The Internal Methionine Codons of Human T-Cellleukemia Virus Type

JOURNAL OF VIROLOGY, Oct. 1990, p. 4914-4921 Vol. 64, No. 10 0022-538X/90/104914-08$02.00/0 Copyright C) 1990, American Society for Microbiology The Internal Methionine Codons of Human T-Cell Leukemia Virus Type II rex Gene Are Not Required for p24rex Production or Virus Replication and Transformation PATRICK L. GREEN,' YIMING XIE,' AND IRVIN S. Y. CHEN' 2* Division of Hematology-Oncology, Department of Medicine,' and Department of Microbiology and Immunology,2 UCLA School of Medicine and Jonsson Comprehensive Cancer Center, Los Angeles, California 90024-1736 Received 21 May 1990/Accepted 12 July 1990

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) have two nonstructural trans-acting regulatory genes, tax and rex, located in the 3' region of the viral genome. The tax gene product (HTLV-I p4O't and HTLV-II p37) is the transcriptional activator of the viral long terminal repeat. The rex gene encodes two protein products, p27rex/p2lrex and p26`ex/p24rex in HTLV-I and HTLV-II, respectively. Rex acts posttranscriptionally to facilitate accumulation of full-length gaglpol and singly spliced env mRNA in the cytoplasm of HTLV-infected cells. Previous studies showed that the first ATG of the rex gene is critical for Rex production and function. The importance of the internal ATGs to Rex function is not known. However, in vitro mutagenesis of the HTLV-I rex gene has provided indirect evidence which suggests that p2lrex, and by analogy HTLV-II p24rex, results from initiation at an internal AUG of the taxlrex mRNA. By using an infectious molecular clone of HTLV-II, we investigated the importance of the internal ATGs of the rex gene on Rex protein production and function. Our results indicate that p24rex of HTLV-II is not initiated at an internal AUG and that the internal methionine codons are not crucial to the function of the rex gene and, ultimately, the transforming properties of the virus.

Human T-cell leukemia virus types I (HTLV-I) and II for interleukin-2 (26), the interleukin-2 receptor (1, 8, 19), (HTLV-II) have been identified as the causative agents of and granulocyte-macrophage colony-stimulating factor (31, some forms of leukemia and chronic neurological disorders 51). Several studies have implicated Tax in the HTLV in humans. HTLV-I causes adult T-cell leukemia in a small oncogenic process (14, 30, 48). percentage of seropositive individuals (18, 33) and, more The rex reading frame produces two proteins of 27 and 21 recently, has been linked with a progressive demyelinating kilodaltons in HTLV-I (p27rex and p21rex) (23) and 26 and 24 syndrome termed HTLV-I-associated myelopathy (32) or kilodaltons in HTLV-II (p26rex and p24rex) (37, 44). Amino- tropical spastic paraparesis (11). HTLV-II has been associ- terminal deletions of Rex coding sequences and specific ated only with rare forms of T-cell leukemia related to point mutations which abolish Rex protein production indi- hairy-cell leukemia (22, 38, 40), and thus its disease associ- cate that Rex acts posttranscriptionally to increase the ratio ation is less substantiated. However, recent studies revealed of incompletely spliced mRNAs (encoding Gag, Pol, and that a significant percentage of intravenous drug abusers in Env proteins) to completely spliced mRNA (encoding Tax the United States and Europe harbor HTLV-II (25, 36, 49), and Rex), resulting in highly regulated viral gene expression indicating that the virus is more prevalent in the population (16, 17, 20). The individual contribution of the smaller rex than first estimated. gene product, if any, has not been determined. HTLV transforms peripheral blood T lymphocytes in The precise relationship of the two rex gene products is vitro, as defined by their continuous proliferation in the not known, although it has been established that both absence of exogenous interleukin-2 (7, 34, 50), even though share the its genome lacks a classical oncogene and does not insert at proteins same carboxy terminus (28). One study specific sites within the host genome (41, 42). Three mRNA which involved in vitro translation of in vitro-synthesized species have been identified for HTLV. A full-length ge- HTLV-I RNA containing specific site-directed mutations in nomic mRNA (8.2 kilobases [kb]) and a subgenomic mRNA the rex open reading frame demonstrated that deletion of an (4.3 kb), in which one intron is spliced out, encode the internal methionine initiation codon resulted in the loss of characteristic retroviral Gag and Pol proteins and Env p2lrex (29). These results suggested that p2lrex is synthesized protein, respectively. A second subgenomic mRNA (2.1 kb), from an internal methionine initiation codon of the doubly which has an additional intron removed, encodes both the spliced taxlrex mRNA. Studies corroborating these results tax and rex gene products via translation in separate over- have not been performed in infected cells, due to the lack of lapping reading frames. The tax reading frame produces a an infectious clone of HTLV-I; thus, the function of the 40-kilodalton protein in HTLV-I (p40ax) and a 37-kilodalton smaller Rex protein has not been elucidated. protein in HTLV-II (p37ax), which localizes to the nucleus By using an infectious clone of HTLV-II, we investigated of infected cells (12, 21, 29, 46). Tax has been shown to the importance of the internal methionine codons of the rex increase the rate of transcription from the viral long terminal gene on p24rex production, viral replication, and transforma- repeat (LTR) (4, 5, 10, 43), as well as from the cellular genes tion. By using site-directed mutagenesis, the internal ATGs in the rex reading frame were systematically mutated. We report here that p24rex is not initiated at an internal methionine codon and that the internal methionine codons are not * Corresponding author. crucial to the function of the rex gene. 4914 VOL. 64, 1990 HTLV-II rex GENE 4915

MATERIALS AND METHODS (determined by the Bio-Rad protein assay), and percentages of ['4C]chloramphenicol acetylation were quantified by scin- Cell lines. 729-6, an Epstein-Barr virus-transformed hu- tillation counting of excised spots. man B-cell line, was maintained in Iscove medium supple- Metabolic labeling and immunoprecipitation. Stable 729 mented with 5% fetal calf serum (FCS) and antibiotics. transfected cell lines were labeled at 106 cells per ml with BJAB, an Epstein-Barr-negative Burkitt's lymphoma cell [35S]methionine-cysteine (Tran35S-label; ICN Biochemicals, line (kindly provided by W. Hall, Cornell University), was Inc. [100 ,uCi/ml]) in methionine-cysteine-free RPMI 1640 maintained in RPMI 1640 medium supplemented with 10% medium supplemented with 10% dialyzed FCS. Cells were FCS and antibiotics. Peripheral blood lymphocytes were labeled overnight at 37°C. Cells were lysed in radioimmuno- isolated by centrifugation over Ficoll-Hypaque of fresh precipitation buffer, and the lysates were ultracentrifuged at blood obtained from normal donors or from leukopacks 100,000 x g for 1 h. The lysates were immunoprecipitated by purchased from the Red Cross. These cells were grown in using either HTLV-II-specific human antisera or antibody RPMI 1640 medium supplemented with 20% FCS and anti- directed against the COOH-terminal tridecapeptide se- biotics. quence encoded by rex (44). Immune complexes were col- Plasmids and mutagenesis. pH6neo, an HTLV-IT infectious lected with protein A-Sepharose (Pharmacia, Inc.), and the proviral clone, LTR-II-CAT, and SV2neo have been de- proteins were separated on sodium dodecyl sulfate-poly- scribed previously (6, 45). Plasmids with the prefix "BC" acrylamide gels. Gels were treated with En3Hance (Dupont, are all derived from the plasmid BC12/CMV/IL-2 (9), which NEN Research Products) for fluorography and exposed to contains a simian virus 40 (SV40) origin of replication and X-Omat R film (Eastman Kodak Co.). the cytomegalovirus immediate early promoter. Proper in- DNA transfer and hybridization. High-molecular-weight sertion of genes downstream of the cytomegalovirus pro- DNA was extracted from stable 729 transfectants or HTLV- moter results in high levels of expression in 729 cells and II-infected BJAB cells and analyzed by the method of peripheral blood lymphocytes. BCHTLV contains the com- Southern (47), as described elsewhere (15). The probe con- plete coding region of HTLV-II (BamHI-BamHI, nucleo- sisted of an HTLV-Il-specific (1,176 bp XhoI-Clal fragment) tides [nt] 361 to 8550) and expresses all viral gene products. 32P-oligo-labeled fragment. BCHTLVCla (Tax- and Rex-), BCHTLVSph (Rex-), and RNA preparation and S1 nuclease analysis. Total cellular BCHTLVrex term (Rex-) contain rex and/or tax mutations RNA was prepared by lysing the cells in 4.7 M urea-1.3% that have been previously described (37). BCHTLV-UT sodium dodecyl sulfate-0.23 M NaCl-6.7 mM Tris (pH contains a deletion in the apparently nontranslated region 8.0)0.67 mM EDTA, followed by three phenol-chloroform between nt 6660 and 6984 (pH6neo-UT contains the identical extractions. RNA was pelleted by ultracentrifugation deletion in the pH6neo vector). BCHTLV-SA contains a through a CsCl pad (30,000 rpm for 16 h), suspended in 10 550-base-pair (bp) deletion in the nontranslated region be- mM Tris, pH 7.6-1 mM EDTA, and the concentration was tween nt 6660 and 7210, which also results in removal of the determined by spectrophotometry. S1 nuclease analysis was splice acceptor signal sequence for taxlrex exon 3, and thus performed with 25 ,ug of total RNA (5 ,ug of sample RNA and is Tax- and Rex- (pH6neo-SA contains the identical dele- 20 ,ug of uninfected cell RNA) and a [-y-32P]dATP-labeled tion in the pH6neo vector). Oligomer-directed site-specific 90-nt oligonucleotide probe for the cap site of HTLV-II (nt mutagenesis was performed in the M13-based vector, Blue- 294 to 383), as described previously (2). script KS+ (Stratagene), which contains the XhoI-SacI fragment of pH6neo (nt 6209 to 8662), by the method RESULTS described by Kunkel (24). The three internal ATGs in the rex reading frame were systematically mutated to ACG, which, Construction of rex internal methionine codon mutants. upon translation, would result in a amino acid substitution of Figure 1 shows the genomic structure of HTLV-II and the threonine for methionine. Mutations were confirmed by single 2.1-kb doubly spliced mRNA which encodes the either dideoxy sequencing or Maxam-Gilbert sequencing trans-regulatory gene products, Tax and Rex, in separate but (27, 39). These mutations were designated Ml (nt 7248, T to partially overlapping reading frames. The well-characterized C), M2 (nt 7338, T to C), and M3 (nt 7389, T to C) and were methionine (Met) initiation codon for the larger Rex species cloned back into the vectors described above (pH6neo and (p26rex) is at nt 5121. The Met initiator codon for the BCHTLV) for characterization. trans-activator protein, p37t", is located downstream at nt Electroporation and CAT assays. Electroporations were 5180. Previous HTLV-1 rex gene mutagenesis studies have performed as described previously by using a Bio-Rad gene suggested that the smaller rex gene product, p2lrex, is pulser and extender (3). Briefly, cells were washed twice initiated at an internal Met codon. The HTLV-II rex gene with phosphate-buffered saline, counted, and suspended in contains three potential internal Met initiation codons lo- RPMI 1640 medium supplemented with 20% FCS and anti- cated at nt 7248, 7338, and 7389 (Fig. 1), the latter of which biotics at a concentration of 2 x 107 cells per ml. A total of corresponds to the one in HTLV-I that was thought to be 5 x 106 cells, together with plasmid DNA, were exposed to responsible for p21rex production (29). a 960-,uF charge with a 250-V potential. Cells were trans- The internal Met codons of the HTLV-II rex gene were ferred to 10 ml of medium and grown at 37°C for 48 h. To systematically mutated to code for threonine (Thr) by site- isolate stable transfectants containing pH6neo-derived directed mutagenesis. Both Tax and Rex have been shown to DNA, cells were plated into 24-well culture dishes (5 x 105 be critical for viral replication and transformation. Since Tax cells per well) under G418 selection (1.5 mg/ml; GIBCO and Rex are encoded in partially overlapping reading frames, Laboratories). Following a 2- to 3-week selection period, informative mutagenesis studies require that the rex muta- wells which grew were pooled, and these mass cultures were tions be silent in the tax reading frame. Therefore, the maintained for further analysis. Cell extracts for chloram- HTLV-IT rex gene mutants, referred to as Ml, M2, and M3 phenicol acetyltransferase (CAT) assays were processed 48 (Table 1), were designed so as not to alter the amino acid h posttransfection as described previously (13). CAT reac- sequence of Tax; the mutations were confirmed by sequenc- tions were standardized for equivalent levels of protein ing (data not shown). 4916 GREEN ET AL. J. VIROL.

15'LTRI- -w m gag pol env UT tax/rex Tax p37 I Rex p26 and p24 2.1 kb mRNA 313449 5044 5183 7214 8751 An

------AUG I ~~~AUG(rex) AUG(tax) (7248) \ 5121 5180 Ml AUG AUG (7338) (7389) M2 M3 FIG. 1. Structural features of the HTLV-II genome and HTLV-II tax/rex mRNA and coding sequence. The complete proviral genome is shown schematically. Boxes depict the LTRs, and vertical lines mark the beginnings and ends of the gag, pol, env, and taxlrex genes and the nontranslated region (UT). Pertinent restriction enzyme recognition sites are listed (B, BamHI; C, ClaI; M, MIuI). The 2.1-kb doubly spliced tax/rex mRNA and the Tax and Rex coding sequences and protein sizes are represented below the genome. The tax AUG (nt 5180) and rex AUG (nt 5121) are indicated. The three internal methionine (AUG) codons (Ml, M2, and M3), which were mutated to code for threonine in this study, and their nucleotide locations in the rex reading frame, based on the HTLV-II pH6neo proviral sequence, are indicated.

Functional analysis of rex mutants by CAT assay. The rex Wild-type and tax and/or rex mutant HTLV-II expression gene Met codon mutants described above were inserted into vectors were cotransfected with LTR-II-CAT into 729 B full-length viral expression plasmids, and their ability to cells, and the functional levels of Tax and Rex were moni- trans activate LTR-linked gene expression was determined tored by measuring the amount of CAT activity (Fig. 2). The in CAT assays. The pH6neo vector contains the complete wild-type constructs (BCHTLV and pH6neo) significantly HTLV-II provirus, and transcription from the native LTR activated the HTLV-II LTR. Comparison of the percent responds to regulation by the trans-regulatory gene prod- acetylation indicated that the cytomegalovirus promoter is ucts, Tax and Rex. BCHTLV contains the complete coding more active than the HTLV-II promoter in 729 cells (88-fold regions of HTLV-II under the transcriptional control of the versus 15-fold over background). A mutation which frame- cytomegalovirus immediate early promoter, resulting in con- shifts both tax and rex (BCHTLVCla) results in no trans stitutive viral gene expression. This vector is analogous to activation above baseline levels, as previously reported (4). SV-HTLV, previously reported using the SV40 early pro- Mutation of the first rex ATG (BCHTLVSph) or a mutation moter (37). resulting in premature termination of rex (BCHTLVrex We previously reported that loss of Rex function results in term), both of which do not alter tax, significantly reduced a decrease in LTR-directed gene expression as measured by CAT activity as compared with the wild type, confirming CAT activity, which can be complemented by addition of previous results that HTLV-II Rex influences LTR-directed Rex in trans (37). This altered LTR activity is due to a gene expression (37). combination of both a Tax effect at the level of transcription Two additional mutants tested and used as positive and and a Rex effect that is posttranscriptional. We used this negative controls throughout these studies are BCHTLV- assay to determine whether our internal rex Met codon UT/pH6neo-UT and BCHTLV-SA/pH6neo-SA, respec- mutants were functional. tively. Both mutants contain deletions in the nontranslated

TABLE 1. Base substitutions in taxlrex coding region s -. .'r.; (f'. 1.1 .e. :-, l: -li -. Mutation name/ Nucleotide Protein amino acid change F__ - - -r, .11 A % nucleotidea base change Rex Tax .5 ::. .g cY Cla/7386 +CG fsb/Ser-79 fsb/Ile-59 SphI5120-5123 -CATG AMet-1 No effect I9 C n'. s/ rex term/7386 C-*A Ser-79--+Stop No effect M1/7248 T_-C Met-33-Thr No effect M2/7338 T-*C Met-63--*Thr No effect M3/7389 T-*C Met-80->Thr No effect 1- In ,. cr>-C u2 lc: hce£114 on AceW C, I.C a Mutations Cla, Sph, and rex term have been previously described (37). The Cla mutation frameshifts both rex (79 amino acids in frame plus 192 amino FIG. 2. Representative CAT assay. LTR-II-CAT and various acids out of frame) and tax (59 amino acids in frame plus 92 amino acids out HTLV-II expression constructs indicated above were introduced of frame). The Sph mutation deletes the first initiator Met codon of rex, into 729 B and percent was measured as described resulting in loss of Rex production and function. The rex term truncates Rex cells, acetylation at amino acid 79. Ml, M2, and M3 were made by site-directed mutagenesis, as in the Materials and Methods. Various mutations (described in the described in the Materials and Methods. Nucleotide and amino acid numbers Materials and Methods) were analyzed in the context of complete are based on the nucleotide sequence and Tax (331 amino acids) and Rex (170 HTLV-II genomes expressed either from their own promoter amino acids) protein sequences of the HTLV-II proviral clone, pH6neo. (pH6neo-derived constructs) or from the cytomegalovirus early b fs, Frameshift. promoter~~Cs~~~~~~~~~~~~~~.(BCHTLV-derived constructs). VOL. 64, 1990 HTLV-II rex GENE 491,

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FIG. 3. Southern hybridization analysis of the 729 stable transfectant cell DNA. Ten micrograms of high-molecular-weight cell FIG. 4. S1 nuclease analysis of 729 stable transfectant RNA DNA, as labeled, was double digested with BamHI and ClaI or with Total cellular RNA was prepared from stable transfectants, as BamHI and MluI (indicated by an asterisk), electrophoresed described in the Materials and Methods. A total of 25 ,ug of RNA (' through a 0.7% ,ug of transfectant RNA, as labeled in the figure, and 20 jig o0 agarose gel, blotted to nitrocellulose paper, and negative control 729 RNA) was subjected to S1 nuclease analysis hybridized with a 32P-labeled, HTLV-II-specific 1,176-bp probe with (XhoI [nt 6209]-ClaI [nt 7384]). The labeled arrows, in base pairs, a [.y-32P]dATP-labeled 90-nt oligonucleotide which spans the indicate approximate sizes of expected HTLV-II-specific fragments transcription start site of HTLV-II. A 70-nt fragment is protected b) and were determined by comparison with HindlIl digests of lambda mRNA synthesized from the cap site. The 90-nt fragment protectec and is due to complete protection of the probe by the 3' terminus of th( SV40 DNAs. RNA due to redundancy of sequences in the LTR. The labelec arrows, in base pairs, are marker sizes (_y-32P-end-labeled pGEM region between env and taxlrex exon 3. However, the SA digested with Sau3A). deletion removes all but four nucleotides of the splice acceptor site for taxlrex exon 3, which should abolish normal splicing of taxlrex mRNA and interfere with production of HTLV-II provirus (data not shown). Comparison of hy- Tax and Rex. When tested in CAT assays, the UT mutant bridization intensities of the predicted size fragments in the shows wild-type CAT activity, and as expected, the SA different transfectants indicates that HTLV-II DNA ic mutant is incapable of trans activation. The CAT activities present in similar copy number (Fig. 3). The 729pH6neoM3 of the rex Met codon mutants, Ml, M2, and M3, are cell line DNA can be distinguished from both the wild- comparable to wild-type levels, indicating that both Tax and type (729pH6neo) and other mutant (729pH6neoM] Rex are fully functional in this assay (Fig. 2). In addition, and 729pH6neoM2) cell line DNAs, since the M3 muta- similar relative activities were seen with the rex Met codon tion resulted in loss of the ClaI restriction site (Fig. 3). mutants in the two vector systems (BCHTLV or pH6neo), The smaller sizes of the hybridizing bands in both indicating that in this functional analysis of Tax and Rex, 729pH6neo-UT and 729pH6neo-SA also confirms the re- vector transcriptional control is not a factor. These results spective deletions of 324 and 550 bp in the nontranslated indicate that mutation of the internal methionine codons for region. Cell lines containing pH6neoM1 and pH6neoM2 the rex gene does not impair the function of the protein as DNA, which cannot be distinguished from wild-type DNA measured in transient CAT assays, whereas mutation of the (pH6neo) by restriction enzyme mapping, were confirmed b) initial Met codon or rex carboxy-terminal truncation abol- direct sequencing of polymerase chain reaction-amplified ishes Rex activity. products of stable transfectant cell DNA (data not shown). Isolation of stable transfectants. To further characterize These results indicate that the stable 729 cell transfectants effects of mutations within the rex gene on aspects of the have integrated full-length HTLV-II proviral DNA and the virus life cycle, including RNA expression, protein produc- constructed mutations are still present following transfectior tion, and budding of infectious virions, stable 729 cell and selection. transfectants containing pH6neoMl, pH6neoM2, and Expression of viral RNA. We investigated the effect oi pH6neoM3, along with positive and negative controls, were these internal Met codon mutations in the rex gene on viral isolated. Stable transfectants are prefixed by "729," fol- RNA expression. Total cellular RNA was isolated from the lowed by the full-length HTLV-II proviral clone with which stable transfectants and subjected to S1 nuclease analysis b) they were transfected (i.e., 729pH6neo is a stable line using a 90-nt oligonucleotide probe spanning the transcrip- transfected with the clone pH6neo). tion start (cap) site. The presence of correctly initiated To confirm the presence of HTLV-II proviral DNA in the HTLV-II mRNA results in protection of a 70-nt band. A stable transfectants, cell DNA was analyzed by nucleic acid 70-nt band of approximately equal intensities was protectec hybridization following digestion with diagnostic restric- in 729pH6neo, 729pH6neoMl, 729pH6neoM2, and 729pH( tion endonucleases (BamHI, ClaI, and MIuI). All stable neoM3 stable transfectant RNA, whereas this band was nol transfectants analyzed contained complete copies of the present in 729pH6neo-SA RNA (Fig. 4). These results art 4918 GREEN ET AL. J. VIROL.

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.. ._ a ...... - FIG. 5. Immunoprecipitation of [35S]methionine-cysteine-labeled stable transfectant cell lines. Cells were metabolically labeled, and cell lysates were prepared as described in Materials and Methods. (A) 729 stable transfectant cell lysate, as labeled, was immunoprecipitated with HTLV-II patient antisera. (B) 729 stable transfected cell lysates, as labeled, were immunoprecipitated with antisera directed against the COOH terminus of the rex gene products. "'C-labeled protein markers were purchased from Amersham Corp., and sizes are listed. consistent with the CAT assay data presented in Fig. 2. Si wild-type 729pH6neo (Fig. 5B). Taken together, these re- nuclease analysis of taxlrex doubly spliced mRNA revealed sults indicate that the internal methionines of the Rex protein similar results.,,..... (data not shown). Thus, the Met to Thr are not important for the functions of Rex identified to date. mutations within the Rex protein do not have an effect on In addition, these results suggest that the smaller rex gene transcription of viral RNA. product of HTLV-II, p24rex, is not initiated at one of the Viral proteins expressed by Rex Met mutants. The stable internal Met codons. transfectants were metabolically labeled with [35S]methio- Production of infectious virus and transformation. We nine-cysteine, and immunoprecipitations were performed on determined the effect of the rex Met codon mutations on cell lysates to determine which viral proteins were being virus production, replication, and transformation. The su- produced. Previous studies indicated that selective point pernatant of the stable transfectants was assayed for the mutations or amino-terminal truncation of the rex gene presence of virus by measuring in vitro reverse transcriptase resulted in the loss of Gag and Env protein expression and, activity. Reverse transcriptase activity was detected in the thus, loss of production and release of infectious virions. supernatant of all stable transfectants, and the activity did Immunoprecipitation of cell lysates with HTLV-II patient not differ by more than twofold (data not shown). These antisera followed by sodium dodecyl sulfate-polyacrylamide results indicate that the stable rex Met codon mutant trans- gel electrophoresis and autoradiography indicated that the fectants release virus into the culture supernatant at levels stable transfectants containing the rex internal Met codon similar to those of the wild-type transfectants. mutations all produced significant levels of Gag (Fig. 5A). In To further demonstrate that the rex internal Met codon this particular experiment, 729pH6neoM3 appeared to pro- mutant viruses were capable of a productive infection, the duce more Gag and 729pH6neoMl produced less Gag than stable transfectants were cocultured with the BJAB cell line. the wild type; however, this variation was not reproducible. BJAB cells were used, since upon infection by HTLV-II, a Therefore, the rex internal Met codon mutants are all func- rapid induction of syncytia with some cytopathicity results. tional and allow Gag protein to be produced. Coculture of the 729 rex Met codon mutant transfectants To determine which rex gene products were being pro- with BJAB cells resulted in syncytium formation with the duced in the stable transfectants, cell lysates were immu- same efficiency as 729pH6neo, and syncytia could be in- noprecipitated with antisera which recognize the carboxy duced with as few as 10 irradiated producer cells (Table 2). terminus of Rex and detect both Rex proteins (p26rex These results indicate that the 729 rex Met codon mutants and p24rex). Both p26rex and p24rex are produced in (Ml, M2, M3) are producing infectious virus. Following 729pH6neoM1, 729pH6neoM2, and 729pH6neoM3, and the coculture of the producer cells with the BJAB cells, the levels of these proteins are comparable to those of the cultures were maintained for 3 weeks by adding fresh BJAB VOL. 64, 1990 HTLV-II rex GENE 4919

TABLE 2. Infection of cells mutations, were confirmed by direct sequencing of polymerase chain reaction-amplified products of BJAB coculture Stable BJA b Primary T-cell Immunofluores- transfectantSa B transformation cence p19 Gag DNA (data not shown). Therefore, we conclude that the rex internal Met codon mutant viruses are stable and infectious 729 - - - in BJAB cells. 729pH6neo + (10) + + To determine whether the rex Met codon mutations al- 729pH6neo-SA 729pH6neo-UT +(10) + + tered the ability of the virus to transform primary T cells, 729pH6neoMl +(10) + + irradiated 729 producer cells were cocultured with primary T 729pH6neoM2 +(10) + + lymphocytes. Three to four weeks following coculture, 729pH6neoM3 +(10) + + stable transformed T cells grew out for all rex internal Met codon mutants (Table 2). These results further confirm that a 729 stably transfected cells were irradiated with 10,000 rads, and 106 cells the were cocultured with 5 x 105 stimulated peripheral blood lymphocytes, or internal methionines of the Rex protein are not crucial to serial 10-fold dilutions of irradiated cells were incubated with 105 BJAB cells the overall function ofRex, and their loss does not hinder the in 24-well culture plates. Cells were fed twice a week with RPMI 1640 ability of the virus to productively infect or transform supplemented with 10% FCS and antibiotics. Syncytia were scored micro- primary T cells in vitro. scopically 3 to 7 days postplating. Transformed cells grew out 3 to 4 weeks postplating. The presence of HTLV was confirmed by detection of HTLV p19 Gag by immunofluorescence in both cases. DISCUSSION b Numbers in parentheses indicate the minimum number of 729 producer cells required for syncytium induction following coculture with BJAB cells. We systematically mutated the Met codons of the HTLV-II rex gene to determine their importance for Rex protein production and function in relation to viral gene cells once a week to compensate for the loss of the cells due expression and transformation. We confirmed that only the to syncytium formation and cytopathicity. first Met codon is required for Rex regulatory function and To determine whether HTLV-II proviral DNA was that mutation of the internal Met codons did not alter this present in the BJAB cells cocultured with the 729 transfec- function. These results also indicate that p24rex translation is tants, cell DNA was analyzed by nucleic acid hybridization not initiated at a Met codon downstream of the initiation of following digestion with diagnostic restriction endonucleases p26rex (BamHI, ClaI, and MluI). All stable BJAB cocultures posi- The results presented in this report, which investigated tive for syncytium formation contained HTLV-II proviral only HTLV-II Rex production and function, are in apparent DNA (Fig. 6). It was also important to confirm the stability conflict with earlier studies investigating Rex protein initia- of the rex Met codon mutations in the BJAB-infected culture tion in HTLV-I. Those experiments identified the initiation and to ascertain whether these mutations reverted in culture. sites for the p27rex and p2lrex proteins and determined that The M3 mutation, which eliminated the ClaI restriction site, p2lrex was initiated at an internal Met codon (29). This Met was still present, as determined by the inability of ClaI to codon, which is conserved between HTLV-I and HTLV-II, digest HTLV-II M3-infected BJAB high-molecular-weight corresponds to M3 in our studies and did not affect HTLV-II DNA. The Ml and M2 mutations, as well as the M3 Met p24rex production. Two possibilities might explain the dis- crepancy. The first interpretation is that production of the Rex protein(s) differs with respect to HTLV-I and HTLV-II. The second possibility is that the HTLV-I results may be an (\. artifact of the experimental system, since conclusions were based on analysis of in vitro-synthesized RNA, followed by translation of this RNA in a rabbit reticulocyte lysate. Those (C (C C) viral constructs have not been tested directly in cells either Q~ Q Q~ Q~ 0:L Q a:: by transfection or infection. <

tional modification. First, p26re and p24re may have the transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. same amino acid composition or backbone and the appear- 14. Grassmann, R., C. Dengler, I. Muller-Fleckenstein, B. Flecken- ance of p26rex is due to glycosylation or phosphorylation. stein, K. McGuire, M.-C. Dokhelar, J. G. Sodroski, and W. A. Haseltine. 1989. Transformation to continuous growth of pri- The second possibility is that p24rex is a degradation or mary human T lymphocytes by human T-cell leukemia virus cleavage product of p26rex. However, our preliminary inves- type I X-region genes transduced by a Herpesvirus saimiri tigations indicate that p24rex is not derived from p26rex via vector. Proc. Natl. Acad. Sci. USA 86:3351-3355. proteolysis. Further analysis of the rex gene will be required 15. Green, P. L., D. A. Kaehler, L. M. Bennett, and R. Risser. 1989. to precisely determine the relationship between p26rex and Multiple steps are required for the induction of tumors by p24rex. Once this is determined, one can address whether Abelson murine leukemia virus. J. Virol. 63:1989-1994. these proteins are functionally distinct by further mutational 16. Hanly, S. M., L. T. Rimsky, M. H. Malim, J. H. Kim, J. Hauber, analysis of the rex gene and insertion into the HTLV-II M. Duc Dudon, S.-Y. Le, J. V. Maizel, B. R. Cullen, and W. C. proviral clone. This will ultimately be beneficial to Greene. 1989. Comparative analysis of the HTLV-I Rex and infectious HIV-1 Rev trans-regulatory proteins and their response ele- understanding the regulation of HTLV gene expression and ments. Genes Dev. 3:1534-1544. its role in disease development. 17. Hidaka, M., J. Inoue, M. Yoshida, and M. Seiki. 1988. Post transcriptional regulator (rex) of HTLV-I initiates expression of ACKNOWLEDGMENTS viral structural proteins but suppresses expression of regulatory We thank laboratory members S. Arrigo, J. Zack, M. Yip, W. proteins. EMBO J. 7:519-523. O'Brien, G. Feuer, and D. Camerini for editorial comments and W. 18. Hinuma, Y., K. Nagata, M. Hanaoka, M. Nakai, T. Matsumoto, Aft for preparation of the manuscript. K.-I. Kinoshita, S. Shirakawa, andI. Miyoshi. 1981. Adult T-cell This work was supported by American Cancer Society grant leukemia: antigen in an ATL cell line and detection of antibodies PF-3369, a Leukemia Society grant, and Public Health Service to the antigen in human sera. Proc. Natl. Acad. Sci. USA grants CA 38597 and CA 32737 from the National Institutes of 78:6476-6480. Health. P.L.G. is an American Cancer Society Fellow. 19. Inoue, J., M. Seiki, T. Taniguchi, S. Tsuru, and M. Yoshida. 1986. 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