Nucleocytoplasmic transport of HTLV-1 RNA is regulated by ... - Nature

Oncogene (1998) 16, 3309 ± 3316  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

Nucleocytoplasmic transport of HTLV-1 RNA is regulated by two independent LTR encoded nuclear retention elements Jason A. King1, Joanna M Bridger2, Martin LoÈchelt3, Peter Lichter2, Thomas F Schulz4, Volker Schirrmacher1 and Khashayarsha Khazaie1,5 Departments of 1Cellular Immunology, 2Organization of Complex Genomes, 3Retroviral Gene Expression, at the German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg 69120, Germany; 4Department of Medical Microbiology and Genetic Urinary Medicine, Royal Liverpool University Hospital, Duncan Building, Liverpool L69 3BX, UK; 5Section for Molecular Diagnosis and Therapy, Department of Surgery, Im Neuenheimer Feld 116, Heidelberg 69120, Germany

Appropriate expression of HTLV-1 genes requires transcriptional transactivation by Tax and post-transcriptional regulation by Rex, both mediated by LTR encoded RNA sequences. Using a combination of deletion mutagenesis, Rex-reporter CAT assays, ¯uorescence in situ hybridization (FISH) and confocal laser scanning microscopy it was established that in the absence of Rex, CAT mRNAs harboring HTLV-1 LTR sequences were unable to leave the nucleus. Deletion of the known U5 encoded cis-acting repressing sequence (CRS) led to a partial release of nuclear retention. A novel regulatory element overlapping the 3' Rex responsive element (RxRE) region was shown to prevent export and expression of these transcripts. Deletion of both the 5' LTR encoded CRS and 3' LTR encoded downstream repressive sequence (3' CRS) led to constitutive mRNA nuclear export and gene expression, independently of Rex. The locations of the two regulatory elements indicate that while the 5' CRS selectively acts to hinder export of unspliced transcripts, the 3' CRS has the capacity to induce nuclear retention of all HTLV-1 transcripts, and therefore could potentially contribute to viral latency in infected cells. Keywords: HTLV-1; RxRE; CRS; nuclear export

Introduction Interaction of the HTLV-1 Rex protein multimeric complex with the RxRE RNA structure facilitates the expression of unspliced and singly spliced viral mRNAs (Bogerd et al., 1992; Seiki et al., 1988; Toyoshima et al., 1990). The mechanism by which the RxRE interaction aects HTLV-1 gene expression is analogous to the mode of regulation aorded by the interaction of HIV Rev and the Rev response element (RRE) (Hope et al., 1991; Malim et al., 1990; Tiley et al., 1992). Both Rex and Rev encode nuclear export functions, de®ned by their activation domains (Fischer et al., 1995; Gerace, 1995; Rehberger et al., 1997; Stauber et al., 1995). Various investigations suggest that the requirement for Rex or Rev function is based on the presence of inhibitory sequences within the

Correspondence: K Khazaie Received 21 November 1997; revised 18 January 1998; accepted 30 January 1998

regulated viral transcripts. These inhibitory elements are proposed to bind to host cell factors, resulting in nuclear retention, mRNA instability and/or inecient translation. Several post-transcriptionally inhibiting elements have been characterized in the gag, pol and env genes of HIV. A 260 base pair region of the HIV pol RNA was reported to prevent translation of unspliced mRNAs (Cochrane et al., 1991). In turn, the HIV Rev was shown to promote both association of poly(A) binding proteins (Campbell et al., 1994) and polysomal association and translation of Rev responsive mRNAs (D'Agostino et al., 1992). A 218 base pair AU rich region of the HIV gag gene was reported to destabilize gag-pol transcripts in the absence of Rev (Schwartz et al., 1992a,b) presumably counteracted by binding of the mRNA to HIV Rev (Felber et al., 1989). Two posttranslational inhibitory regions overlapping the gp120 and the gp41 HIV env gene sequences have been reported, the latter coinciding with sequences coding for the HIV-1 Rev responsive element (RRE) (Brighty and Rosenberg, 1994; Churchill et al., 1996; Nasioulas et al., 1994). At least some of these elements have mRNA nuclear retention functions (Churchill et al., 1996; Zhang et al., 1996). Unlike HIV, so far the only post-transcriptional Cisacting repressive sequences (CRS) identi®ed in HTLV-1 and HTLV-2, do not overlap with coding sequences but rather span the U5 regions of the two closely related viruses (Black et al., 1991; Seiki et al., 1990). Using the HTLV-1 LTR to drive expression of an intron encoding CAT reporter gene, it was shown that sequential deletion of sequences covering the base pairs 620 ± 701 of the HTLV-1 U5 region leads to promotion of Rex independent CAT expression (Seiki et al., 1990). In other studies with HTLV-2, Northern blot analysis of cytoplasmic and of total RNA suggested that a similarly located CRS sequence inhibited the cytoplasmic accumulation of viral mRNA (Black et al., 1991). A common feature of all mentioned negative regulatory elements is their presence within transcripts that require Rex or Rev for their expression. Their removal through splicing yields transcripts which presumably do not require Rex or Rev regulation. We have examined the subcellular localization of HTLV-1 LTR driven CAT mRNAs in the absence of splicing. Release of gene expression from repression was measured by CAT activity, while the sub-cellular location of CAT transcripts was directly visualized using ¯uorescence in situ hybridization (FISH) and confocal microscopy. Our observations indicate that

HTLV-1 RxRE overlaps with a CRS element JA King et al

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the U5 encoded CRS is indeed an RNA nuclear retention element. However, deletion of this sequence alone is not sucient for full release of mRNAs from the nucleus. A further sequence overlapping the 3' RxRE acts synergistically to block export of LTR linked mRNAs. This sequence is expected to be present in all viral transcripts including the doubly spliced mRNAs arising from the pX region, and could therefore potentially contribute to a total shut down of viral gene expression leading to viral latency.

Results The 3' HTLV-1 LTR encodes a downstream cis-repressing function To investigate negative regulatory functions in the LTRs of the HTLV-1 virus, a series of expression vectors were made in which the chloramphenicol acetyl transferase gene (CAT) was expressed from HTLV-1 proviral promoter constructs. The ®rst vector J-2 (Figure 1a) encoded the CAT gene between two complete HTLV-1 LTRs. J-2.CRS71 (Figure 1b) had a deletion in the 5' LTR of nucleotides 576 ± 703

corresponding to the reported CRS (Seiki et al., 1990). J-1.CRS7 (Figure 1c) lacked the 5' CRS and the complete 3' LTR, while J-1 (Figure 1f) lacked the whole 3' LTR. To better de®ne potential downstream sequences associated with repression of gene expression, the 3' LTR was modi®ed to remove the entire RxRE. To this aim, two pairs of primers were used to PCR amplify the U3 and U5 sequences separately. These were then joined together to create an LTR which lacked the RxRE but reconstituted the polyadenylation signals. Replacement of the complete 3' LTR in J-2 and J-2.CRS7 by this modi®ed LTR led to two new constructs lacking the RxRE, namely J2.RxRE7 (Figure 1d) and J-2.CRS7.RxRE7 (Figure 1e). Additional constructs had either an internal polyadenylation signal (Figure 1h and i) upstream of the 3' LTR, or had the 3' LTR replaced by the 3' RxRE (Figure 1g and j). Each of these constructs was transfected alone or together with HTLV-1 Tax and/or Rex expressing vectors into COS7, A293T, Jurkat and HeLa cells. As expected, constructs lacking the CRS had higher Rexindependent expression in CAT assays than those with a complete 5' LTR. This trend was reproducible in all the tested cell lines, with 293T cells showing the

Figure 1 Schematic representation of constructs used in this study. Parental construct with two complete HTLV-1 LTRs ¯anking CAT cDNA (a). Deletion of the Cis-Repressive Sequence from the 5' Long Terminal Repeat (b). Deletion of the CRS and 3' LTR (c). Deletion of the RxRE from the 3' LTR (d). Deletion of the CRS and RxRE (e). Deletion of the 3' LTR (f). Deletion of the 3' LTR but replacement of the RxRE (f). Insertion of an internal SV40 polyA signal just upstream of the 3' LTR (h). Deletion of the CRS and insertion of an internal SV40 polyA (i). Deletion of the CRS and 3' LTR but replacing the RxRE (j). Tax or Rex expressing constructs were included as indicated. Deletion of RxRE left the HTLV-1 polyA signal and site intact and the same distance apart on the RNA as compared to the full length 3' LTR subsequent to RxRE secondary structure formation. To replace the RxRE the appropriate sequences were PCR ampli®ed as an EcoRI fragment ± inserted into the Mun I site just upstream of the SV40 signals


strongest response. The overall trend indicates lack of cell type speci®city with respect to the repressive function (Figure 2, compare J-2 with J-2.RxRE7). However, removal of the 3' LTR resulted in a signi®cantly greater improvement in Rex-independent CAT activity, in all cell lines (Figure 2, see J-1). The maximum CAT activity observed was with constructs lacking the 3' LTR and the 5' CRS (Figure 2, see J1. CRS7). In COS7, A293T and HeLa cells, removal of the RxRE from the 3' LTR produced a signi®cant release of Rex requirement, in part comparable to removal of the CRS element from the 5' LTR (Figure 2, see J2 RxRE7). In Jurkat cells, the in¯uence of the 3' RxRE was only convincingly detectable in constructs also lacking the 5' CRS (Figure 2, see J-2.CRS7. RxRE7). Removal of both of these suppressive elements had an additive eect in the other cell lines (Figure 2, see J-2.CRS7. RxRE7). These results indicate that the downstream repressive element acts in a cell type independent manner and is in part overlapping with RxRE. 5' CRS and 3' CRS act to suppress nucleocytoplasmic transport of mRNA To examine the sub-cellular localization of CAT transcripts, FISH experiments were carried out.

COS7, A293T, and HeLa cells were transiently transfected with appropriate vectors, ®xed and hybridized with speci®c probes against CAT mRNA under partially denaturing conditions, so as to avoid hybridization of the probe to DNA. The frequency of nuclear as compared to cytoplasmic CAT mRNA was visually scored using a ¯uorescence microscope. Quantitative analysis of the transfected cells indicated that the amount of CAT activity measured in the presence of Tax was probably limited by the nuclear export of the corresponding transcripts. Tax promoted expression of CAT mRNA from J-2, but the transcripts remained mainly nuclear (Figure 3a). In accordance with the CAT assay results, full derepression was observed with constructs lacking the 5' CRS and the 3' LTR (Figure 3c). Removal of either the 5' CRS or the 3' RxRE had comparable eects (Figure 3b and d), while removal of both suppressive elements further improved Rex independent export of the CAT transcripts (Figure 3e). Addition of the RxRE to constructs lacking the 3' LTR suppressed nuclear export (Figure 3 compare f with g). In agreement with the results from CAT assays, A293T cells showed the biggest changes in the nuclear cytoplasmic distribution of the transcripts. Confocal microscopy analyses were performed on the 3-dimensionally preserved cells to con®rm the

Figure 2 Reporter CAT assay showing the result of deletion of the 3' LTR, of the 5' CRS, and/or of the 3' RxRE. Jurkat T cells were transfected using the DEAE-dextran method with 3 mg of each HTLV-1 based plasmid and 1 mg of Tax expressing plasmid and/or 0.1 mg of Rex expressing plasmid. Cells were harvested 48 h later for CAT assays. COS7, HeLa and A293T cells were calcium phosphate transfected with 10 mg of each CAT expressing plasmid with 2 mg and 0.2 mg of Tax and Rex expressing plasmids, respectively. The results shown for COS7 cells are from three, A293T cells from two, Jurkat cells from four, and HeLa cells from two representative experiments. CAT assays were kept in the linear range of acetylations. Bars represent experimental variations calculated as standard deviations. The % of maximum expression refers to the CAT signal in the presence of Tax as compared to the presence of Tax and Rex, in each individual case. The highest CAT signals were reproducibly obtained with constructs lacking both the 5' CRS and the 3' LTR

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nuclear or cytoplasmic localization of the transcripts, and the release of mRNA nuclear retention through Rex activity. We have reported apoptotic eects of expression of Tax in susceptible Jurkat T-cell lines (Chlichlia et al., 1995, 1997). Morphological and microscopic analysis did not detect signi®cant apoptosis up to 3 days after transfection of Tax expressing

vectors in the cell lines reported here. In the absence of Rex, transcripts encoding both repressive elements exhibited a dominant localization to nucleoplasm. The visual impression of nuclear localization changed with time after transfection, appearing as punctate intranuclear dots at earlier time points and ®lling the nucleoplasm but excluding the nucleoli at later time

Figure 3 Quantitation of the nuclear and cytoplasmic distribution of CAT encoded mRNAs. Cells were calcium phosphate transfected with 10 mg of each CAT expressing plasmid and 2 mg of Tax expressing plasmids. After 48 h they were subjected to FISH analyses as described in materials and methods. Nuclear or cytoplasmic staining of CAT encoding mRNAs were visually scored using a ¯uorescence microscope. The number of counted cells in each instance are indicated on the right hand panel. Ratio of nuclear to cytoplasmic stainings are shown as bar diagrams

COS

A

B

COS

J-2

C Poly (A)

HeLa Poly (A)

Tax

Tax & Rex

A293T Poly (A)

RxRE Poly (A)

Tax 3 days

Poly (A)

Tax 5 days

Figure 4 Analysis of the eect of Rex on the subcellular localization of the CAT encoding mRNAs using FISH and confocal microscopy, in COS7 cells (A and B), or HeLa and A293T cells (C). Calcium phosphate transfections of 10 mg of each CAT expressing plasmid and 2 mg of Tax expressing plasmids, with or with our 0.1 mg of Rex expressing plasmid, were performed 48 h or at the indicated time points prior to FISH analysis. (A) Subcellular localization of transcripts driven by the J-2 vector encoding two complete HTLV-1 LTRs , in the presence of Tax (a), or Tax and Rex (b). Higher magni®cations showing punctate intranuclear dots visualized 3 days (c), or uniform nuclear staining excluding the nucleolus visualized 5 days (d) after transfection of J2 and pRK7-Tax in the absence of Rex. (B) Expression in the absence of Rex from modi®ed vectors, J-2.CRS7 lacking the CRS (a), J-1.CRS7 lacking the CRS and 3' LTR (b), J-2.RxRE7 lacking the RxRE (c), J-2.CRS7.RxRE7 lacking the CRS and RxRE (d), J-1 lacking the 3'LTR(e), J-1+RxRE lacking the 3' LTR but having the RxRE re-inserted (f). (C) High magni®cations of representative HeLa (a and b) or A293T (c and d) nuclei transfected with the represented vectors, having either two complete LTRs or only a 5' LTR. In A, B, and C panels a and b, propidium iodide was used as a nuclear counter stain to stain total nucleic acids (red). A ¯uorescein-tagged anti-DIG antibody was used to detect the labeled probe (green). The red and green images were acquired simultaneously. Where the two colors overlap a yellow coloration was observed, indicating a nuclear localization of the transcripts. Boxed ®gures are enlargements of marked cells (white arrows). In C panels c and d, cells were counter stained with DAPI and images were acquired using a Photometric cooled charge coupled device (CCD) camera. Images for DAPI and FITC were overlaid in Adobe Photoshop


points after transfection (Figure 4A, c and d respectively). Expression of Rex led to ecient export of the mRNA, as indicated by the cytoplasmic accumulation of transcripts. The exported mRNA also appeared to be released through de®ned routes and to localize into distinct compartments in the cytoplasm (Figure 4A, b). The kinetics of appearance and accumulation of CAT mRNAs did not change following introduction of deletions or re-introduction of RxRE, indicating no big changes in the stability of the mRNAs. These observations are consistent with an mRNA nuclear retention function for the 5' CRS. They also

provide evidence for an additional independent nuclear retention element in the 3' end of the transcripts. The downstream nuclear retention element overlaps with the RxRE. The downstream repressing element acts at the post-transcriptional level in Jurkat T-cells The small cytoplasm of Jurkat T cells made it dicult to con®dently score these cells with FISH. To dierentiate in these cells between a transcriptional and post-transcriptional mode of action for the 3' repressing element, an SV40 polyA signal was

Figure 5 Representative reporter CAT assay showing the result of premature termination of transcription, upstream of the 3' LTR. Jurkat T cells were transfected using the DEAE-dextran method with 3 mg of each HTLV-1 based plasmid and 1 mg of Tax expressing plasmid and/or 0.1 mg of Rex expressing plasmid. Cells were harvested 48 h later for CAT assays. Tax or Rex expressing constructs were included as indicated. CAT assays were kept in the linear range of acetylations. The % maximum acetylation is calculated relative to the combined Tax and Rex induced state. The results were reproduced in two independent assays. (A) CAT activity from control vector with two complete LTRs (a) or the same construct having a premature polyA signal (b), or comparable constructs but lacking the 5'CRS (c and d, respectively). (B) Assays showing CAT activity from a construct with only a single complete 5' LTR (a) or the same construct with the addition of RxRE (b) or comparable construct but lacking the 5' CRS (c and d, respectively)

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introduced downstream of the CAT gene in vectors J-2 and J-2.CRS7, creating vectors J-2.term (Figure 1h) and J-2.CRS7.term (Figure 1i), respectively. It was reasoned that addition of the SV40 polyA signal should not aect transcriptional repression by the still present 3' LTR. On the other hand any potential posttranscriptional suppression caused by the 3' untranslated (U3-R) sequences of the mRNA would be removed by the premature termination of CAT transcripts. Indeed insertion of an SV40 polyA signal inside J-2 or J-2.CRS7 led to enhancements of Rex independent CAT activity from the new vectors J-2.term or J2.CRS7.term (Figure 5A compare a and b, c and d), comparable to that seen with the removal of the 3' LTR in vector J-1 (Figure 5B a). Cloning of the RxRE upstream of the SV40 polyA signal in J-1, and J1.CRS7 suppressed CAT activity from the new vectors, J-1+RxRE and J-1.CRS7+RxRE (Figure 5B, compare a and b, c and d). FISH analysis of transfected COS7 cells showed that these transcripts were mostly nuclear (Figure 4G and Figure 5B, f). The combined results of CAT and FISH analyses indicate that in all cell lines examined the 3' LTR encodes a repressive element that functions at the posttranscriptional level, most likely at the level of mRNA export from the nucleus. Discussion HTLV-1 LTRs were used to construct non-splicing vectors driving the expression of a CAT reporter gene in hematopoietic and non-hematopoietic cell lines. In all the cell lines examined, vectors encoding two complete HTLV-1 LTRs were completely dependent on both Tax and Rex for expression of CAT. However, removal of the 3' LTR, or deletion of the RxRE signi®cantly relieved the requirement for Rex. Similar Rex independent gene expression was obtained by insertion of an SV40 polyA signal immediately upstream of the 3' LTR, implying a post-transcriptional eect. Conversely, replacement of the RxRE into vectors lacking the 3' LTR, suppressed gene expression. In all cases, removal of the reported post-transcriptionally acting CRS element in the 5' LTR enhanced Rex independent CAT expression. Using FISH and confocal microscopy it was established that the release of repression observed in CAT assays correlated with an increased cytoplasmic localization of the CAT mRNA. Removal of the 3' CRS had an even greater eect than deletion of the 5' CRS. Re-introduction of the RxRE into vectors lacking the 3' LTR resulted in the nuclear retention of the corresponding mRNAs. The kinetics of accumulation of CAT mRNA was comparable for all constructs examined, and paralleled the changes in CAT activity. Altogether these observations point to regulation of mRNA transport as being responsible for the alterations in CAT activity. Although we cannot completely rule out additional changes in mRNA stability, the direct examination of subcellular localization of HTLV1 LTR linked transcripts by FISH allows the two repressive sequences (5' CRS and 3' CRS) to be clearly identi®ed as mRNA nuclear retention elements. Our observations indicate that the 5' CRS and 3' CRS are

cooperative mRNA nuclear retention elements. To be appropriately expressed, transcripts encoding these sequences require the mRNA export function of Rex for transport to the cytoplasm. 3' CRS overlaps with the three dimensional RNA structure in HTLV-1 responsible for binding Rex, the RxRE. A similar situation exists in HIV, where in the absence of Rev the RRE functions to inhibit gene expression by blocking nuclear export of the corresponding viral RNAs (Brighty and Rosenberg, 1994; Churchill et al., 1996; Nasioulas et al., 1994). Thus, in both HIV and HTLV-1 the same RNA secondary structures that are responsible for binding Rev or Rex and allowing export of viral RNA, function in the absence of these proteins to retain viral mRNAs in the nucleus. This brings back to light earlier suggestions that binding of Rev to RRE (or Rex to RxRE) may result in displacement of inhibitory host factor (Cullen, 1992; Haseltine, 1991). Several observations suggest that the 3' CRS extends into the U3 region. Thus, removal of the complete 3' LTR was more eective in making CAT expression Rex independent than deletion of the RxRE. On the other hand, replacement of RxRE in vectors lacking the 3' LTR made a greater dierence than removal of RxRE from vectors containing the complete 3' LTR. Thus the 3' CRS is very likely to span a greater part of the 3' untranslated region of HTLV-1 transcripts. Our observations indicate that nuclear retention of HTLV-1 RNA can be completely relieved by deletion of both the 5' CRS and the 3' CRS, leaving the transcripts arising from these modi®ed LTRs solely dependent on Tax for expression. These results emphasize the independent action and the co-operation of the 5' CRS and 3' CRS. Response to Rex by vectors that lacked only the 3' RxRE or the complete 3' LTR indicated which the 5' Rex binding sequence was functional, implying that unspliced genomic transcripts (encoding in HTLV-1 for gag-pol) may bene®t from the additive or synergistic eect of two Rex response elements. It is not clear to what extent the 5' RxRE alone or in combination with other repressive elements may hinder RNA transport in the absence of Rex. Previous reports indicate that expression of unspliced or singly spliced HTLV-1 RNAs is completely dependent upon action of Rex, and that the mechanism of this dependence is essentially identical to that observed with HIV Rev (Hope et al., 1991; Inoue et al., 1986; Malim et al., 1990; Tiley et al., 1992). Rev dependence may be related to the presence of constitutive repressive elements in the viral transcripts encoding structural genes (Brighty and Rosenberg, 1994; Churchill et al., 1996; Cochrane et al., 1991; Felber et al., 1989; Mikaelian et al., 1996; Nasioulas et al., 1994; Schwartz et al., 1992a,b). Such sequences have been reported to act independent of splicing signals (Nasioulas et al., 1994), to over lap in part with the RRE (Brighty and Rosenberg, 1994; Churchill et al., 1996), and to require cooperation with additional viral repressive sequences (Mikaelian et al., 1996). However, the function of 3' CRS may be unique to the requirements of HTLV-1. The HTLV-1 3' CRS is located within the mRNA 3' untranslated region, which is present in all HTLV-1 transcripts. Furthermore, transcripts encoding env do not retain the 5' CRS, which is removed through splicing (Seiki et


al., 1990). Thus, the HTLV-1 env sequences may encode additional repressive elements that cooperate with the 3' CRS to retain these transcripts in the nucleus. Alternatively, the 3' CRS may have a universal repressive function for HTLV-1 gene expression, not limited to the dierential regulation of viral mRNA transport. The location and mode of action of the RxRE could help to explain the eventual universal shut down of HTLV-1 gene expression and establishment of viral latency. The constitutive function of these viral nuclear retention elements suggests that they interact with cellular factors. This opens the possibility that dierential splicing and programmed switches in expression of at least some cellular genes may be regulated through a similar mechanism as described for HTLV-1. Materials and methods Plasmids The HTLV-1 based plasmids used in this study are depicted in Figure 1. HTLV-1 sequences were derived from the HTLV-tat 1 construct (Nerenberg et al., 1987). The 5' LTR of the HTLV-tat 1 construct was modi®ed to restore a full length (755 bp) LTR which included the CRS (Seiki et al., 1990). This LTR was then partially deleted in the U5 region (bp 576 ± 703) to speci®cally remove the CRS. All constructs contained the 750 bp Chloramphenicol Acetyltransferase cDNA, derived from the pBL-CAT2 plasmid (Luckow and Schutz, 1987). Constructs had either a full length 3' LTR (Figure 1a, b, h and i) or a RxRE-deleted (bp 314 ± 573) 3' LTR (Figure 1d and e) or an SV40 polyA signal in place of the 3' LTR (Figure 1c, f and j). In two cases the SV40 polyA signal was inserted immediately downstream of the CAT cDNA (Figure 1h and 1i). HTLV-1 Tax and Rex were expressed from the pRK-7 plasmid (Dr David Goeddel, unpublished data; (Chlichlia et al., 1995, 1997) with CMV immediate early promoters. Transfections Jurkat T cells (10 million) were transfected via the DEAEdextran method with 3 mg of each HTLV-1 based plasmid and 1 mg of Tax expressing plasmid and/or 0.1 mg of Rex expressing plasmid. Cells were harvested 48 h later for CAT assays. COS7, HeLa and A293T cells were calcium phosphate transfected with 10 mg of each CAT expressing plasmid with 2 mg and 0.2 mg of Tax and Rex expressing plasmids, respectively. Cells were again harvested after 48 h for CAT assays. Calcium phosphate transfections of COS7 cells for FISH experiments were carried out on polyL-lysine (4 mg/ml) coated, temperature stable glass microscope slides in `Quadriperm 1' culture plates (Heraeus) using the same amounts of DNA as for CAT assay transfections. Chloramphenicol acetyltransferase assays Cells were harvested, washed with Tris buered saline (pH 7.4), resuspended in 0.25 M Tris pH 7.4 and lysed via three rounds of freeze-thaw fracturing. Cell lysates were then assayed for their CAT enzyme activities in a 1 h incubation with 0.4 mM acetyl Co.A and 14C-labeled chloramphenicol. The reaction mixture was then extracted with ethylacetate and the organic phase loaded and run on a thin layer chromatograph in a chloroform : methanol (19 : 1 v/v) solvent mixture. Percentage acetylation was

calculated using a 14C linear analyzer (Berthold). Percent maximum expression was calculated for each transfection as being relative to the `de-repressed' situation, in which both Tax and Rex are present. CAT assays were standardized by internal b-galactosidase control using an using a CMV promoter driven vector and by protein estimation (BioRad). There was no signi®cant dierence using protein estimations or b-galactosidase for standardization of the assays. Fluorescence in situ hybridization and confocal laser scanning microscopy Forty-eight h post-transfection, glass microscope slides overgrown with COS7 cells were washed three times in PBS and ®xed for 20 min in freshly prepared 4% paraformaldehyde, at room temperature. Cells were washed once in PBS and then permeablized for 20 min in a solution of PBS containing 0.5% Saponin (w/v) and 0.5% Triton X-100 (v/v), at room temperature. Cells were then washed once with PBS and incubated for a minimum of 30 min in 20% (w/v) glycerol, at room temperature (Zirbel et al., 1993). Slides were snap-frozen in liquid nitrogen and then allowed to thaw slowly at room temperature. This procedure permits the three dimentional structure of the cells to be retained. The J-2 plasmid was used as template in a nick translation labeling reaction which incorporated digoxigenin-11dUTP in place of dTTP (Lichter et al., 1990). Labeled probe molecules had an average length of 200 nt, as observed on an ethidium bromide containing 1% agarose gel. Probe DNA (200 ± 400 ng) was precipitated with 7 mg salmon sperm carrier DNA and 3 mg Cot 1 DNA (Boehringer Mannheim), washed twice in 70% ethanol, dried and resuspended in 50% deionised formamide, 26SSC, 10% dextran sulphate and 50 mM sodium phosphate. The probe was then denatured at 758C for 5 min. Cells were treated with 40% formamide and 26SSC (pH 7) for 3 min at 738C to denature only RNA molecules. The probe was added to the cells and hybridization took place at 378C in a humidi®ed chamber for a minimum of 12 h. Cells were washed three times (each 5 min) with 50% formamide/26SSC (pH 7) 428C and three times (each 5 min) with 0.16SSC at 608C to remove non-speci®cally bound probe before being incubated in 46SSC/4% BSA (w/v) for 20 min at room temperature. Probe was visualized using 10 mg/ml fluorescein conjugated sheep anti-DIG antibody (Boehringer Mannheim) in 46SSC/1% BSA. Propidium iodide (2 mg/ ml) was used to visualize the nuclei. Pre- and postdenaturation RNase digestions using 100 mg/ml RNase A (Sigma), con®rmed that FISH signals observed under these partial denaturing conditions were derived from RNA and not DNA (Data not shown). Images were acquired using a Zeiss (LSM 410) confocal laser scanning microscope ®tted with an argon ion laser and a helium neon laser using an oil immersion 636plan-APOCHROMAT lens (Zeiss). Red and green images were simultaneously obtained by excitation of propidium iodide and ¯uorescein at 488 nm. A dierent image acquisition method was used for A293T cells. These cells were counter stained with DAPI and images were acquired using a photometric cooled charge coupled device (CCD) camera. Images for DAPI and FITC were overlaid in Adobe Photoshop.

Acknowledgements We are obliged to Robin Weiss, Rolf FluÈ gel and Louis Gazzolo for continuous advice and encouragement throughout this work. Stelios Psarrass, Fotini Gounari, Katerina Chlichlia and Sabine Rehberger, are greatfully

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acknowledged for their valuable assistance and contributions. Annette Lichtenauer and Esmail Rezavandy are thanked for excellent technical assistance. We also thank and acknowledge Ulrike Mathieu for her work in elucidating the conditions for the in-situ RNA denatura-

tion. We thank Harald zur Hausen for his support and interest. The work was in part ®nanced by the Deutsche Forschungs Gemeinschaft, the Verein zur FoÈrderung der Krebsforschung in Deutschland e.V. and the Schering AG.

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