Effects of Chimeric Mutants of Human ... - Europe PMC

Vol. 65, No. 5

JOURNAL OF VIROLOGY, May 1991, p. 2452-2456

0022-538X/91/052452-05$02.00/0 Copyright © 1991, American Society for Microbiology

Effects of Chimeric Mutants of Human Immunodeficiency Virus Type 1 Rev and Human T-Cell Leukemia Virus Type I Rex on Nucleolar Targeting Signals SATOSHI KUBOTA,' TETSUYA NOSAKA,1 BRYAN R. CULLEN,2 MASATOSHI MAKI,' AND MASAKAZU HATANAKA'*

Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan,' and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 277102 Received 11 June 1990/Accepted 6 February 1991

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus. NcoI DNA fragment of the rex expressor plasmid pKCR27x (13, 30) was substituted with the NOS of Rev-encoding synthetic oligonucleotides. This second chimeric plasmid was termed pH2NOVrex. Mutations were confirmed by a slight modification of the chain termination sequencing method as described previously (27). The oligonucleotides used were synthesized by the phosphoramidite method on an automatic DNA synthesizer (Applied Biosystems model 380B). Antibodies. A rabbit anti-Rex C-terminal peptide polyclonal antibody (for analysis of pH2NOVrex) (29), an affinity-purified rabbit anti-Rev C-terminal polyclonal antibody (for analysis of pH2NOXrev), and serum from a patient with progressive systemic sclerosis (as an antinucleolus antibody) were used (29). For indirect immunofluorescence, fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G antibody and tetramethylrhodamine isothiocyanate-conjugated anti-human immunoglobulin G antibody were prepared as secondary antibodies. Immunofluorescence. COS7 cells were seeded on glass coverslips, and DNA transfection was carried out by a slightly modified calcium phosphate precipitation method (9, 19). After 72 h the cells were permeabilized, incubated with primary antibody, and then stained with secondary antibody as previously described (29). Immunoprecipitation analysis. In a 6-cm culture dish, 1.5 x 105 COS7 cells were prepared a day before transfection. Transfection of DNA into cells was carried out as previously described. In short, 10 jig of each plasmid was introduced into cells by a slightly modified calcium phosphate precipitation method (9, 19). After 48 h of incubation with cysteinefree medium, cells were metabolically labeled with [35S]cysteine for 8 h. Cell extracts were prepared with special RIPA buffer (20) and incubated with antibody overnight at 4°C. Immune complexes were isolated as previously described and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis analysis (20). Assay of Rev and Rex function. To estimate the function of chimeric Rev-Rex protein, two reporter plasmids were used: pBennCATRRE (13), which contains the HIV-1 long termi-

Human immunodeficiency virus (HIV) and human T-cell leukemia virus type I (HTLV-I) are retroviruses that are the primary etiological agents of AIDS and adult T-cell leukemia, respectively (1, 12, 23). They contain characteristic regulatory genes in addition to the gag, pol, env genes which are found in all retroviruses. Among them, the rev gene of HIV and the rex gene of HTLV-I are thought to regulate viral mRNA transport, processing, or stability at posttranscriptional stages (2, 5, 7, 16, 28). Recent studies revealed the functional similarity (10, 13, 26) and predominant nucleolar localization (3, 15, 20, 22, 29) of these gene products. Despite these similarities, Rev and Rex proteins share little homology in primary structure. However, both possess a nucleolar-targeting signal (NOS): in Rev in the middle portion of the amino acid sequence (13) and in Rex at the amino terminus (29). These signals, which consist of 16- or 19amino-acid residues characterized by clustered basic amino acid residues, have the ability to accumulate 3-galactosidase into nucleoli when fused to the P-galactosidase aminoterminal region. Deletions of the rev and rex genes revealed that they are essential for the nucleolar localization and function of Rev and Rex proteins (13, 20). In this report, we constructed chimeric mutants of Rev and Rex to analyze the relevance of nucleolar localization to function.

MATERIALS AND METHODS Cells. COS7 (a monkey kidney cell line) cells were maintained at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and served for DNA transfection experiments. Construction of plasmids. The KpnI-BamHI NOS-encoding DNA fragment of pH2rev (13), which is a eukaryotic rev expression plasmid, was substituted with synthetic oligonucleotides that would express the NOS of Rex instead of that of Rev. The resultant chimeric plasmid was termed pH2NOXrev. Similarly, the NOS of the Rex-encoding SphI*

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FIG. 1. Construction of revlrex chimeric plasmids. Plasmid pH2NOXrev contains rev cDNA whose NOS region was substituted with that of rex. Similarly, pH2NOVrex contains rex cDNA with the rev NOS-encoding region. Nucleotide and deduced amino acid sequences of the exchanged regions are shown. Numbers below the sequence indicate the residue numbers of amino acids counted from the first methionine of Rex or Rev. Newly created and lost restriction enzyme recognition sites are also shown. SV40p represents the simian virus 40 early promoter; polyA indicates the simian virus 40 poly(A) addition region. The double line between SV40p and polyA represents the rabbit P-globin sequence, including its intron and short exon; the cloning site is represented as a solid box. These sequences originate in pKCRH2 (18).

nal repeat (LTR), bacterial chloramphenicol acetyltransferase (CAT) gene, and HIV-1 Rev response element for Rev function, and pSPLB15 (13), which contains the HTLV-I LTR, CAT gene, and 3' U3 region of HTLV-I for Rex function. In each system, tat or tax expression is also indispensable for measuring Rev or Rex function, respectively. Therefore, pH2Ftat (6), a tat expressor, and pKCR40M (30), a tax expressor, were prepared as well. Cells were prepared 1 day before transfection, and chimeric plasmid or control plasmid was cotransfected with pBennCATRRE plus pH2Ftat or pSPLB15 plus pKCR40M by the calcium phosphate precipitation or DEAE-dextran method, with slight modifications (4, 24). The total DNA quantity was adjusted so as to equalize experimental conditions by pKCRH2, a control plasmid that has the same transcriptional and replicational mechanisms as the other expressors (Fig. 1). After 48 h, cells were harvested and the lysate was prepared by three cycles of freeze-thawing, followed by centrifugation to remove debris. CAT activity was measured by an established procedure (8). The autoradiograms were quantitively analyzed with the Dr. Zeineh Soft Laser Scanning Densitometer System (Biomed Instruments Inc.). Each spot was integrated two dimensionally, and the ratio of acetylatedform density to total density was calculated.

FIG. 2. Intracellular localization of Rev, Rex, and Rev-Rex chimeric mutants. COS7 cells were transfected with 10 jig of pH2rev (A and B), pKCR27x (C and D), pH2NOVrex (E and F), and pH2NOXrev (G and H). Intact or chimenc Rev or Rex was detected by anti-Rev (A and G) or anti-Rex (C and E) antibody. Phasecontrast microscopic views of transfected cells are also shown (B, D, F, and H).

RESULTS Rev/Rex chimeric mutants localized in the cell nucleolus. To check the expression of the rev/rex NOS-exchanged genes and to elucidate the subcellular localization of their products, immunofluorescence experiments of COS7 cells transfected by pH2NOVrex or pH2NOXrev were performed. The structures of these plasmids are shown in Fig. 1. Each cDNA possesses the NOS-coding region of the other. Both products clearly localized in cell nucleoli. Rev generally accumulated in cell nucleoli to a greater extent than did Rex, and the distribution patterns of Rev and Rex in cell nucleoli were different (Fig. 2). Both chimeric mutants showed an intermediate pattern between authentic Rev and Rex. The product of pH2NOVrex possessed functional Rex activ-

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FIG. 4. Function assay of pH2NOXrev. (A) Rev system. Cells (5 105) were prepared in a 10-cm tissue culture dish and transfected with a DNA mixture containing 2 ,g of pBennCATRRE, 1 ,ug of pH2Ftat, and 5 ,ug of pH2rev, pH2NOXrev, or pKCRH2 (negative control). Lysate was prepared in 100 RI, and 20 ,ul was subjected to an assay. The acetylating reaction was performed at 37°C for 30 min. The positions of chloramphenicol (Cm) and its acetylated forms (AcCm) are indicated. Lanes: 1, pH2rev; 2, pH2NOXrev; 3, pKCRH2. (B) Rex system. Experimental conditions were as given in the legend to Fig. 3. Lanes: 1, pKCR27x; 2, pH2NOXrev; 3, pKCRH2. x

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12 3 FIG. 3. (A) Functional assay of pH2NOVrex. COS7 cells (2 x 105) were prepared in a 6-cm tissue culture dish and transfected with a DNA mixture containing 3 ,ug of pSPLB15, 2 ,ug of pKCR40M, and 3 Rg of pKCR27x, pH2NOVrex, or pKCRH2 (negative control). Total lysate was prepared in 60 RI, and 7.5 plI was subjected to an assay. The acetylating reaction was performed at 37°C for 20 min. Lanes: 1, pKCR27x; 2, pH2NOVrex; 3, pKCRH2. Positions of chloramphenical (Cm) and its acetylated forms (AcCm) are shown on the left. (B) Immunoprecipitation analysis of the pH2NOVrex product. Cells (1.5 x 10S) were transfected with 10 jig of pKCR27x, pH2NOVrex, or pKCRH2 and immunoprecipitated from metabolically [35S]cysteine-labeled cell lysate. Lanes: 1, pKCR27x; 2, pH2NOVrex; 3, pKCRH2. The arrowhead indicates the specific band identified by anti-Rex antibody. Positions of standard protein molecular weight markers (M) are shown on the right in thousands.

ity. The mutant gene pH2NOVrex produces a Rex protein with the NOS of Rev. This product was functional in our Rex assay system mediated by CAT gene expression (13, 28). Its activity corresponded to the amount of the expressed proteins (Fig. 3). Thus, this chimeric protein functions like the authentic Rex protein. The product of pH2NOXrev inhibited the functions of Rex and Rev. The chimeric plasmid termed pH2NOXrev expressed Rev protein with the Rex NOS. In contrast to pH2NOVrex, the translational product of this chimeric cDNA exhibited no function in the Rev and Rex assay systems (Fig. 4). The loss of function of this mutant may be due to a drastic change in protein conformation, as supported by computer-assisted secondary structure prediction analysis (data not shown), as a result of the substitution of the region corresponding to the NOS, located in the middle portion of the Rev amino acid sequence. However, this nonfunctional mutant inhibited the activity expressed by pKCR27x, a Rex expressor, when cotransfected into COS7 cells (Fig. 5A). An inhibitory effect against Rev was also observed in this mutant (Fig. SB). These inhibitory effects increased in a dose-dependent manner in both experiments (Fig. 5). Interference of nucleolar localization of Rex coincided with the inhibition by pH2NOXrev. To clarify the relationship between nucleolar localization and function of Rex, subcellular localization of Rex in the presence of excess pH2NOXrev was verified by an immunofluorescence study using an anti-Rex antibody that recognizes Rex only. In

the absence of pH2NOXrev, Rex protein localizes predominantly in cell nucleoli. However, in the presence of pH2NOXrev, a large portion of transfected cells showed a different intracellular distribution of Rex. The most typical nucleolar-targeting population decreased greatly, and many B

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FIG. 5. Inhibition of Rex and Rev function by pH2NOXrev. (A) Effect on Rex function. Various amounts of pH2NOXrev were used for cotransfection with 3 ,ug of pSPLB15, 0.5 ,ug of pKCR40M, and 2 ,ug of pKCR27x into 2 x 105 COS7 cells by the calcium phosphate method. Numbers at the bottom line indicate the relative amount of pH2NOXrev to pKCR27x; for example, "3" means that 6 pug of pH2NOXrev was cointroduced into cells with 12 pug of pKCR27x and other plasmids described above. The total amount of plasmid was adjusted to 23.5 pug by adding an adequate amount of the parental expression vector pKCRH2 (Fig. 1). (B) Effect on Rev function. Various amounts of pH2NOXrev were used for cotransfection with 2 pug of pH2rev into 2 x 105 COS7 cells by the DEAE-dextran method. To detect Rev activity, 1.5 ,ug of pBennCATRRE and 0.4 pug of pH2Ftat were also cotransfected in each experiment. Numbers at the bottom indicate the relative amount of pH2NOXrev to pH2rev, as described above. The total amount of plasmid was adjusted to 21.9 ,ug with pKCRH2. The acetylation reaction was performed at 37°C for 30 min. Autoradiograms were quantitively analyzed, and the remaining Rex or Rev activity was calculated. Relative rex or rev function was expressed as the ratio of remaining activity in the presence of pH2NOXrev to the activity in the absence of pH2NOXrev, expressed as a percentage.

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FIG. 6. Typical view of the novel intracellular distribution of Rex in the presence of the pH2NOXrev product. COS7 cells (105 per 3-cm culture dish) were transfected with 2.5 Rg of pKCR27x and 22.5 pg (ninefold excess) of pH2NOXrev by the calcium phosphate method. Transfected cells were doubly stained by rabbit anti-Rex antibody (A) and serum from a patient with progressive systemic sclerosis (C) 72 h after transfection. Immunocomplexes were visualized by fluorescein isothiocyanate-conjugated anti-rabbit antibody (A) and tetramethylrhodamine-conjugated anti-human antibody (C). A view of the same cells by phase-contrast microscopy is also shown (B). The immunofluoresence of cells transfected with 2.5 ,ug of pKCR27x and 22.5 ,ug of pKCRH2 was similar to that shown in Fig. 2C and D (data not shown).

transfected cells represented different staining patterns (Fig. 6). In this case, the expressed Rex protein migrated into nuclei but was apparently expelled from nucleoli. DISCUSSION The NOSs identified by us were able to convey various proteins into nucleoli (13, 29) and were commonly characterized by an extraordinary arginine-rich motif. A previous study revealed that a conserved arginine-rich motif of bacteriophage antiterminators is involved in their specific RNA hairpin structure recognition (14). Given the analogy of the NOS sequence to this conserved motif, there may be some direct relationship between the NOS and specific RNA recognition. Recent reports show that Rev specifically binds to the Rev response element of HIV-1 mRNA, which formed a stable secondary structure (11, 17, 32). Furthermore, the biological activities and in vitro Rev-binding abilities of Rev response element mutants correlated each other (17). Although there is as yet no evidence that Rex interacts directly with HTLV-I mRNA, it is reasonable to presume that a specific RNA-binding event plays a role in the biological function of Rex as well. If Rex protein has an RNA-binding domain(s) in its molecule, the NOS itself might be a candidate. However, the NOS-exchanged Rex mutant (the product of pH2NOVrex) remained functional as Rex protein. This result suggests that the primary NOS sequence itself has little effect on specific RNA recognition (21). If the arginine-rich feature is important in the RNA recognition event, the NOS may play a positive role in the primary approach of Rex to RNA, after which a secondary specific event could take place with a second specificity region. Therefore, the NOS may serve for approaching and attach-

ing to RNA before the specific RNA-Rex binding event, shown in Tat (31).

as

It is known that many mutated genes of rev and rex express a dominant-negative phenotype. Our mutant pH2NOXrev inhibited a Rex function stronger than squelching or titration but less than has been found for dominantnegative mutants. To gain insight into the nature of this inhibitory effect, we examined the subcellular localization of wild-type Rex coexpressed with the pH2NOXrev product, using an anti-Rex antibody. In the presence of pH2NOXrev, intact Rex protein was excluded from cell nucleoli in transfected cells. At the same time, its function was inhibited. The nucleolus is believed to be an organelle of ribosome biogenesis. Not limited to this function, it may affect posttranscriptional regulation of certain mRNAs. One possibility is that it stores some factors that are indispensable for mRNA regulation, or it may provide an extra pathway for unspliced RNA transport to the cytoplasm. We are now investigating the biological significance of the nucleolus. ACKNOWLEDGMENTS We gratefully thank Hiromu Takematsu for preparation of plasmids, Kazunori Hirayoshi and Haruhiko Siomi for microscopy, Masayuki Akayama for antibody purification, and Seiko Nishiguchi and Risa Kubota for typing. This work was supported in part by the Uehara Memorial Foundation, by the Yamada Science Foundation, and by grants-in-aid from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Barre-Sinoussi, F., J. C. Chermann, F. Ray, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. VezinetBrun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk

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