THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 10, Issue of April 5, pp. 4615-4619 1986 Printed in C.S.A.

Activation of a Novel KpnI Transcript by Downstream Integration of a Human T-lymphotropic Virus Type I Provirus*

(Received for publication, August 16,1985)

Takashi Okamoto$, Marvin S. Reitz, Jr.S, Michael F. Clarke$, Linda L. Jagohinskit, and Flossie Wong-StaalS From the $Laboratory of Tumor Cell Bwbgy, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20205 and §Biotech Research Laboratories, Inc., Rockuille, Maryland 20892

A cDNA library was constructed from the HUT102 cell line established from a patient with adult T-cell leukemiaflymphoma and screened for cDNA clones that contain (i) cellular sequences abundantly ex- pressed in HUT102 cells and not in the virus-negative T-cell line HUT78, and (ii) viral long terminal repeat (LTR) sequences either in the 5’ end or in the 3’ end. One such cDNA clone, KT1, was isolated and its nu- cleotide sequence was determined. It contains three regions: a KpnI repeat, a unique cellular region (UCR), and the U3 + R sequence of the human T-lymphotropic virus type I LTR. The arrangement of this clone sug- gests that its RNA transcript was activated by provirus integration in cis, possibly by the activity of a down- stream provirus enhancer. Analysis of HUT102 DNA shows that one allele of the KT1 UCR is rearranged. The expression of the KT1 UCR is unique to HUTlO2. These data are consistent with the idea that the human T-lymphotropic virus type I LTR contains an enhancer which can activate upstream sequences in cis. The possible significance of this finding is discussed.

Seroepidemiological studies have demonstrated that adult T-cell leukemiaflymphoma (ATL’) is causally associated with human T-cell leukemia virus type I (HTLV-I) (1, 2). HTLV- I is a retrovirus which contains, in addition to the normal gag, pol, and enu genes, a unique gene (pX-lor or tat) which is not of host origin (1,3-5). Its protein product appears to activate the promoter of HTLV-I in trans (5) and might also be involved in trans regulation of some cellular genes. The HTLV-I LTR also regulates the transcription of the down- stream viral genes in cis (5). No evidence has been reported for cis activation of cellular genes, either by downstream promotion or orientation-independent enhancement. In order to help ascertain whether cis activation of transcription of cellular genes can occur as a result of HTLV-I infection and subsequent promotion or enhancement by viral regulatory sequences, we examined a cDNA library prepared from an HTLV-I transformed cell line for evidence of chimeric tran- scripts containing both viral and cellular sequences.

- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: ATL, adult T-cell leukemia/lym- phoma; HTLV-I, human T-lymphotropic virus type I; LTR, long terminal repeat; UCR, unique cellular region; RCS, repetitive cellular sequences; kb, kilobase(s); kbp, kilobase pair(s); MMTV, mouse mam- mary tumor virus.

MATERIALS AND METHODS~

RESULTS

Screening and Characterization of Clones Containing Both Cellular and Viral Sequences-We constructed a cDNA li- brary derived from poly(A)+ RNA of an ATL-derived cell line, HUT102, as described (16). Screening of this library by using the homologous cDNA probe revealed that most of the plas- mid clones (about 75%) contained inserts. cDNA synthesized from poly(A)+ RNA of a virus-negative neoplastic T-cell line, HUT78, hybridized to 99.4% of the recombinants containing HUT102 cDNA sequences. From more than 12,000 colonies, we isolated 47 cDNA clones of transcript expressed in HUT102 cells but not in HUT78 cells. Of these, 19 hybridized to the HTLV-I probes. Five of the 19 hybridized to the U3 + R probe, but not to probes for viral structural genes (gag, pol, env, and pX-lor). Four of these clones contained short inserts (C0.5 kilobase pairs (kbp)) and were not analyzed further.

One cDNA clone, KT1, contained an insert of about 1 kbp. Based on the results of hybridization with the MOLT4 ge- nomic DNA and HTLV-I long terminal repeat (LTR) probes, this clone comprised three sets of sequences (Fig. I), a region homologous to U3 and R in the viral LTR, and a cell-specific region 5’ to this which contained repetitive cellular sequences at the 5’ end and a unique cellular region (UCR) between this and the U3 - R sequences.

Determination of the Nucleotide Sequences of KT1 Insert- To characterize the different sets of sequences in KT1, we have determined its nucleotide sequence (Fig. 2A). The 5’ portion of this clone corresponds to the 3’ terminal portion of the human KpnI repeat (29-31). The 5’-most region of the KpnI repeat may have been present but not transcribed by the reverse transcriptase during the cloning procedure. The longest open reading frame potentially codes for only 34 amino acid residues (Fig. 2B), and it seems unlikely that this sequence encodes a functional protein.

Genomic DNA Rearrangement of the KT1 UCR in HUT102”DNA from HUTlOP, MJ (another virus-positive ATL-derived T-cell line), and normal T cells was digested with the restriction enzymes BglII, HindIII, or EcoRI and analyzed by Southern blots with the 32P-labeled Hinff-EcoRI fragment (Fig. 1) (the UCR region) of the KT1 insert (Fig.

Portions of this paper (including “Materials and Methods,” part of “Results,” and Figs. 5 and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-2749, cite the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

4615

4616 Activation of a KpnI Repeat by Downstream HTLV-I Integration

3). A single EcoRI band of 2.4 kbp was detected in all DNAs tested, indicating that the KpnI repeat containing the UCR sequences was present prior to provirus integration. Since there is an EcoRI site at the boundary of this KpnI repeat and the downstream integrated provirus (Fig. l), a rearranged EcoRI fragment band containing UCR and viral sequences would not be detected in HUT102 cells. However, using BglII or HindIII, which cut the provirus once and twice, re- spectively, a rearrangement of one allele of the KT1 gene was evident only in HUT102 (Fig. 3, lanes 2 4 ) . In each case, a band containing viral sequences co-migrated with rearranged

- - < - >

< - > FIG. 1. Restriction map of the KT1 insert. The cDNA library

using poly(A)+ RNA from HUT102 was made essentially as described (15,16). The insert was divided into three sets of sequences: repetitive cellular sequences (RCS) , unique cellular region (UCR) and HTLV-I LTR (U3 + R). Both RCS and UCR fragments used as probes in hybridization studies are marked (U). Horizontal arrows indicate the sequencing strategy. B, BarnHI; E, EcoRI; H , Hinff; K , KpnI; P, PstI; T, TaqI.

10 20 30 40 50 60 A A T ~ ~ ~ A T T A G ~ A ~ \ T A T A ~ c T M T G T A A A T T ~ ~ ~ T A G ~ ~ K ~ 70

~""""""""""""""""""""""""""""""""""- 80 90 LOO I10 I20 I JO 140

A T C A T A T G T A A C C T G C A T G T T G T C C K A T G T K C C T " U T A T A 4 T ~ T K G T

I50 Homolo~ous Region to Human Kpn 1 Repeat ---------X"-

UjCCAmATGTGTATATGTGTTCATGGCATGTKCTAT~TA~Gl l l l lMXCTCl lMAl lG4 I 60 I70 180 190 200 210

220 2 30 24P 250 260 2 70 280 T b A G K T C " T A T C T m T G 8 M T G T A M V .

290 300 310 320 330 340 350 Unlquc Cellular Region (UCR) ......................

T T C T T ~ T T A T m G m C T f f i l l ~ T C A T ~ T A l T f ~

"""""""_ """"""""""""""""""""""""""""""""""-

"""""""""

360 370 380 390 400 410 420 A A P f T C G A T t C C C A t T A c m ~ T ~ T ~ ~ T ~ ~ ~

4 30 440 450 46n 4 70 4 an 490 A c T A C A l l A T C T t A C A A T G A C C C C A W T A T C C C C U G @ 3 C T T ~ C C C ~ ~ """"",C"""""""""""""""""""""""""""""

A l T T C C C C G A A P E A C A A C T C T T C 4 3 X C C C K A C T ~ T C T W & T C T C C C ~

AGCTCAGCACCGGCTCACCCTMXCCCTGACGTGTCCCCCT~TCATbNXTUVCCTCf%G 5 70 580 590 600 610 620 630

500 5 10 520 5 30 540 550 560

- _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ HTLV-1 LTR ("3) ............................

640 650 660 670 680 h90 700 ~AA~~CACCGGMICCACCCA~CCTCCCCATG~GTCA&CCXCCCTCAGGCG~~C&X~OACCCCT

710 720 7 30 740 7 50 760 770 C A C C T C M W W T T T T C A T W Y C & C A T A T W T W ~ T ~ T C T A T ~ T ~

780 790 800 810 820 8 10 840 C A t T T ~ T C G C A T C T C T ~ T T ~ C ~ C X C ~ A c C ~ ~ C A T C ~ ~ f f i

"""""""""""""""""""""""""""""""""""

------------><------------ HTLV-1 LTR ( R ) _____---____________________ 85n

Glll3GTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTWTGCGTCCGCffiTCTWT~lTfA 860 870 880 890 900 910

920 A A t c T U Y ; C T c t A G A c C C ~ G T T C ~ T A c C T T ~ ~ ~ T ~ C

9 30 940 950 960 9 70 980 """""""""""""""""""""""""""""""""""

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

KGCTGTGTGTGCCCCTGTGTGCTWTCTG ( A h 990 1000 I010

""""""""""-"""""

B o 1: yl "p 7 500 am m ax, 9 0 0 1 ~ BM P.W.

.,! ', . ' . . ;, ' ,: ,

Open Reading Runes

. ,; ' , - I . . .

FIG. 2. A, nucleotide sequences of KT1 insert. Regions homolo- gous to human KpnI and HTLV-LTR are indicated. B, positions of methionine codons (bars above line) and termination codons (bars below fine) of the three coding frames.

Bgl I I Hind 111 Eco RI

kbp 1 2 3 4 1 2 3 4 1 2 3 4 -78 _." ~

23.1 - 9.4 - ~

6.6 - 4.4 - 2.3 - 2.0 -

1.35 - 1.08 - 0.87 - 0.60 - ,

I

FIG. 3. Rearrangement of KT1 UCR in HUT102 cells. Southern blots of high molecular weight cellular DNA digested with BglII, HindIII, or EcoRI were hybridized with HTLV-I LTR (lane 1 ) or a HinfI-EcoRI KT1 fragment (lanes 2-4). Sample DNAs: HUT102 (lanes 1 and 2) , MJ (lane 3), and normal human T cells (lane 4) . EcoRI digestion did not show up any extra bands. while BffII or HindIII digestions demonstrated extra bands of 4.6 or 9.3 kbp, re- spectively. These rearranged bands comigrated with one of the bands hybridized with the HTLV-I LTR probe.

KT-1 bands (Fig. 3, lane 1). No other restriction length fragment polymorphism of this region was observed. These results confirmed that the KT-1 clone is not the result of a cloning artifact, but reflects an insertion of HTLV-I proviral DNA adjacent to the UCR and resultant transcription of a cellular KpnI repeat and the UCR terminating at the 5' LTR of the HTLV-I provirus.

Expression of KT1 Sequences in HTLV-I Immortalized Cells-To determine whether the expression of the KT1 se- quences is unique to HUT102 cells, we examined RNA from other HTLV-I immortalized cells. When we hybridized Northern blots of poly(A)+ RNA from the HTLV-I immor- talized cell lines HUTlO2, MJ, ClO/MJ, and 81/66, from the uninfected promyelocytic leukemic cell line HMO, and from normal T cells maintained in short-term culture with inter- leukin 2 using the nick translated insert of KT1, expression of dispersed repetitive sequences were detectable in all of the cells tested (data not shown). We also used the UCR fragment of KT1 as a probe for the same RNA blots. As shown in Fig. 4A, a discrete 5.5-kilobase (kb) band is observed only in HUT102. This band comigrated with one of the bands labeled by the U3 and R LTR probes ( d a t a not shown). The lack of expression of the UCR sequences linked to U3-R in the other HTLV-I transformed cells is consistent with the failure to detect rearrangements of these sequences in the DNA of other cells as well as previous reports on the lack of conserved integration sites for HTLV-I (22).

Levels of expression of the repetitive sequences contained in the KT1 gene were analyzed by RNA dot blot hybridization using a labeled PstI-HinfI fragment of KT1 containing the 3' portion of the KpnI repeat (the RCS probe) and either poly(A)+ or total cellular RNA from these cells. No significant consistent differences were observed (not shown).

Cytoplasmic and nuclear RNA were fractionated from HUT102 cells to determine the relative distribution of KpnI transcripts (Fig. 48). The RCS and the UCR probes were used to examine the expression of KpnI transcripts in general or of the KpnI transcript containing the UCR sequences, respectively. KpnI transcripts were much more abundant in the nucleus than in the cytoplasm. In contrast, transcripts

Activation of a KpnI Repeat by Downstream HTLV-I Integration 4617

A

28s-

18 s -.

1 2 3 4 6 8 7

B UCR RCS N Cy N Cy

10- ' - "-

1 5-

2.5 -

1.25-

rn - a U

2 0.625- 0

0.313-

0.158 -

FIG. 4. A, expression of the KT1-UCR. Northern blots of poly(A)+ RNA ( 5 pg) were hybridized with the nick translated Hinff-EcoRI fragment of KT1. Lanes 1-7 represent the RNA isolated from HUTlO2, HUT78, MJ, ClO/MJ, 81/66, HMO, and normal human T cells respectively. HUT102, MJ, ClO/MJ, and 81/66 are cell lines transformed by HTLV-I. HUT78 is a mature neoplastic T-cell line and shares a similar differentiation stage with the virus-positive T- cell lines. HL60 is a cell line derived from human acute promyelmytic leukemia. B, nuclear localization of KpnI transcripts. RNA dot blot hybridization of nuclear ( N ) and cytoplasmic (Cy) total RNA isolated from HUT102 cells. Two-fold serial dilutions of RNA, starting with 10 pg, were applied to a nitrocellulose paper and hybridized with either labeled Hinff -EcoRI KT1 fragment or the PstI-Hinff fragment.

containing the UCR sequences seemed to be more evenly distributed between these two fractions.

DISCUSSION

We have obtained and analyzed a cDNA clone containing contiguous cellular and viral sequences from an HTLV-I- infected cell line. The cellular sequences are comprised of a KpnI repeat and a unique sequence. This result suggests cis activation of a specific KpnI transcript has occurred by down- stream integration of a HTLV-I provirus, possibly under the influence of the enhancer sequences within the 5' LTR. This is the first report of possible activation of upstream transcrip- tion by the HTLV-I LTR. An alternative possibility is that this may reflect a rare, aberrant transcript of a constitutively expressed KpnI sequence which terminates at the poly(A) signal of the downstream HTLV-I provirus instead of i ts usual internal poly(A) site. A clone isolated from a genomic library of SD cells, a cell line derived from another patient with ATL containing only a single provirus, also contained a KpnI repeat in the 5' flanking cellular sequences 1 kbp from the integrated provirus.3 Studies of the nucleotide sequence of this region and i ts possible transcripts are currently in progress. These two independent findings raise the possibility

S. Colombini, T. Okamoto and F. Wong-Staal, unpublished ob- servation.

that HTLV-I may preferentially integrate downstream from KpnI repeats.

Activation of cellular genes by retrovirus integration has been observed in other systems. Usually, the provirus inte- grates upstream of the cellular gene to be activated and the promotor as well as enhancer sequences of the viral LTR are utilized. However, activation of cellular genes upstream (37) or in opposite orientation (38) from the provirus has also been observed. In these cases, activation is mediated by the enhan- cer sequences alone or in conjunction with the viral transcrip- tional termination signal. Similar data has been reported for Pim-1 (39), a newly identified cellular integration site in a murine T-cell lymphoma induced by mink cell focus-forming virus. The majority of provirus integrations occurred at the 3' end of the Pim-1 gene resulting in transcripts terminated in the 5' LTR.

Preferential integration of provirus near cellular inter- spersed repetitive sequences and their transcriptional activa- tion has also been demonstrated with mouse mammary tumor virus (MMTV)-induced murine T-cell lymphoma. Clones of several integrated MMTV proviruses and flanking cellular DNA contain upstream L1 repeats (a murine homologue of the human KpnI repeat) in the same transcriptional orienta- tion as the integrated proviruses.' The detailed nucleotide sequence of the KpnI repeat itself suggests that most copies are inserts of reverse transcripts, as they have repetitive adenosine-rich tails at the 3' end and short duplications of target sequences flanking the KpnI insert, as do Alu repeats or pseudogenes. These are called retroposons (40). Although the mechanism of provirus integration is not fully understood. it is interesting to speculate that a retrovirus may preferen- tially integrate near retroposons.

Unique retroviral integration sites in neoplasias frequently result in the activation of oncogenes. The KT-1 cell-related region, however, does not so far appear to contain a significant amount of coding potential (although we have not so far succeeded in cloning the 5' 4.5 kb of the KT-1 transcript). Although evidence concerning the biological role of KpnI repeats or their transcripts is inconclusive, it has been sug- gested that they might be involved in coordinate transcrip- tional regulation of cellular genes (41). Several years ago, Davidson, Britten, and co-workers (42) proposed that regu- latory interactions occur within the nucleus between RNA copies of structural genes and complementary repetitive se- quence transcripts by forming RNA:RNA duplexes. KpnI transcripts might fulfill such a role. If this were the case, then aberrant expression of such transcripts might contribute to the role of HTLV-I in leukemogenesis or cell transformation. In either case, the apparent activation of upstream transcrip- tion suggests that enhancement by HTLV-I LTR sequences could be of biological relevance."

Acknowledgments-We thank Dr. R. C. Gallo for encouragement and support throughout this work, Dr. J. Maize1 for secondary struc- ture analysis, and Dr. H. Z. Streicher for critical readinK of the manuscript.

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another KpnI cDNA clone, Kpnl-8 (31). expressed in uninfected human fibroblasts and having an extended 3' end. Within the 3' unique cellular sequences we have found a similar identity of the characteristic structure and of local homology with cellular genes. including one of the HLA gene family.

4618 Activation of a KpnI Repeat by Downstream HTL V-I Integration

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Activation of a KpnI Repeat by Downstream HTLV-I Integration

Tskashi Okamoto*, Marvin S. Reitz Jr.*, Michael F. Clarke*, LinddL. Jagodrinskit and

flos*ie uong-staal'

F ~ o m the *Laborstory of Tumor Cell Bialogy. Developmental Therapeutics Program, Division of

Cancer Treatment, National Cancer Institute, Bethesda. Maryland 20892: and tBiorech Research

laboratories, h e . , Rockville, Maryland

HRTERIiiTS m mmoos

Cell Lines--The cells analyzed include HTLV-I positive human T-cell l iner, HUT102 (6). MJ (7),(7), and 81/66 (8); virus-negative T-cell lines, HUT78 (9) . and MOLT4 (10); nonlymphoid hematopoiefic cell line, EL60 (11); and T cells from healthy individuals cultured

olarkers with HUT102 (6, 9) which suggest that if is in a similar differentiation stage in IL-2-canraining media. HUT78 is a mature neoplastic T-cell line and shares surface

with the virus-positive T-cell lines.

Preparation of Nvcleic Acids-"High molecular weight DNA Yaw prepared from che cultured cells (12). Restriction enzyme digestion was performed according t o the mand~cfurer'~ insrrucrions. Total cellvlar RNA was obtained by the guanidine hydrochloride purification procedure (13). Nuclear and cytoplaarnie RNA sere separated by the methods described by Haniatis el al. (14). Residual DNA was removed by DNase I (RNase free, Phamcia, Sweden) digestion-&rCsCl density uliracenrrifugarion. Poly(&)+ RNA was enriched by oligo(dT)- cellulose c d u w chronafography ( 1 4 ) .

poly(A)C RNA iaolated from HUT102 a6 described previously (15, 16). Transfamed colonies were reolica-olared. and co10oY hvhridizafian was oerfarmed bv rransferrinli colonies onto

ConSfruCtion and Screening of CDNA Library--A cDNA library vas made using cellular

nitroceiluloak fil&rs by the method previously reporfed (17 ; lS ) . 32P-Laieled cDNA probes "eve prepared from HUIlO2, HUT78, and normal T-cell polyedenylared RNA as described (19). HTL-I DNA inserts from the recombinant clones lambda ST ( 2 0 ) . containing a full-length Hmv-I provirus, and c~3Ll (21). containing rhe U3 and R region of the viral large terminal repeat (LTR). were obtained by resfrietion enzyme digestion and labelled by nick translation.

A US specific 30-mer vas eyocherired cuaing the published sequence Of the HTLV-I LTR (3) and NiQk translaled nenomic DNA from MOLT4 cells was also used as a probe for human genomic DNA.

wal 32P-end labeled with polynucleotide kinase. Clones Containing plasmld lnserts were

which also contained viral LTR sequence$ but lacked viral protein coding sequences Yere selected based on hybridiration to the HUT102 but n o t the HUT78 CDNA probes. Pasicive clones

selected f o r further analysis.

. .

a 0.7% agarose gel and analyzed by Southern blotting a3 described (12). RNA samples denatured and elecfromhoreaed on a 2.2 M farmaldehvdell% agarose %el were transferred ro Genescreen

Analysis of Cellular DNA and RNA--DNA restriction enzyme digests were electrophoresed on

region 0 2

4619

5s 3'