Strain-Specific Transcription and Translation of the BamHI Z Area of Epstein-Barr Virus

JOURNAL OF VIROLOGY, Dec. 1986, p.902-909 Vol. 60, No.3 0022-538X/86/120902-08$02.00/0

Copyright X 1986, AmericanSocietyforMicrobiology

Strain-Specific Transcription and Translation of the BamHI Z Area of Epstein-Barr Virus

RUDOLF SEIBL, MANFREDMOTZ, ANDHANS WOLF*

Max vonPettenkofer-Institut, Universityof Munich, 8000 Munich 2, Federal Republic of Germany Received 27 March 1986/Accepted 21 August 1986

The expression of the 1,800-base-pair BamHI Z region of Epstein-Barr virus DNA was analyzed by hybrid-selectedtranslation with several DNA subclones and RNA from different cell lines.Furthermore, large segmentsofthethree reading frames extending in this area were expressed as fusion proteins intoEscherichia coli.Thefusionproteins were partially purified and used to immunize rabbits. These sera were used to confirm ourmapping assignments and to identify the respectiveposttranslationallymodifiedproteins in

in

vivo labeling experiments.Thereadingframe BRLF1(thefirstreading frame starting in the BamHI R fragment in leftward orientation) encoded a93- to96-kilodalton(kDa)protein dependingonthecellline. Themolecularweightof in vivo-labeled proteins was increased relative to that of in vitro-translated proteins, indicating that a posttranslational modffication had occurred. The BZLF1 reading frame encoded a 35-kDa protein. It was posttranslationally cleaved from a 38-kDa precursor in induced B95-8 and induced Raji cells and from a 40-kDaprecursor in induced P3HR1 cells. In Raji cells superinfected with virus derived from P3HR1 cells, the protein seemed to be expressed both from endogeneous Raji genomes and from infecting genomes. The transcripts for the 93- to 96-kDa and the 35-kDaprotein overlapped partially. The serum against the expressed thirdreadingframeBZLF2specifically precipitateda 140-kDaprotein. Thisreading frame contains only 650 nucleotides, and therefore further coding sequences were presumably spliced to BZLF2. The latter is deleted inthe Rajicell line; therefore, the observed 140kDa protein in superinfected Raji cells wasexpressed from infecting P3HR1 genomes.

Epstein-Barr virus (EBV), amember of the herpesvirus family, undergoes limited replication, presumably lifelong, in the oropharynxes ofmost individuals following primary infection (16, 28, 34). ViralDNA can also be demonstrated lifelong in peripheral B lymphocytes of all seropositive individuals. These latently infected lymphocytes acquire the capacityforunlimited growth.Invitro, onlyB lymphocytes can be infected. Cell lines established from such experi- ments, from naturally infected

peripheral lymphocytes,

or from Burkitt lymphoma biopsies, aretheonlyEBV-positive cell culturesystemsavailable. Inthese

permanently growing

cell lines, the virus remains essentially latent and only a limited set of viral genes is expressed. In some cell lines (e.g., B95-8, P3HR1), a low percentage of the cells con- stantly enter the productive cycle, and proteins from the early andlate stagesof virusreplication and infectiousvirus areproduced. The number of cells enteringthelytic cycleis

dramatically

increased after induction with various agents, particularly with phorbol-12-mystrate-13-acetate (35) and butyric acid (10). Some nonproducer cell lines, like Raji,can be stimulated as well

by

these inducers to express viral proteins ofthelytic cycle. TheRajicells are exceptionalin that they produce only proteins ofthe early class. P3HR1 cells have two peculiarities: first, the produced virus is incapable of

transforming

cells and, second, thevirus prep- arations contain a population ofdefective viruses. These defectiveparticles contain fourEBV DNAsegments thatare partially inverted andfused(3, 14, 26). Thedefective DNA (het-DNA)iscorrelatedwith thecapacitytoinduce thelytic cycle from latent genomes, e.g., afterthe superinfection of Raji cells (14, 24). The het-DNABamHI Z area is fused in the opposite orientation to sequences from the BamHI W fragment(3, 6, 26).Transfection ofaclonedsubfragment of

*Correspondingauthor.

the het-DNA containing the fused BamHI W and Z frag- mentsinto latent hybridD98/HR-1 cells(6) orofa standard BamHI Z fragment into Raji cells (30) trans-activates the latent EBVgenome,presumably by proteinsexpressed from the transfectedDNA.

By using hybrid-selected translation with RNA from in- duced P3HR1 cells, we (27) and others

(5)

mapped several proteins to the BamHI Zfragment. Infurtherexperiments, weused RNA frominducedRaji cells and couldmap tothis fragment proteins similartothatfrom P3HR1 cells but with aslightlydiffering molecular weight

(manuscript

inprepara- tion). Itshould be noted that theproteins identified by this method are primary translation products (owing to the absence of

posttranslational

modifications in the in vitro translation system). Monospecific sera ormonoclonal anti- bodiesarethereforenecessary topositively identify which of the in vivo-labeledEBV proteins examinedinseveral labo- ratories intermsoftime-dependentsynthesis, modification, correlation to antigenicgroups, and

sensitivity

toinhibitors areencoded bythevarious genes.

Inthispaper,wedescribe thedetailed examination of the cell-line-specific expression ofthe BamHIZreading frames.

Byusinghybrid-selected translation with several subclones ofthis

region

and RNA from different cell

lines,

we could correlateour

previously

mapped in vitro-translated

proteins

to two ofthe reading frames and obtain basic information about the

transcriptional organization.

The

protein

encoded bythethirdreading frame, which is deletedin EBV- DNAof the Raji strain(22), was not detectable by in vitro transla- tion. We further expressedlarge parts ofthe three

reading

frames inEscherichia coli and immunizedrabbits with the partially

purified proteins.

These rabbit sera were used in

immunoprecipitation

reactions to

assign

the

mapped

pro- teinstothe

single reading

frames andto

identify

therespec- tiveposttranslationally modified

proteins

in invivo

labeling

902

A

^{BamH1 e3}_- 92-96kd.38-40kd^pCZ453

BamHl Z 92-96kd, 38-40kd

BamHl R 92-96kd. 80kd pCeZ 1622

92-96kd. 38-40kd

pCZR 1495 92-96kd,3 8-40kd

a 8 X a + a p H H

I I I I I

BamHl E l92 o

BZLF241

.-

BZLF1

VP93/96 VP35

102II0 O^I

102 103 104 105 106

FIG. 1. Mapof the BamHI Zareawithasummaryof the data.(A)DNAfragmentsused in theexperiments.Thefigure givestheplasmids used forhybrid-selected translation, theproteins mnappedtothem,and thefragmentsof theEBVgenomeusedforproteinexpression.The BamHI e3fragmentwascloned inapUR expressionvector.Thisplasmidwassimultaneouslyused forhybrid-selectedtranslationand for the expressionofpartsof the BZLF2readingframe.FragmentZ453 is contained inapUCvector(pCZ453)andwasused inthis formforhybrid selection. The samefragmentwasalso cloned intoapUR plasmid (pRZ453)for theexpressionofpartsof the BZLF1readingframe.The BamHI Z and Rfragmentscloned inpBR322 (28)andtheplasmidpCeZ1622wereusedonlyforhybrid-selectedtranslation.FragmentZR1495

wasclonedinpUC19 (pCZR1495)and used forhybrid-selected translation;itwasalso cloned inapURvector(pRZR1495)fortheexpression ofpartsof BRLF1 readingframe. Fragments eZ1622 and ZR1495werealso cloned inM13, andthesingle-stranded phage DNA of clones pPeZ1622 andpPZR1495wasused forhybrid-selected translation(see Materials andMethods). (B) OrganizationoftheBamHIZ region.

Promotors ( *), polyadenylation signals (a), open readingframes (), and restriction enzyme sites (B,BamHI; X,XhoI; P, PstI; H, HindIII) were deduced fromsequence data (1). (C)Viral proteins encodedby the threereading frames and extension ofthe transcripts accordingtothe data obtainedby hybrid-selectedtranslation. The endsarepuncturedtoshow that theexact startandendarenotdefined.

experiments. The protein encoded by the third reading frame

wasalso identified by immunoprecipitation with the appro-

priate serum.

MATERIALS ANDMETHODS

RNA manipulations. Tissue culture, RNA preparation, hybrid selection, and in vitro translation were done ^as described previously (27). Briefly, the cells were lysed 2 days after induction (with 40ng of phorbol-12-mystrate-13- acetate per ml plus 3 mM sodium butyrate) with 4 M guanidine isothiocyanate-0.5 M 2-mercaptoethanol. The RNA^wasisolatedby sedimentation throughaCsCl cushion (1.8g/cm3). Forhybrid selection, the DNA wasspotted on

small nitrocellulose filters and hybridized against the total RNA. Bound RNA was eluted by boiling. The selected mRNA was translated in vitro with a rabbit reticulocyte lysate.

Cloning of hybridization probes. The DNAsequenceof the B95-8 strain(1)wasanalyzed withcomputerprograms(7)for restrictionenzymesites and reading frames. The DNAwas

digested with the appropriate restriction enzymes (Boehr- inger Mannheim GmbH, Mannheim, Federal Republic of Germany) and run on agarose gels. Bands containing the desiredsequences wereexcised, eluted electrophoretically, purified and concentrated with Elutip-d columns (Schleicher

& Schuell, GmbH, Dassel, Federal Republic of Germany), ligated with T4 DNA ligase (Boehringer), and used to transform bacteriaas describedby Hanahan (9).

The Sall C fragment of EBV strain M-ABA DNA cloned in pHC79 (23) was digested with HindIlI and XhoI. A 1,495-base-pair (bp) fragment containingpartsof the BamHI Z and R fragments was ligated with Hindlll- and SalI- digested pUC19 (19)togeneratetheplasmid pCZR1495 (Fig.

1). A 1,622-bp fragment covering the BamHI e3 fragment and parts of the BamHI e2 and Z fragments was cloned similarly togenerate theplasmid pCeZ1622. ThelatterWas

digested with HindIII and PstI, and 453 bp of the BamHIZ fragmentwerecloned inpUC19 aspCZ453.

The inserts ofpCeZ1622 and pCZR1495 were cloned in addition in M13mp8 (13). The single-stranded DNA of the bacteriophage particles from the resulting M13 clones pPeZ1622 and pPZR1495 resembles the opposite strands because the fragments in the two clones are in opposite orientation withrespect to their restriction enzyme sites at the ends.

Cloningfor expression ofEBVproteinsas bacterialfusion proteins. The theoretical secondary structure of the EBV proteins superimposed by their hydrophilicity and hydro- phobicitywascalculated withacomputerprogramwritten in

our laboratory following suggestions by E. Golub and G.

Cohen(4). Potentialantigenicsites which arepreferentially

B

C

BB X

I1I1

PI

R

BRLFI 41

.41

VP 140

kb 100 I

101

b

. N

904 SEIBL ET AL.

in hydrophilic ,-turns were included in the expressed por- tions of the EBV proteins (for a detailed description, see reference 17).

The EBV proteins were expressed as fusion proteins with

P-galactosidase

in pUR plasmids (25). The viral reading frames were inserted in frame near the C terminus ofthe

P-galactosidase

reading frame. TheBamHI e3fragment was excised from pCeZ1622 and ligated with pUR278. In the resulting plasmid pRe3-521 (BamHIe3),which was also used for hybrid

selection,

the C-terminal 165 amino acids ofthe 670-bp BZLF2 frame were expressed.The end of theBZLF2 frame is within the BamHI e3 fragment, and therefore the fusion protein ends with theEBVsequence. For the expres- sion of the BRLF1 reading frame, the EBV-derived se- quence of pCZR1495wasexcised withHindlIland XbaI and ligated with pUR288. In the resulting plasmid pRZR1495, again the 3' part of the BRLF1 reading frame is fused in frame to the ,-galactosidase reading frame with a few nucleotides from the pUC vector in between. The last 400 amino acids of the 1,800-bp BRLF1 frame were expressed with the EBV-encoded part again at the end of the fusion protein.

The reading frame BZLF1 contains one probably prefer- ential antigenic site between amino acids 150 and 175. The expression of the appropriate sequences in a pUR vector was only possible after insertion of the EBV-derived se- quences of pCZ453 after aHindIII-XbaIdigest inPINIII Bi (11). From there, the EBV sequences were excised with BamHI and XbaI and ligated withpUR289. In the resulting plasmid pRZ453, the last 330 nucleotides of the 600-bp BZLF1 reading frame were fused to the

P-galactosidase

reading frame with a few nucleotides of PINIII

Bi

in between.

Expression and purification of the fusion proteins. The expressionplasmids weretransformedin E. coliJM103 (12).

This strain represses the transcription of the fusion reading frame byoverproduction ofthe lac repressor. Transcription was induced with 1 mM

isopropyl-p-D-thiogalactopy-

ranosideafter thebacteriahadgrown to an optical density of 0.9. The bacteria were grown for another 2 to 4 h, sedi- mented, digestedwith lysozyme, and sonicated. After addi- tion of3%TritonX-100, thebacteriallysate wassedimented.

Most of the bacterial proteins were dissolved in the super- natant. The expressed fusion proteins werefound predomi- nantly in theinsoluble pellet. The pellet wasdissolvedin 8 M urea-20 mM Tris (pH

8.0)-0.5%

mercaptoethanol and cen- trifuged again. The supernatant with the dissolved fusion proteins was subsequently dialyzed against 6, 4, and 3 M urea. Two bacterial proteins precipitated and could be separated by sedimentation. The 3 M urea supernatant was dialyzedagainst

phosphate-buffered

saline, which led tothe precipitation of a high portion of the fusion proteins as determinedbyacomparison to thesupernatant. The precip- itatedfusionproteins could be isolated bysedimentation and were used forimmunizing therabbits.

Immunoprecipitation, polyacrylamide gel electrophoresis, and Western blotting. Raji cells were superinfected with virusisolatedfromP3HR1 cells asdescribed previously (2).

At 12 hpostinfection, thecells were labeled for 4 h with 50 ,uCi of[35S]methionine per ml. The induced cell lines were labeled 2 days after induction for 16 h with 20

pCi

of [35S]methionineperml. The cells were subsequentlywashed and lysed in immunoprecipitation buffer (1% Triton X-100, 0.1%sodium dodecylsulfate, 0.137 M NaCl, 1 mM

CaCl2,

1 mM MgCl2, 10% glycerol, 20 mM Tris [pH 9.0], 0.01%

NaN3, 1 mM phenylmethylsulfonyl fluoride). The lysate of

106cells wasincubatedwith 5to 10,ul of serumpreadsorbed with an unlabeled protein extract from

107

EBV-negative BJA cells. The immune complexes were bound on protein A-Sepharose beads (Pharmacia, Uppsala, Sweden) washed, eluted by boiling in 2% sodium dodecyl sulfate-3%

sucrose-5% mercaptoethanol-20 mM Tris (pH 7.0)- bromphenol

blue,

andsubjectedtopolyacrylamide gel elec- trophoresis as described previously (2). For Western blot- ting, unlabeled proteins were separated in polyacrylamide gels and electrophoretically transferred onto nitrocellulose (BA85; Schleicher&Schuell). The nitrocellulose wasprein- cubated with a modified Deinhardt solution (4) for 2 hand incubated overnight at room temperature with the 1:50 dilutedserum.Theblots were washed with gelatin buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 150 mM NaCl, 0.25%

gelatin, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate) andincubated for another2h with peroxidase-labeledanti- bodies against humanimmunoglobulins (Dakopatts, GmbH, Hamburg, Federal Republic of Germany). After subse- quently being washed with gelatin buffer, the blot was developed with 0.5 mg of3,3'-diaminobenzidine perml and 0.01%

H202-50

mMTris (pH 7.6).

RESULTS

Fine mapping of the BamHI Z area with hybrid-selected translation. To analyze the complex protein pattern obtained after hybrid-selected translationwith theBamHI Zfragment and P3HR1 RNA (27), we constructed several subclones.

Clones pCeZ1622 and pCZR1495 represent the left and the right half of the BamHI Z fragment, respectively, with adjacent sequences. BamHI-e3 (pRe3-521) isthefragmentto the left ofBamHI-Z and contains mainly sequences fromthe BZLF2 reading frame. Fragment pCZ453 from the central area of the BamHIZfragment contains sequencesfrom the BZLF1 reading frame only.

RNA from induced P3HR1 cells, induced B95-8 cells, induced 883L cells, and induced Raji cells was used for hybrid selection with the previously mentioned plasmids and, in addition, with the BamHI R and Z fragments from strain B95-8 (29). No specific protein abovethebackground could be detected afterhybrid-selected translation with the BamHI e3 fragment (Fig. 2). All other fragments tested hybridized withthe mRNA for aprotein ranging insize from 92 to 96 kilodaltons (kDa) in the different cell lines. With RNA from induced P3HR1 cells, induced Raji cells, and induced B95-8 and 883L cells, 92-, 96-, and94-kDaproteins, respectively, could be translated in vitro. The size differ- ences were reproducible and were clearly visible in many independent experiments (see Fig. 7). Minor bands in the range of 75 to 83 kDa could be detected in all of these hybrid-selected translations, with a similar size distribution among the different cell lines.

The mRNA for the 80-kDa protein mapped previously in theBamHI Rand Kfragments (27) hybridized as expected with theBamHI Rfragment. However, it did not

hybridize

tothe left segment of theBamHIRfragmentcontainedin the plasmid pCZR149S. Therefore, theprotein isencodedbyone of the rightward reading frames in the BamHI R or K fragment, presumably by the BRRF2readingframe, accord- ing to the sequence data. Theprotein couldonlybedetected with RNA from induced P3HR1 and particularly from in- duced B95-8 cells, not from induced 883L cells,

owing

to different responses ofthe individual cell lines to the induc- tion. In the Raji cells, which could also be induced to a reasonableextent, theprotein could notbedetected inmany J. VIROL.

(a 04 u: co cN

a a totalRNA BarnHi R pCZR1495 pCZ453 BamHI Z pCeZI622 Barn H1 e3 RtNA: 1 1 5 1 3 2 4 4 3 2 1 4 3 2 1 4 3 2 1 4 3 2 1 4 3 2 1 4 3 2 1

A.

92/94/9J -80

o0

_ 38

.33

1 <...

W

..

ItF1

do _ R.

_ s

FIG. 2. Hybrid-selected translation withRNAsfrom inducedP3HR1cells(lanes 1), induced B95-8 cells(lanes 2), induced 883L cells(lanes 3), induced Raji cells(lanes 4), and theDNAfragments indicated. Inthe lefttwopanels, single-stranded DNA probesfrom M13phages representing opposite strandswereused.The boundmRNAsandsamples of unselectedRNAincludingRNAfromEBV-negativeBJAcells (lanes 5)ascontrolweretranslated invitrowithreticulocyte lysate and[35S]methionineasradioactive aminoacid. The translationproducts wereimmunoprecipitated withapoolofserafromNPC patientsandseparatedonsodiumdodecylsulfate-polyacrylamide gels. The gelswere dried, fixed, and exposed to 3H-Ultrofilm(LKB Instruments, Bromma, Sweden). Owing tothedifferent numbers of induced cells in the variouscell lines, theintensityof the mapped proteins and of the background proteins varied. WithRNAfrom induced B95-8cells, themost intense signals could be achieved, but the backgroundwasalsoincreasedowingtooverexposure(tomake theverythinsignals withRNA ofinduced 883L cells visible). All of the proteins detectable afterhybridizationwiththeBamHIe3fragmentrepresentbackgroundproteins.

Theprominent 23-kDa protein visible after hybridization with allfragments, especiallywith B95-8RNA, is encodedby theBLRF1 orthe BLRF2readingframe,orboth, in theBamHI Lfragment (R. Seibl and H. Wolf,unpublished results).

pRZR1495 Bam Hi e3

2 3456 7689 2 34 567689 1

4 -- 4

200 *

116 . a

96 *uko..7 =

45 .

68 . - -_

.11

30

FIG. 3. Coomassie-stained electrophoretically separated bacte- rialproteins. Bacteria withoutplasmid (lanes 1), harboring apUR vectorwithout EBVDNAinserted (lanes2), orharboring theEBV expression plasmids withthedifferentreading frames fusedto the ,-galactosidase reading frame (lanes 3)wereinducedtoexpress the proteins controlled by the lac promoter, lysed, and subjected to electrophoresis.

1-Galactosidase

was alsoincluded in the marker protein mixture (116 kDa). The expressed

P-galactosidase

fromthe purepURplasmidcaneasily be identified by comparison with the bacteria lacking the plasmid. The fusionproteins were amixture of proteins translated at fulllength and proteins in which the EBV segmentispresent in amounts varying fromafewaminoacids to nearly full length, as indicated bya smear of proteins above the 116-kDa markerwithaccumulationsatcertainpreferential molecu- lar masses.Thedistribution of the different sizeswascharacteristic for theexpressed amino acidsequences. Inthepurificationprocess, thebacteriallysatewasmade3%withTritonX-100and thesoluble partof thelysate (lanes 4)was discarded. Theinsoluble part was dissolved in8 M ureaand subsequentlydialyzed against3 M urea.

Twobacterial proteins insoluble in8 M(lanes5) and 3 M(lanes 6) ureacouldbeseparated. The fusion proteinsremaineddissolvedin

experiments, indicatingthat the 80-kDaprotein may bealate protein.

From induced Raji, induced B95-8, and induced 883L cells, the mRNA foraprotein of38 kDa hybridized to the plasmids pCZR1495, pCZ453, BamHI-Z, and pCeZ1622.

Occasionally, theprotein wasresolved as a double bandof 38 and 37.5 kDa. With RNA from induced P3HR1 cells, in contrast,adoubleband of 40 and 39 kDa could betranslated after hybridization to the same fragments. To analyze the direction of the transcription, wecloned fragments ZR1495 and eZ1622 into M13mp8 and used single-stranded DNA from phage particles for hybrid-selected translation with P3HR1 RNA. With DNAprobes containing both strands of the respective DNA fragments (pCZR1495 and pCeZ1622), the identical pattern of all the proteins encoded by the BamHI Z fragment could be detected. The single-stranded DNA of the M13 clones represents strands of opposite polarity. pPZR1495 single-stranded DNA, which is in the leftward orientation, did not hybridize to any of the trans- latable mRNAs. With pPeZ1622 single-stranded DNA, a rightward strand, all the proteins could be detected.

Inconclusion, the mRNAs for the 92- to 96-kD and the 38- and 40-kDaproteins both ran in a leftward direction. Only DNA fragments containing sequences from the BZLF1 readingframehybridizedwiththetranscripts for the38- and 40-kDa proteins. Allfragments containing sequences from the BRLF1 reading frame, as well as fragment pCZ453, hybridized with the transcripts for the 92- to 96-kDa pro- teins. Therefore, it was necessary to prepare monospecific seraagainst thepolypeptidesencodedbythe different read-

3 M urea (lanes 7) and could be precipitated by dialysis against phosphate-buffered saline (lanes 8), in whichonly limitedamounts remainedin solution(lanes 9).

im 4m qv ^qw

Anew -cup '4M 4m -. do ow-4!.-

-- k

--.I-

906 SEIBL ET AL.

ing frames to furnish more detailed information on the proteinsencoded by them.

Expression of the open reading frames in bacteria. Large segments of the three open reading frames BRLF1, BZLF1, and BZLF2 were expressed as fusion proteins in E. coli.

Expression and purification of the proteins were controlled by Coomassiestaining (Fig. 3) and Western blotting (Fig. 4) of polyacrylamide gels. The Westernblotswereprobed with a serum pool from nasopharyngeal carcinoma (NPC) pa- tients. The fusion proteins reacted more strongly than did pure

P-galactosidase,

which was similarly expressed from pURplasmids. Therefore, antibodies againstthepolypeptide fusedto the,-galactosidase arepresent in the sera, in high titer in the case ofBZLF2 and BZLF1. All three reading framesare correctly expressed from the pURplasmids in the bacteria andarealsoexpressedfromthe viral DNA in NPC patients because antibodies againstthe proteins are present, asalso shown below by immunoprecipitation ofthe BRLF1 and BZLF1 proteins by the serumpool.

Thefusion proteinswere purified by a simpleand efficient method based upon the relative insolubility of 1- galactosidase fusion proteins. Nearly all the bacterial pro- teins were soluble in the bacterial lysate with 3% Triton X-100. The mainly insoluble fusion proteins were dissolved in 8M

urea-0.5%

mercaptoethanol. Afterdialysisagainst3 M urea,anothertwobacterial proteins could be separated.

Mostof the still-dissolved fusion proteins precipitatedupon dialysis against phosphate-buffered saline,andthis sediment was usedto immunizerabbits.

Identification of the protein encoded by the BZLF2 reading frame. After in vivo labeling of induced P3HR1, induced B95-8, and superinfected Raji cells, a protein of140 kDa could be specifically precipitated by the serum ofa rabbit immunized with the expressed BZLF2protein (Fig. 5). As expected, the protein couldnotbe detected in inducedRaji cells

(data

notshown), since the

reading

frame isdeleted in EBV DNA from the Raji strain (22). Therefore, in

superinfected

Raji cells, the 140-kDa proteinwasexpressed frominfecting P3HR1genomes. Noprotein could be precip- itated from in vitro translations, in accordance with the negative result of the hybrid-selected translations with the

Bam Hi e3 pRZR1495

pRZ453

1 8 4 3 2 8 4 3 2

3 1

9 6

30**

FIG. 4. Wester blot of the bacterallysates andsome purifica- tionstepsdescribed in thelegendtoFig.3.Probesidenticaltothose described in thelegendtoFig. 3wereseparatedinpolyacrylamide gelsandblottedonnitrocellulose.The blot was immunostainedwith apoolofserafrom NPCpatients.

cnC:

X- _

0_

mi c

- - m -CZt

c+

cc CL P

140

FIG. 5. Immunoprecipitation with rabbit serum against the BZLF2protein. Induced P3HR1, induced B95-8, superinfected Raji, and Raji cells were labeled in vivo with [35S]methionine. The negative serum of the rabbit prior to immunization (-) and the immuneserum(+) were usedin parallel forimmunoprecipitation.

An in vitro translation (T) of RNA from induced P3HR1 cells immunoprecipitated with the NPC serum pool (P) was run in parallel as marker. The two prominent proteins in the 140-kDa molecular massrangearethe138-kDaprotein encoded by the BALF2 reading frame (17) and the 143-kDa protein encoded by the BNRF1 reading frame(27).

BamHIe3 fragment. The mostlikely explanation for this is that the BZLF2 protein belongs to the group of proteins which can be moreeffeciently labeled by in vivo translation thanby in vitro translation, which is similartothe situation with the BZLF1 protein. Other proteins, e.g., the BRLF1 protein, behave in the opposite manner. If the BZLF2 protein belonged to the first group, the in vitro translation product would be below the detection limit, because the protein isalready very weakinin vivolabeling experiments.

Identification of the protein encoded by the BZLF1reading frame. The serum of the rabbit immunized with the ex- pressed BZLF1 proteinspecifically immunoprecipitatedthe 40- and 39-kDaproteins fromin vitro translations ofRNA from induced P3HR1 cells and the 38-kDa

protein

from in vitro translations ofRNAfrom induced B95-8 and induced Raji cells (Fig. 6). From extractsof in vivo-labeled induced B95-8,inducedRaji,inducedP3HR1,andsuperinfectedRaji

cells,

a

protein

of 35 kDa could be immunoprecipitated.

Small amounts of the 38- and 40-kDa primary translation products were also detectable. The protein seemed to be

posttranslationally

cleaved. The additional sequencesof the P3HR1 protein seemed to be removed as well. In the

superinfected Raji

cells,both the 38- and the40-kDaprecur- sor protein were visible, indicating that the gene might be expressed from

infecting

P3HR1aswellas

endogeneous Raji

genomes.

Identification oftheprotein encodedbytheBRLF1reading frame.By usingthe serumagainstthe BRLF1

polypeptide,

themappedinvitro-translated

proteins

of96, 94,and 92 kDa with RNAfrominducedRaji cells, inducedB95-8

cells,

and induced P3HR1 cells, respectively, could be precipitated (Fig. 7). From in vivo-labeled induced B95-8 and induced J.VIROL.

co

T V

.. I.

= - a:

cL _

V T

e"

- 1- LO =

V V

CCD

V

P

C._

I P

C3O T

p

440438 4b- _435*_

FIG. 6. Immunoprecipitation with the rabbit serum against the BZLF1protein. Thenegativeserumof the rabbitpriortoimmuni- zation (-) and the immune serum (+) were used in parallel to immunoprecipitate in vitro translations (T) of RNA from induced B95-8 and induced P3HR1 cells andextractsof in vivo-labeled(V) induced B95-8, induced P3HR1, superinfected Raji, and induced Raji cells. An in vitro translation (T) of unselected RNA from induced P3HR1 cellsimmunoprecipitatedwith the NPCserumpool (P) was runinparallel. The serumpoolwasalso usedtoimmuno- precipitate in vivo-labeled superinfected Raji cells, in which the 35-kDabandconsists of more thanonepolypeptide (2). Thesameis truefor the 40-kDa band precipitated by theserumpool from in vitro translations ofRNAfrom induced P3HR1cells(27).

P3HR1cells, proteins with molecularmassesslightly higher than those of proteins from the corresponding in vitro translation experiments were precipitated. The protein seemedtobeposttranslationally modified bytheaddition of an unknown component. In the induced Raji cells, the protein had thesamemolecularmassafter invivoorin vitro translation

according

to sodium dodecyl sulfate-polyacryl- amidegel electrophoresis.

96F _

DISCUSSION

Weanalyzed the transcriptionandtranslationof the EBV BamHI Z areaindifferent celllines. Thisregion is charac- terized by considerable strain-specific protein polymor- phism. These differences enabled us todemonstrate thatin

superinfected

Raji cells, both the endogeneous Rajigenome and the

infecting

P3HR1 genomes (standard or defective) seem tobe expressedintheBamHI Z area.

In contrasttothe other celllinestested, no difference in theexpression of B95-8 and883L

cells

was observed. B95-8 cells have been established by in vitro transformation of marmosetlymphocyteswithvirusderivedfrom the 883L cell line (15). The expression of the

BamHI

Z area remained stablethroughoutthepropagation ofthe celllines. This may indicate that the observed protein polymorphismrepresents

strain-specific

variations alreadypresent in thepatients from

which the cell lines were established and does not reflect consequences ofpropagationincellcultures. Similarstrain polymorphism has been reported for herpes simplex virus (20).

Thethree openreading frames of thisarea wereexpressed as fusion proteins with

P-galactosidase

in E. coli. This approachwaschosenbecause expressionofpureeucaryotic proteinsin bacteria is, in many cases, not efficient, owing to degradation by proteases or incomplete translation. Even in the

P-galactosidase

fusion proteins, the viral segment is frequentlynotsynthesizedatfulllength.Afurtheradvantage of the expression as I-galactosidase fusion protein is the possibility ofrapid and efficient purification by successive precipitation. These protein preparations were used to im- munize rabbits to generate serawhich specificallyrecognize theprotein(s) encoded by the appropriate reading frame.

Thereadingframe BRLF1 encodes aproteinof93 to 96 kDa, depending on the cell line. Additionally, smaller pro- teins in the range of 75 to 83 kDa could be mapped to this reading frame. These presumably represent shorter transla- tionproducts due to truncated mRNAs, which appear in the hybrid-selectedtranslation as indicatedby Northernblotting of selected and unselected RNA (data not shown). The BRLF1 protein is posttranslationally modified, at least in induced B95-8 and induced P3HR1 cells, because smaller proteins were precipitated from in vitro translations than from in vivo-labeled cells. The molecular mass calculated from thesequence of the open reading frame is only 67 kDa.

It cannot be ruled out thatadditional smallcodingsequences from elsewhere on the genome, which would not bedetected under the stringent hybrid selection conditions or other coding sequences in the BamHI Z and R fragments, are fused to BRLF1bysplicing.This seems,however,notvery likely, because similar differences in the calculated molecu- lar mass and that obtained by polyacrylamide gel electro-

T V

T_^_

co LS:a -=

I

T V

a,

V T

.=- Z;( C,,

ccC tL

1r

a: ¹

T T V V

+ + P

492 92

a

FIG. 7. Immunoprecipitation with theserumagainst the BRLF1 protein. The negativeserumof the rabbitpriortoimmunization(-) andthe immuneserum(+)wereusedinparalleltoimmunoprecip- itate invitro translations (T) ofRNAfrom inducedRaji, induced B95-8, and inducedP3HR1cellsandinvivo-labeledextracts(V)of the same cells and of superinfected Raji cells. In vivo-labeled superinfected Raji cells immunoprecipitated with the NPC serum pool(P)were runinparallel.

908 SEIBL ET AL.

phoresis are known from other readingframes.Accordingto our hybridization data, the transcript extends from the BamHI R fragment to the BamHI Z fragment between nucleotides 101750 and 102700. Sequence analysis has re- vealed theexistence of twoTATAboxes nearthe5' endof BRLF1,aswellas twosuitable downstreampolyadenylation sites(1).

Thereading frameBZLF1encodesa35-kDaprotein. The protein is translated intoa38-kDaprecursor and seems to be subsequently cloven. In P3HR1 cells, the precursor has a molecular mass of 40 kDa, whereas the protein identified by invivo labeling is of the normal size. Ininvitro translation assays, the 35-kDa protein was notdetectable, butasecond band of 39 kDa in P3HR1 cells and 37.5 kDa in B95-8 and Raji cells, respectively, was seen. Theprocessing may start in the in vitro translation assays but cannot be completed there. The calculated size ofthe protein, according to the sequenceofBZLF1,is only21kDa. Therefore, it is possible that other reading frames contribute to this protein. The transcript doesnothybridizetothe

neighboring fragments

of BamHI-Z. One of thepolyadenylation sites discussed for the BRLF1 transcript may be used.

The reading frame BZLF2 encodes a 140-kDa protein;

however, the calculatedsize of the protein translated from BZLF2 is only25 kDa. Therefore, other coding sequences arepresumably splicedtothis frame. The5'end of the amino acid sequence encoded byBZLF2consists ofa

hydrophobic

domain which fulfills the criteriaofasignal peptideconsen- sussequence(21, 31, 32). Signal peptidesareresponsible for the vectorial translation of secretory and membrane pro- teins. The 140-kDa EBV membrane protein, reported from severallaboratories (8, 18, 33), maybe

partially

encodedby BZLF2.

With the

specific

sera

against

the

proteins

encoded

by

the BamHI Z area modification, time-dependent

synthesis

and cellularlocalization of the

proteins

are now accessible for examination, and their role in the trans-induction of latent EBVgenomes can be studied.

ACKNOWLEDGMENTS

We thankWolfgang Jilgforhelpfuldiscussions and immunization of the rabbits.

Thisworkwas supported by grantWo227/4 from the Deutsche Forschungsgemeinschaft.

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