BRIEF COMMUNICATION Differential expression of alternatively

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Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines. A Cereseto1, Z Berneman1,2, I Koralnik1, J Vaughn1, G Franchini1 and ...
Leukemia (1997) 11, 866–870  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

BRIEF COMMUNICATION Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines A Cereseto1, Z Berneman1,2 , I Koralnik1, J Vaughn1, G Franchini1 and ME Klotman1,3 1

Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Human T cell leukemia/lymphotropic virus (HTLV) is a complex 9 kb human retrovirus with at least eight alternatively spliced mRNAs expressed from the 39 or pX region of the genome. These mRNAs allow for the expression of novel proteins from the previously recognized pX open reading frames I and II in addition to Tax, Rex and p21rex encoded from orf III and IV. These alternatively spliced messages have been detected using reverse-transcriptase polymerase chain reaction (RT/PCR) amplification in HTLV-I-transformed T cell lines as well as in peripheral blood mononuclear cells (PBMC) from infected patients with and without disease. To gain insight into the role of these alternatively spliced mRNAs in pathogenesis, we developed a semi-quantitative non-PCR-based RNase protection assay to detect and quantitate their presence in HTLVI-infected cells. Analysis of RNA from HTLV-I-infected cells established from patients with adult T cell leukemia (ATL) as well as tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and both IL-2-dependent and IL-2-independent HTLV-I-infected cell lines by RNase protection has confirmed the existence of all of the alternatively spliced messages in each cell line analyzed. However, the relative quantity of each message was significantly different among these lines suggesting that splice site utilization is an important viral regulatory pathway. Keywords: HTLV-I; alternative splicing; adult T cell leukemia (ATL)

Introduction Human T cell leukemia/lymphotropic virus (HTLV), the causative agent of adult T cell leukemia (ATL) (reviewed in Ref. 1) as well as the slowly progressive neurological disease, tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/ HAM)2 is a complex 9 kb human retrovirus. The degree of complexity of the viral genome has recently been recognized with the detection by several groups of at least eight alternatively spliced mRNAs expressed from the 39 or pX region of the genome.3–6 These mRNAs allow for the expression of novel proteins from the previously recognized pX open reading frames I and II in addition to Tax, Rex and p21rex encoded from orf III and IV. The alternatively spliced messages include: the doubly spliced pX-tax/rex with the coding capacity for Tax, Rex and p21rex ; the singly spliced pX-orf I encoding a protein, p12, and the doubly spliced pX-rex-orf I coding for p12 and with the potential for encoding a 152 amino acid protein (Rex-orf I or Rof), the singly spliced pX-orf II encoding p13; the doubly spliced pX-tax-orf II encoding a larger protein (p30, Tax-orf II or Tof) which utilizes the initiation codon of Tax; and the singly spliced pX-p21rex, a monocistronic message encoding p21rex of unknown function. In vitro transfection

studies have demonstrated the presence of p30, p13 and p12 in addition to the previously recognized Tax, Rex and p21rex proteins.3,5,7 A unique splice site has also been detected 17 bases downstream from the splice acceptor for the envelope gene, however, no unique proteins are generated from this alternative splicing.4 These alternatively spliced messages have been detected using reverse-transcriptase polymerase chain reaction (RT/PCR) amplification in HTLV-I-transformed T cell lines as well as in peripheral blood mononuclear cells (PBMC) from infected patients with and without disease.4,5,8–10 The latter observation provides direct evidence of continued virus expression in the host raising the possibility that viral proteins expressed from alternatively spliced mRNAs may chplay a role not only in the initiation of diseases like ATL, as previously proposed, but also a role in the maintenance of disease. To gain insight into the role of these alternatively spliced mRNAs in pathogenesis, we developed a semiquantitative non-PCR-based assay to detect and quantitate their presence in HTLV-I-infected cells. Analysis of RNA from HTLV-I-infected cells established from patients with ATL as well as TSP/HAM by RNase protection has confirmed the existence of all of the alternatively spliced messages and demonstrated that splice site utilization may represent an important viral regulatory pathway.

Materials and methods

Cell lines Several previously described IL-2-dependent and IL-2-independent HTLV-I-infected T cell lines were utilized in these studies. They included the IL-2-independent lines C10/MJ established from a patient with ATL (who subsequently developed TSP/HAM),11 NS1 established from a healthy seropositive individual,12 C8166-45 established from a patient with ATL and containing defective proviruses13 and LAF, established from a patient with TSP/HAM.5 The IL-2-dependent cell lines, N1185 and N1186, were established by cocultivation of PBMCs from two patients with ATL with cord blood cells.4 The cells were maintained in RPMI with 10% FCS with the addition of Il-2 (20 units/ml, Boehringer Mannheim, Indianapolis, IN, USA) for the IL-2-dependent cell lines.

RNA preparation Correspondence: ME Klotman, Mt Sinai School of Medicine, 1 Gustave L Levy Pl., New York, NY 10029, USA Present addresses: 2University of Antwerp VIA, Antwerp, Belgium; 3 Mt Sinai School of Medicine, New York, NY 10029, USA Received 20 May 1996; accepted 21 February 1997

Total cellular RNA was prepared from the infected cell lines by the guanidinium thiocyanate-acid phenol method of Chomczynski and Sacchi.14

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Protection probes The cDNA of the unique alternatively spliced mRNAs were originally reverse transcribed, amplified and cloned into the pBluescript vector (Stratagene, La Jolla, CA, USA) as previously described.4,5 Probes used in the RNase protection assay were generated from these vectors by in vitro transcription after linearization of the vectors with EcoRI. The probes generated as well as the location and size of the protected fragments, the specific alternatively spliced mRNA detected by the probes and the protein(s) expressed from each mRNA are indicated in Figure 1. A control probe to detect 18S ribosomal RNA was synthesized from pT7 RNA 18S (Ambion, Austin, TX, USA). The 18S probe has been used in each experiment to normalize the RNA loading. Radioactively labeled cRNA probes were generated by in vitro transcription utilizing the T7 Maxiscript (Ambion) with 50 mCi of 32P-UTP in each reaction for the generation of the HTLV-I probes and 3 mCi of 32 P-UTP diluted with unlabeled UTP for generation of the 18S ribosomal probe. Labeled RNA was separated from unincorporated nucleotides with a sepharose G50 chromatography column (5 prime-3 prime, Boulder, CO, USA).

RNase protection assay The RNase protection assay was performed as described.15 Briefly, 5 × 105 c.p.m. of the specific probe resuspended in hybridization buffer (40 mM PIPES, pH 6.4, 0.4 mM MnCl, 1.0 mM EDTA, 20% formamide) were annealed to 10 mg of total heat denatured cellular RNA in 30 ml of hybridization buffer by heating at 60°C for 16 h. RNase digestion was performed with 37 mg/ml of RNase A and 736 U/ml of RNase T1 (RNase Cocktail, Ambion) in 350 ml of ribonuclease digestion buffer (10 mM, Tris-HCl, pH 7.5, 300 mM NaCl and 5 mM EDTA). This reaction was incubated for 60 min at 30°C. Control experiments were done using 10 mg of tRNA in place of cellular RNA and the reaction was stopped with 10 ml of 20% SDS and 2.5 ml of 20 mg/ml proteinase K. After phenol/chloroform/isoamyl alcohol extraction and ethanol

precipitation, the RNA was analyzed on a denaturing polyacrylamide/urea gel. The gel was analyzed and bands quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Results RNA from the cell lines C10/MJ and LAF were analyzed with all of the above described probes. All eight alternatively spliced pX messages previously identified and cloned by RTPCR were detected in the RNA from both the cell line established from the patient with ATL (C10/MJ) as well as the cell line established from the patient with TSP-HAM (LAF) (Figure 2). RNA from the C10/MJ and LAF cell lines were analyzed by RNase protection assay and full length protected bands quantitated relative to 18S ribosomal RNA after PhosphorImager analysis. Due to differences in the sequence of each probe, the PhosphorImager units of each protected fragment were divided by the number of labeled nucleotides (UTP) present in the corresponding probe. To better compare the results, values were normalized with respect to the lowest PhosphorImager value obtained (pX-rex-orfI for both C10/MJ and LAF cell lines) (Table 1). In the C10/MJ cell line, expression of pX-tax-orfII was consistently found to be at least three times greater than any of the other mRNAs. In a similar manner, analysis of expression of the pX-specific mRNAs from LAF indicates that expression of p21rex was at least three times greater than any of the other analyzed transcripts (Table 1). Notably, each cell line analyzed had a different pattern of expression of the mRNAs. While pX-tax-orfII was predominant in C10/MJ, it was only moderately expressed in LAF. This mRNA has a coding capacity for a unique 241 amino acid protein of unknown function (p30) which localizes to the nucleolus.5,7 In contrast, the monocistronic singly spliced message coding for p21rex, highly expressed in LAF, was moderately expressed in C10/MJ. At least two proteins of HTLV-I have been associated with cell transformation. A number of studies have shown that expression of Tax, either alone or in the presence of other

Figure 1 Alternatively spliced HTLV-I pX mRNAs. The single line at the top of the figure represents the full length HTLV-I genomic RNA with the indicated U3, R and U5 regions as well as the major open reading frames. Below are the exons of the alternatively spliced pX mRNAs with the location of the splice donors and splice acceptors indicated (+1 representing the RNA initiation site). The arrows indicate the 59 and 39 ends of the splice junction probes used in the RNase protection assay. Also noted is the size of the expected full length protected fragment for each probe and the potential protein coding capacity for each of the unique mRNAs.

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Figure 2 Semi-quantitative RNase protection for the alternatively spliced pX mRNAs in the HTLV-I-transformed cell lines, C10/MJ and LAF. Probes spanning the unique splice sites are indicated above each lane and the top band in each lane represents the full length probe and protected fragment. The small bands in each lane represent overlapping regions of alternatively spliced mRNAs recognized by shorter sequences in the probe. A probe specific for 18S ribosomal mRNA was used as well and is indicated by the arrow. Size markers (M) included a combination of radiolabeled in vitro synthesized RNA probes of known length. Table 1 Relative expression of the pX alternatively spliced mRNAs in HTLV-I-transformed cells

C10/MJ (ATL)

LAF (TSP/HAM)

pX-tax-orf II

22.5

pX-orf II pX-orf I env D17 p21 rex pX pX-D17 pX-rex-orf I

8 5 3.5 2 2 1 1

p21rex

47

pX-orf II pX-orf I env D17 pX-tax-orf II pX pX-D17 pX-rex-orf I

14 11.5 9 6 4.5 2 1

Values indicate the fold increase of expression of each transcript with respect to the lowest value. Intensity of each band representing the transcript was quantitated by PhosphorImager analysis and normalized for 18S signal in each lane. Obtained numbers have been corrected for the number of radioactive nucleotides (UTP) present in each probe used for the analysis. Values represent the average of three different experiments.

viral proteins, results in transformation in vitro and is associated with the development of tumor growth in vivo.16–20 More recently, the protein, p12, has been shown to have weak oncogenic potential when expressed in the presence of the bovine papilloma virus protein, E5.21 Furthermore it has been shown that p12 binds to the b and gc chains of the IL-2 receptor.22 Therefore, we investigated

whether the IL-2-independent growth of HTLV-I-transformed cells correlated with the level of expression of the alternatively spliced mRNAs (pX, pX-orf I and pX-rex-orf I) encoding the potential viral transforming proteins (Tax and p12). The levels of other viral pX transcripts (p21rex and pX-tax-orfII) that have been found to be highly expressed in the two cell lines above, were also analyzed. The IL-2-dependent, HTLV-I-infected T cell lines N1185 and N1186 were compared to the IL-2-independent T cell lines C10/MJ, NS1 and C8166-45. In these experiments, the five cell lines were analyzed each time with a single probe and the level of expression compared among the cell lines. Results, shown in Figure 3, indicated a significant variation in the level of expression of each alternatively spliced mRNA among the cell lines analyzed, which was confirmed by PhosphorImager analysis (not shown). However, there was no clear correlation between the level of expression of any of the pX transcripts analyzed and the IL-2 growth requirements of the T cell lines. In fact, each cell line had a unique pattern of expression of the alternatively spliced mRNAs independent of the IL-2 dependence.

Discussion It is now clear that the human retroviruses HTLV-I and HTLVII have complex patterns of alternative splicing similar to the human retroviruses associated with immunodeficiencies, HIV1 and HIV-2. This complex splicing results in mono- and poly-

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Figure 3 Semi-quantitative RNase protection assay of individual alternatively spliced pX mRNAs in IL-2-dependent (+) and IL-2-independent (−) HTLV-I-transformed cell lines. (a) and (b) are separate protection assays utilizing the probes indicated. The probe specific for 18S ribosomal mRNA is indicated by an arrow. Size markers (M) were a combination of radiolabeled in vitro synthesized RNA probes of known length.

cistronic mRNAs with the coding capacity for unique viral proteins in addition to the structural proteins. Our work, utilizing a number of cell lines and a non-PCR-based semi-quantitative assay, clearly demonstrates that these mRNAs represent a significant amount of the total viral RNA expressed as demonstrated by the predominance among the pX messages of the doubly spliced pX-tax-orf II in C10/MJ and the singly spliced message, p21rex, in LAF mRNA. It appears that in each cell line there is a unique pattern of expression of these alternatively spliced mRNAs relative to each other. These data suggest that, although the splice sites are conserved, the efficiency of splice site utilization varies among the HTLV-Itransformed T cell lines. One determinant of splice site utilization may be the presence of defective viruses. A common occurrence in HTLV-Iinfected cell lines in vitro and in primary infected cells in vivo is the presence of both normal and deleted proviruses.23–30 It has been suggested that the high expression of the singly spliced pX-p21rex mRNA primarily originates from defective proviruses and that the protein is expressed from this monocistronic message rather than from the full-length doubly spliced pX mRNA.31 If this is the case, such defective viruses are very common, in that this mRNA was detected in all of the cell lines we analyzed as well as in PBMCs from infected patients, as we and others previously reported.4,5 Although the mechanisms of action and the role of the transregulatory proteins Tax (p40tax) and Rex (p27rex) in the viral life cycle have been demonstrated, the functions of these other pX-encoded proteins remain largely unknown. The protein p21rex can be detected in HTLV-I-infected cells and RNA detected from PBMC of infected individuals.4,5,10 The additional proteins, p12, p13 and p30 have been detected in transfected cell lines and localized to the cellular endomem-

branes, the nucleus and the nucleolus respectively.3,5,7 p12 shares sequence homology with the E5 protein of bovine papillomavirus and enhances the in vitro transforming properties of this protein, suggesting that it may function in the transformation process associated with HTLV-I infection. 21Furthermore, p12 binds the b and gc chains of the IL-2 receptor22 suggesting its possible role in the process that transforms an IL-2-dependent HTLV-I T cell line into an IL-2-independent one. The level of expression of the p12-encoding mRNAs, pXorf I and pX-rex-orf I did not correlate with the IL-2 growth requirements in the cell lines we studied. However, this does not preclude a role for this protein in the transformation process, perhaps through complex interactions with other viral (Tax) as well as cellular proteins. The application of this sensitive and specific semi-quantitative assay for measuring alternatively spliced pX mRNAs to direct patient samples should provide insight into the role that virus expression plays in the pathogenesis of HTLV-I-associated diseases. References 1 Gallo RC. Human T-cell leukemia-lymphoma virus and T-cell malignancies in adults. Cancer Surv 1984; 3: 113–159. 2 Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calendar A, de The G. Antibodies to human T-lymphotropic virus type I in patients with tropical spastic paraparesis. Lancet 1985; 2: 407– 409. 3 Ciminale V, Pavlakis GN, Derse D, Cunningham CP, Felber BK. Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I. J Virol 1992; 66: 1737–1745. 4 Berneman ZN, Gartenhaus RB, Reitz MS Jr, Blattner WA, Manns A, Hanchard B, Ikehara O, Gallo RC, Klotman ME. Expression of

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alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers. Proc Natl Acad Sci USA 1992; 89: 3005–3009. Koralnik IJ, Gessain A, Klotman ME, Lo Monico A, Berneman ZN, Franchini G. Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I. Proc Natl Acad Sci USA 1992; 89: 8813–8817. Orita S, Saigo A, Takagi S, Tanaka T, Okumuna K, Aono Y, Hinuma Y, Igarashi H. A novel alternatively spliced viral mRNA transcribed in cells infected with human T cell leukemia virus type 1 is mainly responsible for expressing p21X protein. FEBS Lett 1991; 295: 127–134. Koralnik IJ, Fullen J, Franchini G. The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments. J Virol 1993; 67: 2360–2366. Kinoshita T, Shimoyama M, Tobinai K, Ito M, Ikeda S, Tajima K, Shimohtono K, Sugimura T. Detection of mRNA for the tax1/rex1 gene of human T-cell leukemia virus type I in fresh peripheral blood mononuclear cells of adult T-cell leukemia patients and viral carriers by using the polymerase chain reaction. Proc Natl Acad Sci USA 1989; 86: 5620–5624. Hirata M, Ikematsu H, Nakashima K, Hayashi J, Kashiwagi S. Higher expression levels of alternatively spliced pX mRNA in human T lymphotropic virus type I asymptomatic carriers positive for antibodies to p40tax protein. J Infect Dis 1995; 172: 1098– 1102. Orita S, Takagi S, Saiga A, Minoura N, Araki K, Kinoshita K, Kondo T, Hinuma Y, Igarashi H. Human T cell leukaemia virus type 1 p21X mRNA: constitutive expression in peripheral blood mononuclear cells of patients with adult T cell leukaemia. J Gen Virol 1992; 73: 2283–2289. Markham PD, Salahuddin SZ, Kalyanaraman VS, Popovic M, Sarin P, Gallo RC. Infection and transformation of fresh human umbilical cord blood cells by multiple sources of human T-cell leukemia–lymphoma virus (HTLV). Int J Cancer 1983; 31: 413–420. Hall WW, Kaplan MH, Salahuddin SZ, Oyaizu N, Gurgo C, Coronesi M, Nagashima K, Gallo RC. In: Blattner WA (ed). Human Retrovirology: HTLV. Raven Press: New York, 1990, pp 115–127. Salahuddin SZ, Markham PD, Wong-Staal F, Franchini G, Kalyanaraman VS, Gallo RC. Restricted expression of human T-cell leukemia–lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology 1983; 129: 51–64. Chomzynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156–159. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds). Current Protocols in Molecular Biology. John Wiley and Sons: New York, 1993. Grassman R, Dengler C, Muller-Fleckenstein I, Fleckenstein B, McGuire K, Dokhelar MC, Sodroski JG, Haseltine WA. Transformation to continuous growth of primary human T-lymphocytes by human T-cell leukemia virus type I X-region genes transduced by herpesvirus Saimiri vector. Proc Natl Acad Sci USA 1987; 86: 3351–3355. Neremberg M, Hinrichs SH, Reynolds RK, Rhoury G, Jay G. The

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24 25

26

27 28 29

30

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tat gene of human T-lymphotropic virus type I induces mesenchymal tumors in transgenic mice. Science 1987; 237: 1324–1329. Hinrichs SH, Neremberg M, Reynolds K, Khoury G, Jay G. A transgenic mouse model for human fibromatosis. Science 1987; 237: 1340–1343. Tanaka A, Takahashi C, Yamaoka S, Nosaka T, Maki M, Hatawaka M. Oncogenic transformation by the tax gene of HTLV-I in vitro. Proc Natl Acad Sci USA 1990; 87: 1071–1075. Grossman WJ, Kimata JT, Wong F-H, Zutter M, Ley TJ, Ratner L. Development of leukemia in mice transgenic for tax gene of human T-cell leukemia virus type I. Proc Natl Acad Sci USA 1995; 92: 1057–1061. Franchini G, Mulloy JC, Koralnik IJ, LoMonico A, Sparkowski JJ, Andresson T, Goldstein DJ, Schlegel R. The human T-cell leukemia/lymphotropic virus type I p12 protein co-operates with the E5 oncoprotein and binds the 16-kilodalton subunit of the vacuolar H+ ATPase. J Virol 1993; 67: 7701–7704. Mulloy JC, Crowley RW, Fullen J, Leonard WJ, Franchini G. The human T-cell leukemia/lymphotropic virus type I p12 protein binds the interleukin-2 b and g chains and affects their expression on the cell surface. J Virol 1996; 70: 3599–3605. Kobayashi N, Konishi H, Sabe H, Shigesada K, Noma T, Honjo T, Hatanaka M. Genomic structure of HTLV (human T-cell leukemia virus): detection of defective genome and its amplification in MT2 cells. EMBO J 1984; 3: 1339–1343. Konishi H, Kobayashi N, Hatanaka M. Defective human T-cell leukemia virus in adult T-cell leukemia patients. Mol Biol Med 1984; 2: 273–283. Aldovini A, De Rossi A, Feinberg MB, Wong-Staal F, Franchini G. Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA. Proc Natl Acad Sci USA 1986; 83: 38–42. Hiramatsu K, Yoshikura H. Frequent partial deletion of human adult T-cell leukemia virus type I proviruses in experimental transmission: pattern and possible implication. J Virol 1986; 58: 508–512. Sakurai H, Kondo N, Ishiguro N, Mikuni C, Ikeda H, Wakisaka A, Yoshiki T. Molecular analysis of a HTLV-I pX defective human adult T-cell leukemia. Leukemia Res 1992; 16: 941–946. Bhat NK, Adachi Y, Samuel KP, Derse D. HTLV-1 gene expression by defective proviruses in an infected T-cell line. Virology 1993; 196: 15–24. Orita S, Kobayashi H, Saiga A, Kubota R, Osame M, Igarashi H. A spontaneous point mutation in the human T-cell leukemia virus type 1 pX gene leads to expression of a novel doubly spliced pXmRNA that encodes a 25-kD, amino-terminal deleted rex protein. DNA Cell Biol 1994; 13: 353–364. Kira J, Koyanagi Y, Yamada T, Itoyama Y, Tateishi J, Akizuki S, Kishikawa M, Baba E, Nakamura M, Suzuki J et al. Sequence heterogeneity of HTLV-I proviral DNA in the central nervous system of patients with HTLV-I-associated myelopathy. Ann Neurol 1994; 36: 149–156. Orita S, Kobayashi, Aono Y, Saiga A, Maeda M, Igarashi H. p21X mRNA is expressed as a singly spliced pX transcript from defective provirus genomes having a partial deletion of the pol-env region in human T-cell leukemia virus type 1-infected cells. Nucleic Acids Res 1993; 21: 3799–3807.