S.However, aside from thesmaller poly-peptidesdescribedabove, noadditional
transla-tion products were observed. From these
re-sults, it seemed probable that the P105-related proteinswerethemajor, ifnot theonly, transla-tionproducts ofthePRCII genome. Inaddition, itappeared thatthetransforming protein, which has been referred to previously as a single species (P105), actually consists oftwo major electrophoreticallydistinctcomponentsof 110K
and105K.
Pl10 and P105 are both present in PRC
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[image:6.491.56.451.77.469.2]PRC II 773
A B
9
*0
c
9
a
0 *
9 E
9
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FIG. 4. Tryptic peptidemapping of in vivo- and in vitro-synthesized P110 and P105. PRCII-transformedCEF werelabeled with [35S]methionine andimmunoprecipitated with anti-p19 serum. Heat-denatured PRC II 70S virion RNA was translated in a messenger-dependent lysate as described in the legend to Fig. 3. The P110 and P105 proteins were identified by preparative gel electrophoresis, removed from the gel, and digested with trypsin. The products were resolved on cellulose thin-layer plates by electrophoresis at pH 4.7 toward the cathode, with the origin on the left, followed by ascending chromatography from bottom to top. (A) In vivo-synthesized P110 and P105. (B) In vitro-vivo-synthesized P110. (C) In vitro-vivo-synthesizedP105.
transformed, nonproducer clones. Itwas
impor-tant to determine whether P110 and P105were
synthesized independently from distinct RNA
genomes in transformed cells or whether one
protein was produced by modification or
proc-essing of the other. If P110 and P105 were
synthesized from different genomic RNAs, in-fection ofCEF withlimiting dilutions ofaPRC IIvirus stock should have resulted in the
segre-gation of two separate genomes. To test this possibility, we preparednonproducer clonesby infecting CEF with limiting dilutions of the PRC
II virus stock. These cells werethan plated in
softagar,and individualfociwereisolated after
10 days and after 2 weeks. Each clone was
grown up separately, labeled with
[35S]methio-nine and
32p;,
and immunoprecipitated withanti-p19serum.Fromatotal of nine focipicked,one
was completely negative for the expression of either helper or transformation-specific viral proteins. This focus presumably represented a
clump of cells none of which had been infected andconveniently actedas anuninfected control (Fig. 6, lane 8). Among the remaining eight clones, one had clearly received both a helper virus genome and atransforming genomesince
both the P105 complex (P110 and P105) and
Pr769'9werepresent(Fig. 6, lane4). However,
seven of thefoci picked consisted ofclones of
nonproducercellssince Pr769'9wasabsent from
these preparations. In each of these seven
clones, itwasevident thatP110 and P105 were
present in approximately equimolar quantities.
Therefore, itseemedunlikelythatP110andP105
were synthesized from different RNAs present
in our PRC II virus stock. From these
experi-ments, wetentatively concluded that P110 and
P105 shared a common origin in that a single species of the PRC II transforming genome in nonproducer cells appeared to express both proteins.
P105, the primary translation product of the
PRC II genome, is a precursor toP110. Wewere
interested in determining whethera
precursor-product relationship existed between the P110 and P105proteins observed in vivo. Toaddress this question, PRC II-transformed CEF were
pulse-labeled for 5minwith
[35S]methionine
and then chased with medium containingan excessof cold methionine for varying time intervals. The cells were then lysed, immunoprecipitated with anti-p19 serum, and analyzed by SDS-polyacrylamide gel electrophoresis. As Fig. 7 shows, the major translation product observed witha5-min pulsewasthe lowermember of the doublet, P105. The upper component, P110, begantoappear aftera 10-minchase, and after 30minof chase timeP110and P105werepresent
inessentiallyequimolaramounts.This ratiowas
also observed under conditions of long-term
labeling,as shown inFig. 7, lane S. Inaddition, pulse-chase experiments in whichthe chase was
performed for periods as long as 20 h revealed
thatP110and P105 had roughly equal half-lives
(data not shown). These results suggested that
approximately 50% of the newly synthesized
P105 in transformed cells was modified
post-translationally togive rise to P110.
Toinvestigatethepossibilitythat P110
result-edfrom the modification ofa specific region of
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ADKINS, AND
1 2 3
180
- 110-105
76
-._m
.Wu
FIG. 5. Comparison of in vivo- andin vitro-synthe-sizedP110 and P105by polyacrylamide gel electropho-resis. PRC II-transformed CEFwerelabeled for16 h with [35S]methionineasdescribed in thetext.The cells were washed, lysed, and immunoprecipitated with anti-p19 serum. Heat-denatured PRC II 70S virion RNAwastranslatedinamessenger-dependentrabbit
reticulocytelysateatafinalconcentration of25,ug/ml
inthepresenceof[35S]methionine. The proteinswere
resolvedon a12.5%polyacrylamide gel.Lane1,
35S-labeled PRC II CEF immunoprecipitated with
anti-p19 serum; lane 2, [35S]methionine-labeled in vitro
translationproductsofPRCII70S viralRNA;lane3, [35S]methionine-labeled in vitro translation products withnoaddedRNA.
P105,wesubjected35S-labeled P110 and P105to
partial digestion with S. aureus protease V8.
Figure 8 showsthat most of the smallerdigestion
productswereidentical for P110 and P105.
How-ever, the largest partial digestion products of
theseproteins didnotcomigrateand had
appar-ent molecular weights of83,000and 77,000for
P110andP105, respectively.This result
suggest-edthat the modification of P105whichgenerated
P110probably occurredwithina77Kportionof
the protein. Preliminary evidence from the V8
digestion patterns of P110, P105, and Pr76919 labeledatthe aminoterminus by in vitro
synthe-sis with [35S]formyl
methionyl-tRNAf
me,sug-gested thatthemodifiedregiondoes notinclude
the extremeaminoterminus.
We attempted to test whether this
modifica-tionwasthe resultofglycosylation by using(i)
treatment ofimmunoprecipitates with
endogly-cosidase H and (ii) treatment of PRC II-trans-formed CEF with tunicamycin. In neither case
did we detect achange in the relative
representa-tion of P110 andP105 (data not shown). This was
ingood agreement with the results obtainedby
Neil et al. (24), whose attempts to labelP105
with[3H]glucosamineor[3H]mannosewere
un-successful. Although we could not exclude the
possibility that some sugars are present in
link-ages not affected by endoglycosidase H or
tuni-camycin treatment, it seemed unlikely thatthe
observedchange inelectrophoretic mobilitywas
due toglycosylation.
It has been reported that P105 is
phosphory-lated in vivoat several sites and contains both
phosphoserine andphosphotyrosine(5, 26). This
suggested the possibility that additional phos-phorylation ofaportion of the P105 molecules resulted in an increase in apparent molecular weight, producing the protein which we called P110. In general, we observed that P110 from 32P-labeled PRC II-infected CEFwasmore high-ly phosphorylated than P105on amolarbasis. A preliminary analysis of the phosphoamino acid
contents ofseparated P110 and P105 indicated
thateach protein contained both phosphoserine and phosphotyrosine. However, the ratios of phosphoserine to phosphotyrosine appearedto
be different in that P110was morehighly phos-phorylated at tyrosine residues than P105. We
arepresentlycontinuingthese investigations by examining the phosphotryptic peptides of P105 and P110.
Although the precise nature of the modifica-tion is still unclear, we concluded that P110 arises by a post-translational modification of P105 and that thismodification occurs withina 77K portionof P105.
Kinase activity associated with the in vitro
translationproducts of PRCIIviral RNA. PRC II
P105synthesized in vivoand immunoprecipitat-ed with either anti-p19 serum or RSV TBR
serum has been shown to be associated with
protein kinase activity, which results in phos-phorylation of P105 or the heavy chain of immunoglobulin G or both (5, 26). We were
interested indetermining whether the P105
com-plex (thatis,P110 andP105)synthesizedin vitro wouldlikewise demonstratephosphotransferase activityin the immunecomplex.Toexplorethis possibility, PRC II denatured 70S virion RNA
wastranslatedinvitro in the rabbitreticulocyte
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[image:8.491.100.198.77.416.2]PRC II EXPRESSION 775
1 2 3 4 5 6 7 8 9 10
4mep
#VWV smv.. 'V"-,3 L. ol
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1 2 3 4 5 6 7 8 9 10
110 5
105
...e
:..._ _- ___.- _M _._f 40 M OW4
a-35S 32p
FIG. 6. P110and P105immunoprecipitatedfrom chicknonproducercellsinfected with PRC II.Subconfluent
culturesofCEFwereinfectedwith serial dilutions of the PRC II virusstock for 1 h at37°C.The cellswerethen
trypsinized, replatedin 1.2%agar,andplacedat37°Cfor 1 to 2 weeks.Individual fociwerepickedwitha
drawn-outPasteur pipette andtransferred to 17-mm dishes containing5 x 10i uninfected CEF. Subsequently, the culturesweretransferredtoduplicate plates;oneof theseplateswaslabeledfor 16 h with[35S]methionine,and theotherwaslabeled with32p;for 16 h asdescribed in thetext. Lanes 2through 10 containanti-p19 serum immunoprecipitatesfrom cultures of nine individualpickedfoci. Lane1containsananti-p19serum immunopre-cipitateofacontrol culture of uninfectedcells.
lysate.WhenTBR serum wasused to
immuno-precipitate the in vitro products in a buffer
containing NP-40 as the only detergent,
phos-phorylation of the heavy chain of
immunoglob-ulin wasobserved (Fig. 9, lane 1). The control
reaction, in which no RNA was added to the
translation mixture, showed a small degree of
phosphorylation of theheavychain. This
residu-alactivitywasprobably duetothe fact that the
TBRserumusedcross-reacted withendogenous
pp6OC-srcin therabbitreticulocyte lysate. When
immuneserum waspreabsorbed with
detergent-disrupted virus, immunoprecipitation of the
P105 complex synthesized in vitro was
pre-vented. Under theseconditions,
immunoprecipi-tation of the endogenous pp6(C-src was not
affected. The level of incorporation into the
immunoglobulin heavy chain was the same in
this case(Fig. 9, lane 2)asin theprecipitations
where no RNA was added (Fig 9, lane 3).
Quantitation of the amount ofradioactivity in
theheavy chain in eachcaserevealed afive- to
sevenfold enhancementofproteinkinase
activi-tyin thesample in which P105waspresentin the
precipitate compared with the controls. A
phos-phoamino acid analysis oftheheavychain
phos-phorylated in immunoprecipitates of in
vitro-synthesized P110 and P105 revealed that
tyrosinewastheonlyacceptoramino acid in this
reaction(datanot shown).
It is possible thatour conditions for in vitro
translationwerenotoptimalfor thesynthesis of
anenzymatically active P105 complex.Wewere
not able to detect appreciable kinase activity
Pr76-VOL.41, 1982
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[image:9.491.64.431.75.415.2]S
4 0 10 30 60
"I P
120+
contained SDS and deoxycholate or when the kinase buffer contained magnesium instead of
manganese as the source of divalent cations, although these conditions worked well with in vivo-synthesized P105.
Thisdemonstrationofphosphotransferase
ac-0 1 10 300
A B A B A B A B
d
[image:10.491.57.247.75.492.2]AAW
FIG. 7. Pulse-chase analysis of P110 andP105 in PRCII-transformedCEF. Five identical 35-mmplates
ofPRC IT-transformed CEFwerepulse-labeledfor 5 min with 400,Ciof[35S]methionineperml in medium lacking methionine. The labeling medium was then replacedwith the chasemedium,which containedan excessof cold methionine. The chasewascarriedout
for0, 10, 30, 60, or120 min asindicated above the respectivelanes. At theend of eachchaseperiod,cell extracts werepreparedandimmunoprecipitated with anti-p19 serum as described in the text. Lanes S contains immunoprecipitates from PRC TI-trans-formed CEF labeled for 16 h with [35S]methionine.
Lane Pcontains animmunoprecipitate from PRC
II-transformed CEF labeledfor 16 h with32P;.
when immunoprecipitations of in
vitro-synthe-sized proteins were performed with anti-p19
serumorwithimmunoprecipitationbufferwhich
FIG. 8. Partial proteolytic digestion of P110 and P105 with S. aureus protease V8.
[YS]methionine-labeled PRC TI-transformed CEF were
immunoprecip-itated with anti-p19 serum, and P110 and P105 were
identifiedon a12.5%preparative acrylamide gel.The samples were prepared as described in the textand
weresubjectedtoproteolysiswith0,1, 10,and 300ng
of S. aureus protease V8 as indicated. The partial proteolytic digestionproductswereresolvedon a15%
acrylamide gel. LanesA, P110;lanesB,P105.
me
P110
-P105 Pr76..
_ -. .4
_MA .S...s
A*
,.-
83-77-..
a
J. VIROL.
Prl8O
4w
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W a*'iww-.:
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[image:10.491.261.448.143.576.2]PRC II EXPRESSION 777
HcC-
is
X [image:11.491.97.195.74.386.2]1 2 3
FIG. 9. Kinase activity associated with the in vitro translationproducts of PRC1170S virionRNA.
Heat-denatured PRC II 70S virionRNAwastranslated ina messenger-dependent rabbit reticulocyte lysate, and
thesamplewasdiluted withNP-40bufferand immuno-precipitated in thepresence (lane2)orabsence(lane 1)
of detergent-disrupted RSV virions. Protein kinase activity was assayed in immune complexes as de-scribed in thetext. Lane 1, PRCII70S virion RNA,
unblocked TBR serum; lane 2, PRC II 70S virion RNA, blocked TBR serum; lane 3, no RNA, un-blockedTBR serum. HC, indicates the immunoglob-ulinheavy chain, whichwasvisualized by staining.
tivity associated with in vitro-synthesized P110
and P105 supported the hypothesis that the
tyrosine-specific kinase activityobserved in
im-munecomplexescontaining in vivo-synthesized
proteinswas anintrinsicenzymatic property of
P110orP105orboth.
DISCUSSION
PRC IIand FSVcontainvery closely related
transformation-specificsequencesandmayhave
been generated by independent recombinations with the same or related cellular genes (35). However, these viruses are distinct from one
another in that the FSV putative transforming
protein (P140) is approximately 35,000 daltons larger than PRC II P105 and contains two or
three fps-specific [35S]methionine-labeled tryp-tic peptides that are not found in P105 (5, 25). These observations may be explained in several ways. One possibility is that both PRC II and FSV contain
fps-related
sequencesofsimilaroridentical lengths but that in the case ofFSV, moreof this region is translated. Another possi-bility is that the amount offps-related sequence obtained during the recombinational event or
maintained thereafter is different for FSV and PRC TI. In this paper, we have shown by a direct comparison of PRC IT and FSV virion RNAs that the FSV genome is approximately 800 bases larger than the PRC II genome. This difference in size between the PRC II and FSV RNA genomes is consistent with the molecular weight differences previously observed between P140 and P105. It would be interesting todetermine whether the FSVinformation that isnotfoundin PRC II is of viral or cellular origin and where these additional sequences map relative to the 5'- and 3'-ends of the acquired cellular se-quences.
Ithasbeen reported thatthe unique region of FSV RNA is partially homologous to the ge-nomesof ST-FeSV andGardner-ArnsteinFeSV (35).SincePRC II and FSV arerelated,we were able to use a molecular clone of ST-FeSV DNA as aprobeforPRC II-related RNA sequences in transformed cells. The fact that hybridization was detected directlydemonstrated that PRC II is atleast partially homologous toST-FeSV and enabled us to determine that the PRC II genome isexpressed in transformed CEF as a single full-length RNAspecies of 4.0 kb. It is important to point out the possibility thatadditional species of PRC II-specific RNA might be present in transformed cells, but such species were not detected with any of the probes that we used. Therefore, itseemsverylikelythattransformed cells do notcontainPRCII-specificsubgenomic mRNA's. The 4.0-kb RNA in PRC II-trans-formed CEFcomigrateswith the smallerof the two RNAs found in PRC II virion RNA. In addition, the 4.0-kbPRC II intracellular RNA is approximately the sizeexpectedforanRNAof 25to27S,which we have shown to be thepeak sedimentationvalue invirion RNApreparations for the messenger activity for P110 and P105. Therefore, it is probable that a single PRC IT-specific RNA or two slightly different RNAs
having similar molecular weightsare translated in vivo to yield P110 and P105. The absence of any smaller, subgenomic RNAsin transformed cells makes it unlikely that transformation-spe-cific proteins other than P110 and P105 are
expressed as primary translation products ofthe
PRC TI genome. Indeed, in vitro translation of poly(A)-containing RNAs prepared from dena-tured 70S PRC II virion RNA failed to reveal 41,
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778 ADKINS, HUNTER, AND
any PRC II-specific polypeptides aside from P110 and P105. Since this technique has been usedsuccessfully toidentifycandidates forthe transformingprotein of RSV (7, 27, 28), thelack of invitromessengeractivityforPRCII-specific polypeptides otherthan P110 and P105 supports the hypothesis that these proteins are the only transformation-specific proteins synthesized in vivo.
We attempted toidentify both the differences and thesimilaritiesbetween P110and P105. The tryptic peptide maps of[35S]methionine-labeled P110 and P105synthesizedin vitro were indistin-guishablefrom one another and were identical to the mapsofthe invivo-synthesizedproducts. A side-by-side comparison by one-dimensional polyacrylamidegel electrophoresis demonstrat-ed thecomigration ofthe in vivo- and in vitro-synthesized P110and P105 proteins. However, the ratio of
[35S]methionine
label incorporated intoP110andP105 was notthesameinvivoand in vitro.Theproductssynthesized in vitro repro-ducibly showed apreferential labeling ofP105. In contrast,when PRCII-transformed cellswere labeledtosteadystatewith[35S]methionine
and immunoprecipitated with anti-p19 serum, P110 and P105 were labeled to approximately the same extent, suggesting a 1:1 molar ratio be-tween the two proteins. We considered two possible explanations which could account for thesynthesis both in vivoand invitroof thetwovery closely related proteins, P110 and P105. Thefirstpossibilitywasthat P110andP105were
synthesized from differentRNAspeciespresent in our uncloned stock of PRC II. If present, these different RNA species could represent either independent recombination events be-tween the helper virus genome and the same
cellular sequences or,
alternatively,
a diver-gence of the parental transforming PRC II genome afterthe recombinationalevent.What-ever the potential origin, we reasoned that it should bepossibletoseparatedifferentgenomes byisolating transformedclones madeby infect-ing CEF with limiting dilutions ofour PRC II stock. Clones prepared in this manner were
examinedforthe presence of P110 and P105 in theabsence of the other.Allof thenonproducer clonesanalyzedcontained both P110 and P105. This resultsuggestedthat thetwoproteinswere
probably not synthesized from different RNA species. The alternative explanation was that P110 and P105 were related to each other in a
precursor-productfashion (e.g.,by post-transla-tional modification or proteolytic cleavage). This
possibility
was tested by analyzing the accumulation of P110 and P105 in transformed cellswhich had been subjectedto abriefpulse with[35S]methionine
and then chased for in-creasing periods of time. Thisexperimentdem-onstrated that P105 is the primary translation product of the PRC II genome and that within 10 to 15min after synthesis of P105, some portion ofthe P105 molecules is converted to P110 by post-translational modification. From these ex-periments, we concluded that the PRC II genome is expressed in transformed CEF in the form of two very closely related proteins, P110 and P105, which are present at steady state in roughly equimolar amounts, and that P110 is derived by post-translational modification of P105.
The fact that the tryptic peptide maps of [35S]methionine-labeled P110 and P105 were identical can be accounted for by any one of three possible explanations. The modification may be on atryptic peptide which lacks methio-nine. Alternatively, the P110 population may be heterogeneouswith respect to the sites of modi-fication so that only a small percentage of the P110 molecules are modified at an individual tryptic peptide. In this case, the modified pep-tide would make a minor contribution to the total map. It isalso possible thatthe modifica-tion might be destroyed during preparation of the samples for peptide mapping or that the modification might not result in a change of migrationinthissystem. Althoughwehave not been able todistinguish amongthese possibili-ties,wehave shown that thepartial proteolytic digestion patterns of [35S]methionine-labeled P110 and P105, generated with S. aureus prote-aseV8,aredifferent.Thisanalysis allowedus to assign at least one region ofmodification to a
77Kdaltonportion oftheprotein.
Wehavenotbeen successful inidentifyingthe typeofmodification whichproduces P110. The available evidencearguesagainst glycosylation, but it is still possible that sugar residues are presentin a formwhich has not been detected yet.Themostlikelypossibility is that phosphor-ylation of P105 produces P110, perhapsat spe-cific tyrosine residues.We arecurrently examin-ingthephosphotryptic peptide mapsofP110and P105to testthis idea.
We have observed that the gag-related pre-sumptive transforming proteinsencodedbyFSV (P140), ST-FeSV (P85), and Y73(P90) arealso present in transformed cells as two electropho-retically distinct forms (Beemon, unpublished data). Itisnotclear whetherafunctional signifi-cance can be assigned to the generation and maintenance of two very closely related pro-teins. However, it is conceivable thatonlyone
of the two forms has an associated enzymatic activity orthat eachof the formscan be local-ized to a different subcellular compartment.
Us-ingcellfractionationtechniques,we are
current-lyinvestigatingthe latterpossibility.
Several laboratories have reported the
exis-J. VIROL.
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tenceofa
tyrosine-specific protein
kinase activi-ty inimmunoprecipitates
containing
invivo-synthesized
PRCIIP105(5,
26).
The substrates for thiskinaseactivity
includeP105ortheheavy
chain ofimmunoglobulin
orboth,
depending
ontheantiserumusedfor
immunoprecipitation.
Al-though
these data suggest that PRC IIP105is theprotein
kinaseresponsible
for phosphotransfer-aseactivity
in theimmunecomplex,
thepossibil-ity
that theactivity
is due to an associated cellularprotein
kinase whichimmunoprecipi-tateswithP105 hasnotbeenruledout. We have demonstratedinthis
study
thatimmunoprecipi-tatesof the invitrotranslation
products
of PRC II 70S virion RNA likewise contain aprotein
kinase
activity capable
ofphosphorylating
theheavy
chainofimmunoglobulin
attyrosine
resi-dues.Thisactivity
could be detectedonly
ifNP-40buffer and TBRserumwereused for
immuno-precipitation.
Little if anyphosphorylation
of P105 orP110wasobserved,
suggesting
that the invitro-synthesized
proteins
arepoorsubstrates forphosphorylation.
Although
lesslikely
in thiscase, it is
still
possible
thattheprotein
kinaseactivity
that we have observed is due toco-precipitation
of invitro-synthesized
P110 and P105withanendogenous
kinase from the rabbitreticulocyte
lysate
usedfortranslation. It will be necessarytodemonstratekinaseactivity
inpuri-fied
preparations
of P110 and P105 toassign
definitively
anenzymatic
functionto one orboth of theseproteins.
Nonetheless,
our data areconsistent with the
proposal
that the PRC II genome encodes atyrosine-specific protein
ki-nase.
ACKNOWLEDGMENTS
WethankE. A.McNellyforexcellent technicalassistance,
D. Bolognesiforantisera,C. Sherrforprovidingthe A
ST-FeSVclone,and J. A.Cooperand B. M.Seftonforhelpful
discussionsandforcriticallyreadingthemanuscript.
Thisworkwassupported byPublicHealthServicegrants CA-17096andCA-23896from theNationalCancerInstitute. B.A.wassupportedbyaPublic Health Servicepredoctoral
traininggrant from the National Institutes of Healthto the
UniversityofCaliforniaatSanDiego.
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