JOURNAL OF VIROLOGY, Dec. 1994, p. 7927-79320022-538X/94/$04.00+0Copyright C) 1994, American Society for Microbiology

The Activated Mlvi-4 Locus in Moloney Murine LeukemiaVirus-Induced Rat T-Cell Lymphomas Encodes

an Env/Mlvi-4 Fusion ProteinCHRISTOS PATRIOTIS AND PHILIP N. TSICHLIS*

Department of Medical Oncology, Fox Chase Cancer Center,Philadelphia, Pennsylvania 19111

Received 16 June 1994/Accepted 30 August 1994

A genomic DNA probe derived from the region immediately 3' of the clusters of integrated proviruses in theMlvi-4 locus detects a 5.5-kb mRNA transcript which is specifically expressed in normal rat thymus and spleen.The same probe detects two tumor-specific mRNA transcripts 2.5 and 10 kb long, both of which are expressedonly in tumors carrying a provirus in the Mlvi-4 locus. Sequence analysis of two cDNA clones (LE3a and B1.l)of the 2.5-kb tumor-specific mRNA, obtained from two independent tumors (6889 and Bi), revealed that theyare both derived from hybrid env/Mlvi-4 mRNA transcripts. The splicing of env to Mlvi-4 sequences linked a

cryptic splice donor site at nucleotide position 6397 of the viral genome with a splice acceptor site in the regionimmediately 3' of the integrated provirus. The mRNA that gives rise to cDNA clone B1.1 terminates 1,005 bases3' of the splice acceptor site without additional splicing. The mRNA that gives rise to cDNA clone LE3aterminates in the same site but undergoes differential splicing of an 81-base-long intron. The resulting mRNAscontain 247-amino-acid (clone B1.1) or 226-amino-acid (clone LE3a) open reading frames sharing 221N-terminal amino acids, ofwhich 207 are derived from the viral env gene and 14 are derived from Mlvi-4. RNaseprotection assays using 6889 tumor cell RNA and a probe derived from the cDNA clone LE3a detected bothmRNA transcripts. More abundant of the two, however, was the one encoding the putative 247-amino-acidprotein. Transient transfections of a construct expressing the RNA transcript defined by clone Bl.1 into D17cells led to the expression of an Env/Mlvi-4 fusion protein with an apparent molecular mass of 33 kDa. Giventhat cells with provirus insertions in the Mlvi-4 locus are selected and that retroviral env gene products may

have profound effects in the biology of hematopoietic cells, we suggest that the detected fusion proteins maycontribute to the growth of T-cell lymphomas.

Oncogenesis by retroviruses that lack an oncogene dependson insertional mutagenesis of cellular genes involved in theregulation of cellular proliferation and differentiation (14, 24).One of the genes targeted by provirus integration is the c-mycproto-oncogene. Clusters of independent provirus insertionhave been detected in a variety of retrovirus-induced lymphoidneoplasms both 5' and 3' of this gene. Two such clusters ofprovirus integration, identified in this laboratory, were mappedat 27 kb (Mlvi-4) (25) and -300 kb (Mlvi-llpvt-1) (13, 26) 3' ofc-myc. Provirus insertion in both loci in Moloney murineleukemia virus (MoMuLV)-induced rat T-cell lymphomasaffected the expression of c-myc by cis-acting mechanisms (16).Moreover, provirus insertion in the Mlvi-4 locus was linked tothe expression of abundant 2.5- and 10-kb tumor-specific RNAtranscripts hybridizing to a probe derived from the regionimmediately 3' to the Mlvi-4 provirus (25). The same Mlvi-4probe detected a 5.5-kb mRNA transcript in normal thymusand spleen (25).The experiments in this report were undertaken to deter-

mine the structure and coding potential of the tumor-specificMlvi-4 mRNA transcripts. The results suggest that the 10-kbRNA may be a noncoding readthrough transcript containingboth viral and cellular sequences, while the 2.5-kb RNA is a

heterogeneous population of MoMuLV/Mlvi-4 hybrid mRNAtranscripts encoding 226- or 247-amino-acid Env/Mlvi-4 fusionproteins.

* Corresponding author. Mailing address: Fox Chase Cancer Cen-ter, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: (215)728-3635. Fax: (215) 728-2741.

MATERIALS AND METHODS

Tumors and cell lines. The MoMuLV-induced rat T-celllymphomas and the cell lines derived from them have beenpreviously described (15). D17 canine osteosarcoma cells werekindly provided by D. Kaplan (Frederick Cancer ResearchFacility, Frederick, Md.) and were maintained in Dulbecco'smodified Eagle's medium supplemented with 10% calf serum,penicillin (50 U/ml), and streptomycin (50 pg/ml).cDNA cloning and sequencing. Total cellular RNA was

isolated by the guanidine thiocyanate-acid-phenol method (2).Polyadenylated RNA was selected by affinity chromatographyin oligo(dT)-cellulose (Boehringer Mannheim) as instructedby the manufacturer. cDNA libraries were constructed frompoly(A)-selected RNA derived from tumors Bi and 6889. Priorto cDNA synthesis, the RNA derived from tumor 6889 was sizefractionated in a linear 10 to 30% sucrose gradient containing0.5% sodium dodecyl sulfate (SDS). Centrifugation of thegradients was carried out in an SW41 Beckman ultracentrifugerotor at 210,000 x g for 15 h (22). cDNA synthesis was carriedout by using oligo(dT)12_18 primers and a cDNA cloning kit(Pharmacia) as instructed by the manufacturer. The synthe-sized double-stranded cDNA was ligated to the EcoRI site ofthe bacteriophage lambda vector lambda ZaplI (Stratagene)by using EcoRI-NotI adaptors. The ligation products were invitro packaged, and the resulting phage particles were propa-gated in Escherichia coli XL-1 blue (Stratagene) or LE392 (22).

Mlvi-4 cDNA clones identified by plaque hybridization werein vivo excised. The 5' and 3' ends of the resulting pBluescriptplasmid clones and sequential deletions of two of them (B1.1and LE3a, derived from tumors Bi and 6889, respectively)

7927

Vol. 68, No. 12

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from

7928 PATRIOTIS AND TSICHLIS

LOc- D

'aCl)

a)

CD m

FIG. 1. Northern (RNA) blot analysis of polyadenylated RNAfrom normal rat spleen and thymus and from the rat T-cell lymphomasBi and 6889. The blot was hybridized to the Mlvi-4 genomic probepLE18 (25).

121

241

361

481LE3aBl.1

601LE3aB1.1

721LE3aBl.1841

LE3aB1.1

961LE3aB.1

1081LE3aB1.1

1201LE3aBl.1

1321

1441

1561

1681

1801

1921

2041

were sequenced by using T3 and T7 sequencing primers andthe dideoxy-chain termination method employing Sequenaseversion 2.0 (U.S. Biochemical) (22). The products of thesequencing reactions were analyzed in 4 and 6% polyacryl-amide-8.3 M urea gels.RNase protection. RNase protection assays were carried out

as previously described (1). Briefly, a nearly full-length cDNAclone, obtained as described above, designated LE3a, was usedas a template to synthesize in vitro a cold (unlabeled) senseand a 32P-labeled antisense env/Mlvi-4 RNA (see Fig. 4A). Thesense RNA was synthesized by using T3 RNA polymerase andcontains 100-base-long plasmid-derived sequences (PDS) at its5' end and 30-base-long PDS at its 3' end. The antisense RNAwas synthesized by using T7 RNA polymerase and contains88-base-long PDS at its 5' end and 32-base-long PDS at its 3'end (see Fig. 4B). The in vitro-synthesized, 32P-labeled anti-sense RNA (106 cpm) was annealed to 5 p,g of polyadenylatedRNA derived from 6889 cells or the in vitro-synthesized senseLE3a RNA. Annealing was carried out for 16 h at 50°C in abuffer containing 80% deionized formamide, 40 mM pipera-zine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 6.7), 0.4 MNaCl, and 1 mM EDTA. Annealing was followed by onephenol-chloroform and one chloroform extraction and ethanolprecipitation. The precipitated RNA was resuspended in 100mM LiCl-200 mM NaCl-10 mM Tris-HCl (pH 7.5)-5 mM

r--R r- LE3a R -,I U5GCGCCAGTCCTCCGATTGACTGAGTCGCCCGQGTACCCGTGTATCCMATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGiTGATTGAC

r--o-EflvTACCCT_ CGGTTTCTTGGCCTCOGGGATSsGGGGCCCCTGCCCAGGGA CCCGACCCACCACCGGG^GC TCACTTAC AGGCTCTCTACTTAGTCCACCGiAA

r-o Bl.l1GTCTGGAGACCTC;TGGCGGCAGCCTACAGAChTGGACCGCGTTACTCACCCTTACOGAGTlGGAAATGTGGGTCCGCCGACCCGACTAACTAGAACCT

CGCTGGCAGCTTGGRTACTvA^AGAGGCCGCCACGTGAAGGCT

TCTAG;ACTGACATGGC GCGTTCAACGCTCTCAACCTAAATAGTAACGGAGCCCTAATCCCCTTAATTCTTCTGATGCCGGGTQTCGTCGCOM A R S T L S I P L h N K V N P R G P L I P L I L L M L R G V S T A S P G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

GCTCAGTCCTAGCRATTACCCaTAACCAATGGAGASTCGGGTAGCGTTGGGT CTCTGGCACACCTCTG_S S P H O V Y 1f z r w E V T N G D R E T V N A T S G N H P L N T N N P D L 7 P D

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ATTTATGTATGTTAGCCCACCATGGACCTTrGCTAATCATCCCCT T C T T CAGTPC M L L A H H G P S Y V G L E Y Q S P F s S P P G P P C C S G G S S P G C S R D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ACTGCGAAACTTTAACCTCTACCCCTCGGTGCAAATCGACGCTCAAGCTAGACCAG ACACTCATAAATCAATATGAGITTTTAGCCCGCCC E E P L T S L T P R C N T A W N R L E L D Q T T H K S N E G F r V C P G P H R

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

GCCCCCGAGAATCCAAGTCATGTGGGGGTCCAG^cACrGTACCTTCTACGTGCCTCTCATCTGGGTTCACTCACACrTAAP R E S K S C G G P D S F Y C A Y I G C E T T G R A Y N K P S S S W D F I T V N. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I Mlvi-4ACAACAATCTCACCTCTGACCAGGCTCCAGGATTCTCCTCATGGTTGTCGAAGGTCTCGCAN NL r S D Q A V Q D S P H G L S K V L Q L IQ-- - - -

....... . . . . . . . . . . . . . . . G S H C R L H A G F E L R V I R

GCCTTTAcTchAlTcTTcATGTT;ATTCTCAGCCT TTCAAMGCAACCAGACACCCT- - - - - - - - - - - D F H E A*L S Q D S K F R N Y *

CACATACTGGACCCCCTACTCATGGTGCACTTrCTTGAACTTTCGTGAAAACTTCTT CTCTGCCTGTlT&CAAG;TGTCTCCTTCT GCCAGACACCTCCTCTTGACCCCAACTCT

TGGCCTATTA&GCTCATTCTCCAAATTCACTTGRGAOATGTAT CCTTCTCTGTCCCTAGAGGGG CCCATCTGAACCCTAATTCAGCCCTGGATAMCTCACCTAAMAGTAATAGA

GGGGGAACGGTAAAACCo rA C M TAT G TTG;CGGTTACGTCG TGCTaGGAGTCCTGGGCTGAGCTT

CCCAGAATTGAGTCATCACTTCACT TACAACTTTT ATCCTACACTCAG _AT;MWAAATTCAGCTGCTTACTATATGCTCGGCCCAGCTCTAGTGTACCTCAAC

TCCTGAGATGGTCACTGTAACTTGAATGTGGCGACTGTCCCTATCACTATGCTGTTAGGMACGAGGCATA AA; AAAAACTGGCACAAGGTCACTCATCAAATAAG

AGCTrGGCTTCAGATTATTGTCAlATTCTCSTTATTTGrrATTCTTACATGTATATATATTGAATTAAGCTCTGTGA

. GAAAGSHCRLHAGFELRVIRCTA

120

240

360

480

600

720

840

960

1080

1200

1320

1440

1560

1680

1800

1920

2040

2119

FIG. 2. Nucleotide and deduced amino acid sequences of the full env/Mlvi-4 hybrid mRNA transcripts. The 5' ends of the cDNA clones LE3aand B1.1, the 5' end of R, the junction between R and U5, the beginning of the viral env gene, and the env/Mlvi-4 junction are indicated with arrows.

The amino acids encoded by the viral env gene are shown in italics, while the amino acids encoded by the Mlvi-4 sequences are shown in romantype. The first 220 amino acids encoded by the mRNAs defined by the cDNA clones LE3a and B1.1 are identical. The identity between the two

sequences is indicated by the dots in the B1.1 line. The first 14 Mlvi-4 amino acids, shared by the proteins encoded by the two mRNAs, are

underlined. The boxed sequence defines the intron which is differentially spliced out in the LE3a-encoded mRNA. The interrupted line shownunder the boxed sequences in the LE3a line corresponds to the amino acids missing from the protein encoded by the LE3a mRNA. The two

putative glycosylation sites are shown in boldface. The asterisks correspond to termination codons.

J. VIROL.

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from

Env/Mlvi-4 FUSION PROTEINS 7929

EDTA and digested with a mixture of RNase T1 (5 ,g/ml) andRNase A (50 ,ug/ml). The products of the RNase digestionwere analyzed in 4% polyacrylamide-8.3 M urea gels.

Transfections. A eukaryotic expression construct (pLE3ahy/J4Q) which was predicted to direct the expression of hybridMoMuLV/Mlvi-4 transcripts identical to the most abundantMlvi-4 transcripts detected in 6889 and B1 cells (see Fig. 2 and3) was constructed as follows: a nearly full-size envlMlvi-4hybrid cDNA, designated LE3ahy, was constructed by insert-ing a PstI-HindIII internal fragment derived from an incom-plete Bl-specific envlMlvi-4 cDNA clone (B1.1) in the corre-sponding sites in cDNA clone LE3a. The KpnI site in the Rregion of the MoMuLV long terminal repeat (LTR) in thepBR322-based vector pJ4Q (20) was reconstituted by insertingan XbaI-KmnI fragment derived from an intact MoMuLV LTRinto the XbaI site of the pJ4fQ LTR and the KpnI site of thepolylinker of this vector. The reconstituted KpnI site in theLTR and the BamHI site of the polylinker were used forintroduction of the LE3ahy cDNA. The resulting construct(pLE3ahy/J4Q) (see Fig. 5A) or the pJ4Q vector were thentransfected transiently into D17 canine osteosarcoma cells bycalcium phosphate precipitation using standard procedures(12). Briefly, 10 jig of the corresponding plasmid DNA wasmixed with lx BES [N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid]-buffered solution, pH 6.95 (BBS)-125 mM CaCl2in a final volume of 500 RI for 15 min at room temperature.One hour prior to the transfection, the cells (-106 per60-mm-diameter dish) were given 4.5 ml of fresh completemedium, and the DNA-CaCl2-BBS suspension was addeddropwise. The cells were then incubated at 35°C in 3% CO2overnight (at least 16 h). The cells were finally washed twicewith Dulbecco's phosphate-buffered saline at room tempera-ture and reincubated with fresh complete medium for anadditional 48 h at 37°C and 5% CO2. At 48 h from the time oftransfection, the cells were analyzed for expression of Env/Mlvi-4 hybrid protein.

Immunoprecipitation and SDS-polyacrylamide gel electro-phoresis. At 48 h from the time of transfection, D17 cellstransfected with plasmid pLE3ahy/J4Q or pJ4Q were labeledfor 4 h with [35S]methionine (250 uCi/ml; ICN). The cells weresubsequently lysed in 500 RI of radioimmunoprecipitationassay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl,1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mMphenylmethylsulfonyl fluoride, 2 ,ug of leupeptin [Sigma] perml, 2 jig of aprotinin [Sigma] per ml). Immunoprecipitations ofthe Env/Mlvi-4 hybrid protein from the transfected cell ex-tracts were carried out with a goat anti-Rauscher murineleukemia virus gp7O antiserum (Microbiological Associates)according to standard procedures (19). Briefly, the cell lysateswere centrifuged at 12,000 x g for 10 min at 4°C andprecleared with 25 RI of 50% protein A-agarose beads inRIPA buffer. The precleared lysates were then incubated with1 RI of anti-gp7O serum (1:500) and 40 RI of 50% proteinA-agarose beads at 4°C for 4 h. Following low-speed centrif-ugation (4 min at 5,000 x g), the pelleted agarose beads werewashed three times in a buffer containing 50mM Tris-HCl (pH7.5), 5 mM EDTA, 5% sucrose, 1% Nonidet P-40, and 0.5 MNaCl and once in RIPA buffer at 4°C. The washed immuno-precipitates were resuspended in SDS-polyacrylamide gel elec-trophoresis loading buffer (0.75 M Tris-HCl [pH 6.8], 1 Murea, 100 mM dithiothreitol, 10% SDS, 50% glycerol, and0.15% bromophenol blue), boiled for 5 min, and electropho-resed in 0.1% SDS-12.5% polyacrylamide gels.

A SMoMuL V Miv,-4

5'-- [21- 3'

B B 1.1

II(Env) E-r- III (Mlvi-4)

...Q D S P H G L S K V L a L Q a 8 H C R L H

A G F E L R V R L S a P S K F R N Y -

CCATQAAGCTTA..

C LE3a

ET* Illb...CATcCTCAlG lll=l_l x 1A1o1 ..

a D S P H G L S K V L a L Q D F H E A '

FIG. 3. Structure of the tumor-specific env/Mlvi-4 hybrid tran-scripts. (A) Linear diagram of the rearranged Mlvi-4 locus. S.D, envsplice donor site. (B) Splicing events giving rise to the long envlMlvi-4hybrid transcript represented by cDNA clone B1.1. The lower portionshows the nucleotide and deduced amino acid sequences at theenv/Mlvi-4 junction. (C) Structure of the short mRNA transcriptrepresented by cDNA clone LE3a. Exon Illa represents the first 43bases of cell-derived sequences (underlined) which are present in boththe long and short envlMlvi-4 hybrid transcripts. Exon IlIb representsthe 3' cellular sequences attached to the splice donor of exon IIIafollowing differential splicing. Splicing of exon Illb to exon lIla isresponsible for a switch in the reading frame. Therefore, the proteinsencoded by the two transcripts have different C-terminal ends.

RESULTScDNA cloning of the 2.5-kb tumor-specific Mlvi-4 mRNA

transcript and sequencing of cDNA clones. Four of fortyanalyzed MoMuLV-induced rat T-cell lymphomas contain a

provirus in the Mlvi-4 locus. Using probe pLE18 from theregion 3' of the integrated provirus (25), we demonstrated lowsteady-state Mlvi-4 RNA levels in thymocytes and spleen cellswith a normal uninterrupted locus. Provirus insertion is asso-ciated with the enhanced expression of two 2.5- and 10-kbaberrant Mlvi-4 RNA transcripts (Fig. 1). To characterize thesetranscripts, we obtained cDNA clones of them by screeningcDNA libraries constructed from polyadenylated RNA derivedfrom the T-cell lymphomas Bi and 6889 as described inMaterials and Methods. Screening the 6889 library with thepLE18 Mlvi-4 probe yielded 12 1.2- to 3.6-kb-long cDNAclones. DNA sequence analysis revealed that the 3' end, whichwas shared by all clones, mapped 2.85 kb 3' of the site ofprovirus integration. The 5' ends, although unique for eachclone, were all of viral origin. In nine clones, the Mlvi-4sequences were linked to the 3' LTR, which was flanked at its5' end by the positive-strand primer binding site and env.

These clones may define 5' truncated cDNA copies of read-through viral RNA transcripts. The only candidate RNAtranscript to give rise to these cDNA clones was the 10-kbMlvi-4 RNA. The remaining three clones were cDNA copies ofspliced MoMuLV/Mlvi-4 hybrid mRNA transcripts. The lattermay have been derived from the 2.5-kb mRNA. Sequenceanalysis (Fig. 2 and 3) of a nearly full-length cDNA clone(LE3a) of a spliced mRNA revealed that (i) the 2.5-kb RNAtranscript initiates at the 5' LTR of the provirus integrated inthe Mlvi-4 locus and is spliced to the env gene by utilizingnormal retroviral splicing signals and (ii) the sequences encod-ing the 207-amino-acid N terminus of Env are spliced onto the

VOL. 68, 1994

II(EnvJ <- Illia(Mlvi-4J

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from

7930 PATRIOTIS AND TSICHLIS

4--

B ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-f

IIII

I ~ ~~~II.._..

-* ., v

.A .;@A A.,4

C

1 35 3

1 078 -

872 -

a b c C.i

FIG. 4. RNase protection assay. (A) The LE3a cDNA clone inpBluescript SK(-) was used as a template to in vitro synthesize a coldsense [T3(+)RNA] and 32P-labeled antisense [T7(-)RNA] env/Mlvi-4RNA, using T3 and T7 RNA polymerases, respectively. B, BamHI; E,EcoRI; C, ClaI. (B) The T3(+)RNA contains 100-base-long PDS at its5' end and 30-base-long PDS at its 3' end. The T7(-)RNA contains 88bases of PDS at its 5' end and 32 bases of PDS at its 3' end. 32P-labeledT7(-)RNA was annealed to poly(A)+ 6889 RNA or cold T3(+)RNAas described in Materials and Methods. (C) Lane a, the band indicatedwith the upper arrow represents the undigested, 32P-labeled T7(-)RNA. Lane b, the band indicated with the middle arrow represents the32P-labeled T7(-)RNA annealed to T3(+)RNA and digested withRNase. PDS are removed by this treatment, which leads to a 58-basesize reduction (from 88 to 30 bases) of the 32P-labeled T7(-)RNA.Lane c, annealing of the 32P-labeled T7(-)RNA to poly(A)+ 6889 cellRNA and RNase digestion gives rise to two major 1,113 and 923-base-long bands. Overexposure of the same in lane d revealed a minor band120 (88 + 32) bases shorter than the band seen in lane a. Interpreta-tion of these data is shown in panel B. The protected minor bandshown in lane d represents an mRNA which is colinear with cDNAclone LE3a. The low intensity of the protected band in this lanesuggests that this mRNA is present at low abundance in the tumorcells. The sizes of the two major 1,113- and 923-base-long bands are

consistent with the hypothesis that they are produced by protection of

flanking Mlvi-4 sequences, using a cryptic splice donor site. Theresulting mRNA transcript terminates at a site 1,005 bp 3' ofthe splice acceptor site without additional splicing or afterdifferential splicing of an 81-bp intron. The differentiallyspliced mRNA transcript encodes a 226-amino-acid hybridprotein composed of an NH2-terminal domain (207 aminoacids) encoded by the env gene and a C-terminal domain (19amino acids) encoded by Mlvi-4. The nondifferentially splicedmRNA encodes a 247-amino-acid protein. The two proteinsshare 221 N-terminal amino acids, of which 207 are derivedfrom env.RNase protection. The 6889 tumor-specific cDNA clone

LE3a was used as a template for in vitro synthesis of a coldsense [T3(+)RNA] and a 32P-labeled antisense [T7(-)RNA]envlMlvi-4 RNA, using T3 and T7 RNA polymerases, respec-tively (Fig. 4A). The T3(+)RNA contains 100-base-long PDSat its 5' end and 30-base-long PDS at its 3' end. TheT7(-)RNA contains 88-base-long PDS at its 5' end and32-base-long PDS at its 3' end. The 32P-labeled T7(-)RNAwas annealed to poly(A)+ 6889 RNA or the cold T3(+)RNA.Following treatment with RNase T, and RNase A, the pro-tected RNA hybrids were analyzed in polyacrylamide-urea gelsas shown in Fig. 4C. The band indicated with the upper arrowin lane a represents the undigested, 32P-labeled T7(-)RNA(2,131 bases). The middle arrow shows the 32P-labeled RNAwhich was protected following annealing to T3(+)RNA anddigestion with RNase. This treatment removes 58 bases of PDSfrom the 32P-labeled T7(-)RNA and gives rise to a 2,073-base-long RNA band. Annealing of the 32P-labeled T7(-)RNA to poly(A)+ 6889 RNA protects two major 1,113- and923-base-long bands (Fig. 4C, lane c). Longer exposure of thesame gel revealed a minor band, indicated with the lowerarrow, 120 bases shorter (1,953 bases) than the band seen inlane a. The protected minor band in lane d represents anmRNA which is colinear with the cDNA clone LE3a. The lowintensity of the protected band in lane d suggests that thismRNA is present in low abundance in 6889 tumor cells. On theother hand, protection of the two major 1,113- and 923-base-long bands (lane c) is consistent with the hypothesis that themost abundant mRNA transcript differs from the mRNAtranscript defined by cDNA clone LE3a in that it may carry anunrelated insert at nucleotide position 1113 (Fig. 4B). ThemRNA transcript specified by cDNA clone B1.1 contains an81-base-long insert (exon Illa) in this position. We concludetherefore, that the mRNA defined by clone B1.1 and encodingthe larger 247-amino-acid protein is likely to represent themost abundant env/Mlvi-4 mRNA transcript in tumors with aprovirus in the Mlvi-4 locus.The 2.5-kb RNA encodes a 33-kDa env-derived protein. A

nearly full-length env!Mlvi-4 cDNA clone, LE3ahy, corre-sponding to the most abundant env/Mlvi-4 transcript, detectedin Mlvi-4-positive tumor cells, was constructed as described inMaterials and Methods. LE3ahy was then introduced as aKpnI-BamHI insert in the eukaryotic expression vector pJ4Qafter the KpnI site in the LTR sequences in the vector wasreconstituted (Fig. 5A). The resulting expression construct,pLE3ahy/J4Q, is predicted to direct the expression of anmRNA transcript identical to the most abundant Mlvi-4 tran-script in 6889 and Bi cells. This construct was transientlytransfected in D17 canine osteosarcoma cells by calcium

the T7(-)RNA by the long env/Mlvi-4 transcript defined by clone B1.1.Therefore, the B1.1 DNA clone is likely to represent the mostabundant env/Mlvi-4 transcript in the tumor cells.

A

J. VIROL.

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from

Env/Mlvi-4 FUSION PROTEINS 7931

A

AnAr

B

50-

32-

pLE3ahy/J4Q2pJ4Q 1 2 3

FIG. 5. Construction of an envlMlvi-4 expression construct andexpression of the fusion protein in D17 cells. (A) The full-size cDNAof the long Env/Mlvi-4 transcript (LE3ahy) was constructed by trans-ferring an internal PstI-HindIII fragment (nucleotides 1140 to 1246)from the incomplete B1.1 cDNA into the LE3a clone. The LE3ahycDNA was then inserted as a KpnI-BamHI insert in the pJ4Q vector.Prior to this, the KpnI site, previously eliminated from the vectorMoMuLV LTR, was reconstituted. N, NheI; K, KpnI; B, BamHI; S.D,env splice donor site; SV40, simian virus 40; tIVS, intervening se-

quences. The 5' end of env and the env/Mlvi-4 boundaries are indicatedby arrows. ATG and TGA define the translation initiation andtermination codons. (B) Immunoprecipitation of the Env/Mlvi-4 fu-sion protein from lysates of D17 cells transfected with pJ4Q1 alone orwith the pLE3ahy/J4fQ expression construct, as indicated. Before lysisand immunoprecipitation, the cells were metabolically labeled with[35S]methionine. The immunoprecipitations were carried out with ananti-Rauscher murine leukemia virus gp7O rabbit polyclonal antibody.Sizes are indicated in kilodaltons.

phosphate precipitation. The cells were metabolically labeledwith [35S]methionine 48 h after transfection, and followinglysis, they were used for immunoprecipitation with an anti-Rauscher murine leukemia virus gp7O polyclonal antibody.The results (Fig. SB) showed that following transfection of thepLE3ahy/J4Ql construct, D17 cells express a protein with an

apparent molecular mass of approximately 33 kDa. The pre-dicted size of the protein product encoded by the env/Mlvi-4hybrid mRNA after the removal of the signal peptide is -24kDa. The difference between the predicted and the apparent

molecular weight of this protein is probably due to glycosyla-tion at the two putative glycosylation sites (Fig. 2) character-istic of retroviral Env proteins.

DISCUSSION

Earlier studies had shown that provirus insertion in theMlvi-4 locus activates the c-myc proto-oncogene by a cis-actingmechanism (16). The data presented in this report suggest thatin addition to the activation of c-myc, provirus integration inMlvi-4 may contribute to retrovirus oncogenesis by inducingthe synthesis of viral-host hybrid RNA transcripts encodingaltered envelope glycoproteins.Tumor induction and progression by nonacute retroviruses

depends in part on proliferative signals mediated by theprotein product of the retrovirus env gene (5, 18, 23, 24). Therole of retrovirus-encoded envelope glycoproteins in oncogen-esis was originally suggested by the isolation of enveloperecombinant viruses from the preleukemic thymus of mousestrains with a high incidence of leukemia (7, 8). This role wasconfirmed by testing the oncogenic potential of natural or invitro-constructed envelope recombinant viruses (3, 6, 10, 21).To explain how the retrovirus envelope glycoprotein mayinfluence oncogenesis, several hypotheses were proposed andtested. One of these hypotheses suggests that the envelopeglycoprotein interacts with growth factor receptors and stimu-lates the transmission of proliferative signals to the nucleus.Strong evidence in support of this hypothesis was provided bythe finding that the defective envelope glycoprotein of spleenfocus-forming virus (SFFV) binds to the erythropoietin recep-tor and promotes the growth of erythroid precursor cells (4,17). Additional support was provided by the finding that minkcell focus-forming (MCF) viruses may alter the growth factorrequirements of growth factor-dependent lymphoid cell lines.In our earlier studies, we had shown that infection with MCFviruses rendered an interleukin-2 (IL-2)-dependent T-cell lym-phoma line IL-2 independent (23) and that IL-2 independenceis associated with the rapid selection of cells expressing IL-9and carrying a provirus in a locus (Gfi-2) which encodes theIL-9 receptor (5). Moreover, Li and Baltimore have shown thatMCF virus infection renders an IL-3-dependent lymphoid cellline IL-3 independent and that this transition is dependentupon the expression of the IL-2 receptor (18). These datacombined suggested that the envelope glycoprotein of theMCF viruses interacts with the IL-2 receptor and that thisinteraction initiates signals that alter the growth requirementsof the infected cells.The glycoprotein encoded by the env/Mlvi-4 hybrid mRNA

transcripts may modulate the growth factor requirementsand/or the rate of proliferation of the affected cells. The fusionproteins between the MoMuLV env gene product and Mlvi-4may be analogous to the defective gp55 envelope glycoproteinof SFFV. In the case of gp55, it has been suggested thatC-terminal truncation of gp7O due to deletion of its transmem-brane and cytosolic domains unmasks the ability of this proteinto interact with the erythropoietin receptor (4, 9, 11). TheEnv/Mlvi-4 fusion proteins retain the N-terminal domain ofthe MoMuLV Env glycoprotein, and they may be structurallyanalogous to the SFFV gp55. The structure of these proteinsand the reproducible selection of cells that express themsuggest that, similarly to the SFFV gp55, they may triggergrowth factor receptors expressed by the MoMuLV-inducedT-cell lymphomas and may stimulate cellular proliferation.The detection of env/Mlvi-4 hybrid mRNA transcripts in the

Mlvi-4-positive tumors raises two additional questions thatneed to be addressed. (i) Is the reproducible utilization of the

VOL. 68, 1994

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from

7932 PATRIOTIS AND TSICHLIS

splice donor site at position 6397 of the viral genome indicativeof differential splicing that may give rise to so far undetectedviral transcripts? (ii) Do the cellular sequences at the 3' end ofthe hybrid mRNA transcripts define the 3' end of the Mlvi-4gene which is selectively expressed in normal thymus andspleen? Moreover, if that is the case, do these sequences giveclues about the identity of this gene? To address this question,we carried out computer-assisted comparisons between the40-amino-acid C-terminal domain, encoded by the mRNAdefined by the cDNA clone B1.1, and the protein sequences inthe GenBank database. These comparisons revealed that theputative Mlvi-4 gene defined by these sequences is unique. Theclosest homologs of the 40-amino-acid domain were proteinkinase STE7 (32.5% identity and 57.5% similarity in thesequence between amino acids 409 and 457) and other mem-bers of the family of serine-threonine protein kinases (data notshown).

In conclusion, although the relationship of the Env/Mlvi-4fusion protein to the product of the normal Mlvi-4 generemains undetermined, the results presented in this reportsuggest that one of the effects of provirus integration in theMlvi-4 locus is the synthesis of MoMuLV Env/Mlvi-4 fusionproteins. The potential interaction of these fusions with cellu-lar proteins may initiate mitogenic signals that contribute tooncogenesis. Additional questions raised by these results,including the potential role of differential splicing in theregulation of viral gene expression and the potential identity ofthe normal Mlvi-4 gene, will be addressed in future studies.

ACKNOWLEDGMENTS

We thank Susan Bear for making available her Northern blots ofnormal and neoplastic cells. We also thank David Kaplan (FrederickCancer Research Facility) for providing the D17 canine osteosarcomacells.

This research was supported by Public Health Service grantsCA51893 and CA56110. Additional support was provided by PublicHealth Service grant CA06927 and by an appropriation from theCommonwealth of Pennsylvania to the Fox Chase Cancer Center. C.P.was a fellow of the Leukemia Society of America, Inc.

REFERENCES1. Bellacosa, A., K. Datta, S. E. Bear, C. Patriotis, P. A. Lazo, N. G.

Copeland, N. A. Jenkins, and P. N. Tsichlis. 1994. Effects ofprovirus integration in the Tpl-l/Ets-1 locus in Moloney murineleukemia virus-induced rat T-cell lymphomas: levels of expression,polyadenylation, transcriptional initiation, and differential splicingof the Ets-1 mRNA. J. Virol. 68:2320-2330.

2. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroform ex-traction. Anal. Biochem. 162:156-159.

3. Cloyd, M. D., J. W. Hartley, and W. P. Rowe. 1980. Lymphomo-genicity of recombinant mink cell focus-forming murine leukemiavirus. J. Exp. Med. 151:542-549.

4. Ferro, F. E. Jr., S. L. Kozak, M. E. Hoatlin, and D. Kabat. 1993.Cell surface site for mitogenic interaction of erythropoietin recep-tors with the membrane glycoprotein encoded by Friend erythro-leukemia virus. J. Biol. Chem. 268:5741-5747.

5. Flubacher, M. M., S. E. Bear, and P. N. Tsichlis. 1994. Replace-ment of interleukin-2 (IL-2)-generated mitogenic signals by amink cell focus-forming (MCF) or xenotropic virus-induced IL-9-dependent autocrine loop: implications for MCF virus-inducedleukemogenesis. J. Virol. 68:7709-7716.

6. Green, N., H. Hiai, J. H. Elder, R. S. Schwartz, R. M. Khiroya,C. Y. Thomas, P. N. Tsichlis, and J. M. Coffin. 1980. Expression ofleukemogenic recombinant viruses associated with a recessivegene in HRS/J mice. J. Exp. Med. 152:249-264.

7. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. 1977. Anew class of murine leukemia virus associated with development ofspontaneous lymphomas. Proc. Natl. Acad. Sci. USA 74:789-792.

8. Hiai, H., P. Morrissey, R. Khiroya, and R. S. Schwartz. 1977.Selective expression of xenotropic virus in congenic HRS/J (hair-less) mice. Nature (London) 270:247-249.

9. Hoatlin, M. E., S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak,and D. Kabat. 1990. Activation of erythropoietin receptors byFriend viral gp55 and by erythropoietin and down-modulation ofthe murine Fv-2' resistance gene. Proc. Natl. Acad. Sci. USA87:9985-9989.

10. Holland, C. A., J. W. Hartley, W. P. Rowe, and N. Hopkins. 1985.At least four viral genes contribute to the leukemogenicity ofmurine retrovirus MCF 247 in AKR mice. J. Virol. 53:158-165.

11. Kabat, D. 1989. Molecular biology of Friend viral erythroleuke-mia. Curr. Top. Microbiol. Immunol. 148:1-42.

12. Kingston, R. E., C. A. Chen, and H. Okayama. 1993. Introductionof DNA into mammalian cells, p. 9.1.1-9.1.9. In F. M. Ausubel, R.Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith,and K. Struhl (ed.), Current protocols in molecular biology. JohnWiley & Sons, New York.

13. Koehne, C. F., P. A. Lazo, K. Alves, J. S. Lee, P. N. Tsichlis, andP. V. O'Donnell. 1989. The Mlvi-1 locus involved in the inductionof rat T-cell lymphomas and the pvt-1/mis-1 locus are identical. J.Virol. 63:2366-2369.

14. Kung, H.-J., C. Boerkeel, and T. H. Carter. 1991. Retroviralmutagenesis of cellular oncogenes: a review with insights into themechanisms of insertional activation. Curr. Top. Microbiol. Im-munol. 171:1-25.

15. Lazo, P. A., A. J. P. Klein-Szanto, and P. N. Tsichlis. 1990. T-celllymphoma lines derived from rat thymomas induced by Moloneymurine leukemia virus: phenotypic diversity and its implications. J.Virol. 64:3948-3959.

16. Lazo, P. A., J. S. Lee, and P. N. Tsichlis. 1990. Long distanceactivation of the c-myc proto-oncogene by provirus insertion inMlvi-1 or Mlvi-4 in rat T-cell lymphomas. Proc. Natl. Acad. Sci.USA 87:170-173.

17. Li, J. P., A. D. D'Andrea, H. F. Lodish, and D. Baltimore. 1990.Activation of cell growth by binding of Friend spleen-focus-forming virus gp55 glycoprotein to the erythropoietin receptor.Nature (London) 343:762-764.

18. Li, J. P., and D. Baltimore. 1991. Mechanism of leukemogenesisinduced by mink cell focus-forming murine leukemia viruses. J.Virol. 65:2408-2414.

19. Makris, A., C. Patriotis, S. E. Bear, and P. N. Tsichlis. 1993.Genomic organization and expression of Tpl-2 in normal cells andMoloney murine leukemia virus-induced rat T-cell lymphomas:activation by provirus insertion. J. Virol. 67:4283-4289.

20. Morgenstern, J. P., and H. Land. 1990. A series of mammalianexpression vectors and characterization of their expression re-porter gene in stably and transiently transfected cells. NucleicAcids Res. 18:1068.

21. Oliff, A., K. Signorelli, and L. Collins. 1984. The envelope geneand long terminal repeat sequences contribute to the pathogenicphenotype of helper-independent Friend viruses. J. Virol. 51:788-794.

22. Patriotis, C., A. Makris, S. E. Bear, and P. N. Tsichlis. 1993.Tumor progression locus-2 (Tpl-2) encodes a protein kinaseinvolved in the progression of rodent T cell lymphomas and in Tcell activation. Proc. Natl. Acad. Sci. USA 90:2251-2255.

23. Tsichlis, P. N., and S. E. Bear. 1991. Infection by mink cell focusforming (MCF) viruses confers interleukin-2 (IL-2) independenceto an IL-2 dependent rat T cell lymphoma line. Proc. Natl. Acad.Sci. USA 88:4611-4615.

24. Tsichlis, P. N., and P. A. Lazo. 1991. Virus-host interactions andthe pathogenesis of murine and human oncogenic retroviruses.Curr. Top. Microbiol. Immunol. 171:95-171.

25. Tsichlis, P. N., J. S. Lee, S. E. Bear, P. A. Lazo, C. Patriotis, E.Gustafson, S. Shinton, N. A. Jenkins, N. G. Copeland, K. Hueb-ner, C. Croce, G. Levan, and C. Hanson. 1990. Activation ofmultiple genes by provirus integration in the Mlvi-4 locus in T-celllymphomas induced by Moloney murine leukemia virus. J. Virol.64:2236-2244.

26. Tsichlis, P. N., P. G. Strauss, and L. F. Hu. 1983. A commonregion for proviral DNA integration in MoMuLV-induced ratthymic lymphomas. Nature (London) 302:445-449.

J. VIROL.

on March 4, 2016 by P

enn State U

nivhttp://jvi.asm

.org/D

ownloaded from