JOURNAL OF VIROLOGY, Apr. 1990, p. 1803-1807

Vol. 64, No. 4

0022-538X/90/041803-05$02.00/0 Copyright C) 1990, American Society for Microbiology

NOTES Effects of a Highly Basic Region of Human Immunodeficiency Virus Tat Protein on Nucleolar Localization HARUHIKO SIOMI, HISATOSHI SHIDA, MASATOSHI MAKI, AND MASAKAZU HATANAKA*

Institute for Virus Research, Kyoto University, Kyoto 606, Japan Received 28 September 1989/Accepted 22 December 1989

Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization, we constructed fusion genes encoding serially deleted segments of Tat joined to the amino-terminal end of the Escherichia coli 0-galactosidase molecule. We show that the basic region of Tat was sufficient for nuclear localization but not for nucleolar localization. Addition of three amino acids (59, 60, and 61) of the Tat sequence at the C-terminal end of the basic region was neccesary for the chimeric I-galactosidase to localize in the nucleus as well as in the nucleolus. We demonstrate that a short amino acid sequence (G-48 RKKRRQRRRA HQ N-61), when fused to the amino terminus of 0-galactosidase, can act as a nucleolar localization signal.

Human immunodeficiency virus type 1 (HIV-I), the causative agent of acquired immunodeficiency syndrome, is a highly regulated retrovirus; gene expression of the virus is controlled by several trans-acting genes as well as cis-acting regulatory sequences located within the viral long terminal repeat (LTR) and elsewhere in the viral genome (28). Of the trans-acting proteins, both Tat and Rev are essential for virus growth, but their precise functions remain to be defined. It has been proposed that the Tat protein accelerates the rate of virus production at one or more levels of control, such as RNA transport, RNA stability, and translation efficiency, as well as having effects on transcription (28). Hauber et al. (11) demonstrated that Tat is predominantly located in the nucleolus. Other trans-regulatory proteins of human retroviruses, Rex of human T-cell leukemia virus type I (HTLV-I) (26) and Rev of HIV-I (7, 20), have recently been shown to be located predominantly in nucleoli. Rex and Rev augment the production of viral structural polypeptides by increasing the cytoplasmic concentration of the intron-containing env and gag-pol mRNAs (6, 7, 13, 16, 24, 27). These facts suggest the possibility that this nucleolar event may be involved in the regulation of human retrovirus gene expression. At present, we know very little about how certain proteins accumulate in the nucleolus, although evidence for active transport of proteins to the nucleus and for the functioning of nuclear transport signals (3) in relation to cellular transport machinery (5, 12, 18, 19) has accumulated. Identification of a nucleolar localization signal of Tat would contribute to our understanding of the molecular mechanism of nucleolar targeting and further Tat-mediated trans-activation. Tat possesses a stretch of basic amino acid residues which is highly conserved among various HIV-I isolates, and a highly basic amino-terminal sequence of Rex of HTLV-I has been shown to constitute a nucleolar accumulating signal (26). To examine whether the basic region of Tat is crucial for nuclear and nucleolar migration, we constructed recombinant plasmids encoding for hybrid proteins with Tat at the *

amino terminus and 3-galactosidase at the carboxy terminus. We also used a vaccinia virus (VV) expression vector system to achieve a high level of transient gene expression in the VV-infected cells (2, 17, 26). In this VV system, Tat was also localized predominantly in the nucleolus (4). To express the chimeric genes which harbor 5' portions of the tat gene fused in frame to lacZ, the Sall fragments from pFtat (4) were inserted into the BamHI-SmaI site present on plasmid p7.5C40X (26). Resultant plasmid pV7.5Ftat, which contains the VV 7.5 promoter (2) followed by the entire tat-coding sequence and the HTLV-I long terminal repeat, was digested at its unique SmaI site, and then exonuclease III and Si nuclease digestion (9) were performed to create deletions in the 3' portion of the tat-coding region. To determine exact nucleotide endpoints, clones were sequenced by the dideoxy method of Sanger et al. (22). After ligation of a Sall linker (5'-GGTCGACC-3'), the Hindlll-Sall fragments containing the 3'-deleted tat-coding regions and VV 7.5 promoter were ligated into pMC-LTR (26), which contains the Escherichia coli lacZ gene followed by the HTLV-I long terminal repeat. This resulted in seven plasmids which produce seven hybrid proteins having 41, 49, 50, 52, 55, 58, or 61 amino-terminal residues fused to ,B-galactosidase (Tat-p-galactosidase fusions) (Fig. 1). We examined the subcellular localization of the fusion proteins described above by indirect immunofluorescence by using rabbit anti-p-galactosidase antibody (Fig. 2). The fusion protein expressed from pV7.5tat(1-61)lacZ32 was observed predominantly in the nucleus, as well as in the nucleolus. The fusion proteins expressed from both pV7.5tat(1-58)lacZ30 and pV7.5tat(1-52)lacZ25 were localized mainly in nuclei but were absent from nucleoli, although some cytoplasmic staining was also observed. These results strongly suggest that the basic region may be involved in nuclear transport of the Tat protein. In contrast, cells transfected with pV7.5tat(1-55)lacZ18, pV7.5tat(1-50)lacZ34, and pV7.5tat(1-41)lacZ1 lacked any localized staining and exhibited fluorescence throughout the cell, as did cells transfected with pV7.5tat(1-49)lacZ33 (data not shown). Although Tat(1-55)LacZ contains the basic stretch of

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RKKRRQR, its nuclear accumulation was inefficient. The possible explanation may be that the Tat portion of the fusion protein was prevented from folding properly by its association with 3-galactosidase, leading to the inability to be associated with some element of the nuclear transport system. The behavior of Tat(1-58)LacZ, which contains the entire lysine-arginine-rich region of Tat, was absent from the nucleolus of transfected cells. However, a fused protein with only three additional amino acids of Tat, Tat(1-61)LacZ, was targeted to the nucleolus as well as the nucleoplasm. Therefore, these results show that this basic region is not sufficient for nucleolar migration and additional amino acids are required for efficient nucleolar localization, when fused to 3-galactosidase. Next, we tested whether the short peptides, including the basic region of Tat, can direct P-galactosidase to the nucleolus. A duplex DNA linker containing the translational initiation codon followed by the codons for Tat was chemically synthesized with 5' SphI and 3' Sall overhangs and ligated between the SphI and Sall sites of the plasmid pGOM3 (26) containing the VV 7.5 promoter, the gene encoding ,-galactosidase, and the HTLV-I long terminal repeat, to give rise to the plasmid pV7.5tatBAAlacZ (Fig. 3A). pV7.5tatBAAlacZ or the control plasmid pV7.5NlacZ was transfected into CV-1 cells, and the subcellular localization of the polypeptides was examined by indirect immunofluorescence. Figure 3B shows that the polypeptide expressed from pV7.5NlacZ has predominantly a cytoplasmic distribution. In sharp contrast, the fusion protein TatBAAlacZ is seen to accumulate in the nucleolus within the nucleus, although the fusion protein is also partly seen in the cytoplasm. These data showed that the putative Tat nucleolar localization signal sequence GRKKRRQRRRAHQN is functional in targeting a cytoplasmically localized heterologous polypeptide to the nucleolus. Our experiments showed that the first 52 of the 86 amino one

acids of Tat were sufficient to localize the fused 3-galactosidase to the nucleus. This result is in accord with a recent finding that a ,3-galactosidase fusion protein that contained a 5-amino-acid sequence (GRKKR) derived from the basic region of Tat at the amino-terminal end of ,3-galactosidase accumulated within the nucleoplasm and was excluded from the nucleolus (21). The RKKR sequence is homologous to the nuclear localization signal of simian virus 40 large T antigen (14). The requirement of nonconserved Tat sequences at the carboxyl end of the basic region in order to generate sequences that are able to localize ,-galactosidase to the cell nucleolus provides an important hint for postulating models for the interaction of this basic stretch with a nucleolar constituent. It is reasonable to assume that the basic stretch will be functional only if it is exposed properly on the surface of a protein and that its function is sensitively affected by the structural environment within which it is present in any given protein. The amino acid residues 48 to 61 of Tat contain the sequence RKKRRQRRR. A similar sequence of two stretches of basic amino acids flanking a glutamine residue is present in a nucleolar localization signal of the Rex protein of HTLV-1 (26). The HIV Rev protein contains a similar highly basic region with a predominance of arginine residues (27). Glutamines also play a role in the sequence of Rev protein. The amino acid sequence (R-35 QARRNRRRRW RERQ R-50) of Rev could actually replace the basic region of Tat (4), and when glutamines were fused to the amino terminus of 3-galactosidase, they also acted as a nucleolar localization signal (15). Although the characteristic feature of the nucleolar localization signals in the three proteins is apparent, it may not require exact primary sequences, as shown by the comparison of the three sequences and the mutational analysis of Rex and Tat (10, 26). Protein sequences that bear little sequence homology have been reported to manifest identical biological functions in other

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VOL. 64, 1990 cases. For example, signal sequences that direct the insertion of proteins into the endoplasmic reticulum bear little, if any, sequence homology (1, 29); the same is true for signal sequences of mitochondrial proteins (23) and also for signal sequences of nuclear proteins (3). Therefore, it is possible that the three sequences share an underlying structural trait and may participate in the same mechanism for nucleolar localization. Although the experiments reported here do not directly probe the mechanism of nucleolar accumulation, a catalog of nucleolar targeting sequences from trans-regulatory proteins of human retroviruses may provide a clue for further investigation. We thank Koreaki Ito for anti-3-galactosidase serum, Hiromu Takematsu and Satoshi Kubota for technical assistance, and Hifumi Maeda for excellent secretarial assistance. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan. LITERATURE CITED 1. Briggs, M., and L. Gierasch. 1986. Molecular mechanisms of protein secretion: the role of the signal sequence. Adv. Protein Chem. 38:109-180. 2. Cochran, M. A., C. Puckertt, and B. Moss. 1985. In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J. Virol.

54:30-37. 3. Dingwall, C., and R. A. Laskey. 1986. Protein import into the cell nucleus. Annu. Rev. Cell Biol. 2:367-390. 4. Endo, S., S. Kubota, H. Siomi, A. Adachi, S. Oroszlan, M. Maki, and M. Hatanaka. 1989. A region of basic amino acid cluster in HIV-I tat protein is essential for trans-acting activity and nucleolar localization. Virus Genes 3:99-110. 5. Featherstone, C., M. K. Darby, and L. Gerace. 1988. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J. Cell Biol. 107:1289-1297. 6. Feinberg, M. B., R. F. Jarrett, A. Aldovini, R. C. Gallo, and F. Wong-Staal. 1986. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46:807-817. 7. Felber, B. K., M. Hadzopulou-Cladaras, C. Cladaras, T. Copeland, and G. N. Pavlakis. 1989. Rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc. Natl. Acad. Sci. USA 86:1495-1499. 8. Gerace, L., and B. Burke. 1988. Functional organization of the nuclear envelope. Annu. Rev. Cell Biol. 4:335-374. 9. Guo, L.-H., and R. Wu. 1983. Use for DNA sequence analysis and in specific deletions of nucleotides. Methods Enzymol.

100:60-96. 10. Hauber, J., M. H. Malim, and B. R. Cullen. 1989. Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein. J. Virol. 63:1181-1187. 11. Hauber, J., A. Perkins, E. P. Heimer, and B. R. Cullen. 1987. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc. Natl. Acad. Sci. USA 84:6364-6368. 12. Imamoto-Sonobe, N., Y. Yoneda, R. Iwamoto, H. Sugawa, and T. Uchida. 1988. ATP-dependent association of nuclear proteins

1807

13.

14.

15.

16.

17.

18. 19.

20.

21.

22. 23. 24.

25. 26.

27.

28.

29.

with isolated rat liver nuclei. Proc. Natl. Acad. Sci. USA 85:3426-3430. Inoue, J., M. Yoshida, and M. Seiki. 1987. Transcriptional (p40X) and post-transcriptional (p27x-11) regulators are required for the expression and replication of human T-cell leukemia virus type I genes. Proc. Natl. Acad. Sci. USA 84:3653-3657. Kalderon, D., W. D. Richardson, A. F. Markham, and A. E. Smith. 1984. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature (London) 311:33-38. Kubota, S., H. Siomi, T. Satoh, S. Endoh, M. Maki, and M. Hatanaka. 1989. Functional similarity of HIV-1 rev and HTLV-I rex proteins; identification of a new nucleolar-targeting signal in rev protein. Biochem. Biophys. Res. Commun. 162:963-970. Malim, M. H., J. Hauber, S.-Y. Le, J. V. Maizel, and B. R. Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature (London) 338:254-257. Nam, S. H., M. Kidokoro, H. Shida, and M. Hatanaka. 1988. Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease. J. Virol. 62: 3718-3728. Newmeyer, D. D., and D. J. Forbes. 1988. Nuclear pore binding and translocation. Cell 52:641-653. Richardson, W. D., A. D. Mills, S. M. Dilworth, R. A. Laskey, and C. Dingwall. 1988. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52:655-664. Rosen, C. A., and W. A. Haseltine. 1988. Selective regulation of HIV gene expression by the art gene product, p. 23-36. In F. B. Robert, Jr., B. R. Cullen, and F. Wong-Staal (ed.), The control of human retrovirus gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Ruben, S., A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W. A. Haseltine, and C. A. Rosen. 1989. Structure and functional characterization of human immunodeficiency virus tat protein. J. Virol. 63:1-8. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. Schatz, G. 1987. Signals guiding proteins to their correct locations in mitochondria. Eur. J. Biochem. 165:1-6. Seiki, M., J. Inoue, M. Hidaka, and M. Yoshida. 1988. Two cis-acting elements responsible for posttranscriptional transregulation of gene expression of human T-cell leukemia virus type I. Proc. Natl. Acad. Sci. USA 85:7124-7128. Shida, H. 1986. Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology 150:451-462. Siomi, H., H. Shida, S. H. Nam, T. Nosaka, M. Maki, and M. Hatanaka. 1988. Sequence requirements for nucleolar localization of human T-cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell 55:197-209. Sodroski, J., W. C. Goh, C. Rosen, A. Dayton, E. Terwilliger, and W. Haseltine. 1986. A second post-transcriptional transactivator gene required for HTLV-IlI replication. Nature (London) 321:412-417. Varmus, H. 1988. Regulation of HIV and HTLV gene expression. Genes Dev. 2:1055-1063. von Heijne, G. 1985. Signal sequences: the limits of variation. J. Mol. Biol. 184:99-105.