Nuclear Localization of Human Immunodeficiency Virus Type 1 Integrase Expressed as a Fusion Protein with Green Fluorescent Protein

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1 Virology 258, (1999) Article ID viro , available online at on Nuclear Localization of Human Immunodeficiency Virus Type 1 Integrase Expressed as a Fusion Protein with Green Fluorescent Protein Wim Pluymers, Peter Cherepanov, Dominique Schols, Erik De Clercq, and Zeger Debyser 1 Rega Institute for Medical Research, Katholieke Universiteit, Leuven, Belgium Received January 11, 1999; accepted March 23, 1999 Lentiviruses in general and the human immunodeficiency virus type 1 (HIV-1) in particular have the ability to integrate their genome stably into the chromosome of nondividing cells. Integration of HIV cdna is mediated by the viral integrase (IN). Apart from its catalytic activity, this enzyme seems to play an important role in the transport of the HIV preintegration complex into the nucleus of nondividing cells. We studied the karyophilic properties of IN by constructing an N-terminal fusion protein of HIV-1 integrase and green fluorescent protein (GFP-IN). Transient expression of GFP-IN in various mammalian cell lines was demonstrated by fluorescence microscopy, flow cytometry, and Western blotting. Although wild-type GFP was localized throughout the cell, GFP-IN was localized predominantly in the nucleus. Nuclear localization of GFP-IN was also obtained after transient transfection of the cells arrested in the G 1 /S phase of the cell cycle. These results provide compelling evidence for the karyophilic properties of the HIV-1 integrase Academic Press INTRODUCTION Integration is an essential step in the retroviral replication cycle. The only viral enzyme required for efficient integration of the retroviral cdna is integrase (IN), a protein of 32 kda encoded by the 3 -end region of the pol gene (for a review, see Brown, 1997). The enzyme is produced by protease-mediated cleavage of the gag-pol precursor during virion maturation. The IN recognizes specific sequences in the long terminal repeat elements of the viral cdna. The terminal 15 bp of the long terminal repeat is necessary and sufficient for site-specific cleavage and integration. A highly conserved dinucleotide CA repeat immediately upstream of the cleavage site is critical for enzymatic activity (Sherman and Fyfe, 1990; La Femina et al., 1992). In the first step of the integration reaction, termed 3 -end processing, two nucleotides are removed from each 3 -end to produce new 3 -hydroxyl ends (CA-3 OH). This reaction occurs in the cytoplasm, within a large viral nucleoprotein complex referred to as the preintegration complex (Miller et al., 1997). After entering the nucleus, the processed viral doublestranded DNA is joined to host target DNA. The joining reaction involves the coordinated 4- to 6-bp staggered cleavage of the target host DNA and the ligation of processed CA-3 OH viral DNA ends to the 5 phosphate ends of the cleaved target DNA. Gap repair, although not well understood at this time, probably is accomplished by host-cell DNA repair enzymes, although IN might carry out a cleavage ligation reaction (Chow et al., 1992). 1 To whom reprint requests should be addressed at Minderbroedersstraat 10, B-3000 Leuven, Belgium. Fax: zeger. debyser@uz.kuleuven.ac.be. Oligonucleotide-based assays have been designed to mimic both processing and joining reactions in vitro (Bushman et al.,; 1990; Katz et al., 1990). These assays have been instrumental in elucidating the biochemistry of the reactions carried out by IN. In contrast to other retroviruses, the lentiviruses such as the human immunodeficiency virus type 1 (HIV-1) have the capacity to replicate efficiently in nondividing and differentiated cells; thus nondividing mucosal dendritic cells and terminally differentiated macrophages play important roles in the pathogenesis of HIV infection (Finzi and Siciliano, 1998). Lentiviral integration requires transport of the preintegration complex through the nuclear membrane pores via an active, energy-dependent process (Bukrinsky et al., 1992). The role of various viral proteins in this process is not well understood. At least three components of the HIV-1 preintegration complex seem to play a role in nuclear transport: Vpr (Heinzinger et al., 1994; Fouchier et al., 1998; Popov et al., 1998), matrix protein (Bukrinsky et al., 1993; Gallay et al., 1995), and IN (Gallay et al., 1997). These different proteins interact with each other and with the cellular import machinery. The nuclear localization of Vpr and matrix protein is already well defined. The karyophilic properties of IN are not well established. IN of avian sarcoma virus was expressed in eukaryotic cell lines as such (Morris-Vasios et al., 1989) or as a C-terminal fusion protein with -galactosidase (Kukolj et al., 1997). A stable murine cell line expressing Rous sarcoma virus IN has also been reported (Mumm et al., 1992). In these instances, IN was found localized in the nucleus. Conflicting data have been presented with respect to HIV-1 IN. An HIV-1 IN -galactosidase fusion was expressed in /99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.

2 328 PLUYMERS ET AL. the cytoplasm (Kukolj et al., 1997), whereas a fluorescein-conjugated recombinant protein in which IN was fused to the C-terminus of glutathione S-transferase, injected into the cytoplasm of COS cells, localized almost exclusively in the nucleus (Gallay et al., 1997). The interaction between IN and karyopherin- was required for nuclear import and was mediated by the recognition of a bipartite nuclear localization signal in the IN protein (Gallay et al., 1997). We constructed a fusion protein of HIV-1 IN and green fluorescent protein (GFP-IN) to address directly the karyophilic properties of HIV-1 IN. The presence of GFP enables direct visualization of the expressed protein by fluorescence microscopy. In accord with the data of Gallay et al. (1997), we found that GFP-IN was localized predominantly in the nucleus of transiently transfected cells. Our results demonstrate that HIV-1 IN has the propensity to migrate into the nucleus. Expression of GFP-IN RESULTS Transient transfection of 293T cells with the plasmid pegfp-in resulted in the expression of GFP-IN as analyzed by fluorescence microscopy (Fig. 1). As a control, expression of GFP from the plasmid pegfp-c2 was verified in parallel. With both plasmids, 293T cells could be transfected with a transfection efficiency of about 75%. The percentage of fluorescent cells correlated with the transfection efficiency as measured by parallel transfections of a -galactosidase reporter plasmid (data not shown). However, in all cells examined, the intensity of the fluorescent signal was much lower in cells expressing the fusion protein in comparison with cells expressing GFP. To prove that the fluorescence in the cells transfected with pegfp-in was really due to the presence of the fusion protein and not to GFP itself, we performed a Western blot of a lysate of transiently transfected 293T cells (Fig. 2). A 60-kDa protein was detected with antibodies to HIV-1 IN (lane 4 in Fig. 2A), whereas in the lysate of the cells transfected with pegfp-c2, no specific bands were detected. The antibody recognizes recombinant his-tagged HIV-1 IN (about 33 kda) (lane 2 in Fig. 2A). When the same lysates were analyzed with polyclonal antibodies directed against GFP, a protein of 27 kda was detected in the cells transfected with pegfp-c2 (lane 3 in Fig. 2B). The fusion protein GFP-IN was not detected in this blot. Probably the affinity of the antibodies to GFP is not high enough to detect the low-level expression of GFP-IN. Nuclear localization of GFP-IN in HeLa, OST/TK, and 293T cells The expression of GFP-IN and GFP was visualized in different cell lines by fluorescence microscopy (Fig. 1). We used HeLa (carcinoma), OST/TK (osteosarcoma), and 293T cells (transformed embryonic kidney cells) for this purpose. Although GFP was present in both the cytoplasm and the nucleus of the three cell lines examined, the GFP-IN fusion protein was predominantly located in the nucleus. The presence of GFP in the nucleus is likely due to diffusion [because the protein has a molecular size (27 kda) below the exclusion limit of the nucleopore], but the nuclear localization of GFP-IN points to karyophilic determinant(s) in the HIV-1 IN. The presence of a fluorescent label allowed us to determine the kinetics of GFP-IN production and nuclear transport. At 24 h post-transfection, GFP-IN was predominantly localized in the cytoplasm. At 48 h post-transfection, nuclear localization of GFP-IN was evident. Closer investigation revealed that GFP-IN was not distributed homogeneously in the nucleus because in some cells, the protein seemed to be condensed into discrete loci (Fig. 1, right). In rare cells, spots of intense fluorescence were observed in the cytoplasm as well, most probably as a result of precipitation of GFP-IN expressed at high levels. Localization of GFP-IN in nondividing cells The presence of GFP-IN in the nuclei of transfected cells may be the result of the DNA-binding activity of IN in cells during mitosis and nuclear membrane disassembly. To rule out this possibility, transfection was performed in arrested 293T and OST/TK cells. Cells were arrested in the G 1 /S phase of the cell cycle by aphidicholin treatment before transfection. Again, GFP-IN was almost exclusively localized in the nucleus of transfected cells (data not shown). Expression of HIV-1 IN quantified by FACS analysis Attempts to express detectable levels of HIV-1 IN by cloning RRE behind IN and coexpressing Rev have failed so far. The expression of HIV-1 IN as a fusion construct with GFP was a strategy for expressing HIV-1 IN in eukaryotic cells that worked in our hands. The expression of HIV-1 IN is likely hampered by instability sequences that are known to exist in the transcribed mrna of the IN gene (Schneider et al., 1997). The following experiment illustrates the destabilizing effect of the IN mrna on GFP expression. HeLa cells were transfected with pegfp-in or pegfp-c2 by electroporation, and the percentage of fluorescent cells was determined by flow cytometry (Fig. 3). Although transfection efficiency, expressed as percentage of fluorescent cells, was similar (39% versus 41% or 33% versus 32%, in the two experiments shown in Fig. 3), mean fluorescence intensity in the analyzed cells was about fivefold lower in the cells expressing GFP-IN compared with the cells expressing GFP.

3 NUCLEAR LOCALIZATION OF HIV-1 INTEGRASE 329 FIG. 1. Expression of GFP and GFP-IN in different cell lines. Transient expression of GFP (left) and GFP-IN (right) in HeLa, OST/TK, and 293T cells as visualized by fluorescent microscopy 48 h post-transfection.

4 330 PLUYMERS ET AL. FIG. 2. Transient expression of GFP and GFP-IN in 293T cells. Lysates were made of 293T cells transiently expressing GFP or GFP-IN and analyzed by Western blotting. Lanes 3 and 4 each represent 24,000 cells. (A) Polyclonal antibodies directed against his-tagged HIV-1 IN were used. Lane 1 indicates rainbow protein molecular weight marker; lane 2, 5 ng of his-tagged HIV-1 IN; lane 3, lysate of cells transfected with pegfp-c2; and lane 4, lysate of cells transfected with pegfp-in. (B) Polyclonal antibodies directed against EGFP were used. Lanes 2 4 indicate the same as in A. DISCUSSION We provide conclusive evidence for the nuclear localization of HIV-1 IN, expressed as a N-terminal fusion protein with GFP in HeLa, OST/TK, and 293T cells. This result is at odds with the finding of the cytoplasmic localization of a C-terminal fusion protein of HIV-1 IN with -galactosidase expressed transiently in HeLa-Tat cells (Kukolj et al., 1997). Because the same authors also reported on the nuclear localization of a similar avian sarcoma virus IN -galactosidase fusion, the size of the fusion protein cannot be a limiting factor for nuclear import. However, fusion to the C-terminus of IN might prevent correct folding and interfere with the interaction of the bipartite nuclear localization signal of HIV-1 IN and karyopherin- (Gallay et al., 1997). HIV-1 IN contains three putative NLS sequences: KELKK 160, KRK 188, and KELQKQITK 219 (Myers et al., 1992). The two latter sequences were proposed to constitute a bipartite signal that binds to karyopherin- (Gallay et al., 1997). We constructed an N-terminal fusion product with IN that therefore should not interfere with the C-terminal NLS sequence. Nuclear import of a fluorescein-conjugated glutathione- S-transferase IN fusion protein was demonstrated previously (Gallay et al., 1997). In this experiment, however, IN was not expressed endogenously, but instead recombinant protein was injected in the cytoplasm of COS-7 cells. Here we show that IN, expressed in various mammalian cell lines, is imported into the nucleus. The size of GFP (27 kda) is below the exclusion limit (40 kda) of the nuclear pore complex (NPC). This implicates that GFP can enter the nucleus without active transport and will be distributed throughout the cell. The size of GFP-IN (60 kda) is well above the exclusion limit of the NPC. Passive diffusion of the protein into the nucleus is prevented by the NPC; therefore, without a specific transport mechanism through the NPC, the protein would be excluded from the nucleus. Nevertheless, we observed an accumulation of GFP-IN in the nucleus that points to (1) an active transport through the NPC and to (2) a functional NLS in the IN protein. The distribution of GFP-IN in the nucleus was not homogeneous, and the protein appeared to concentrate at discrete loci. High-density fluorescent spots that were observed in the cytoplasm of some cells may represent intracellular precipitates of GFP-IN. FIG. 3. Quantification of GFP-IN expression using FACS analysis. HeLa cells transiently expressing GFP or GFP-IN or control HeLa cells (MOCK) were fixed and analyzed by flow cytometry. An analysis of two separate representative experiments (top and bottom) is shown. The mean fluorescence intensity and the percentage of fluorescent cells are indicated in each histogram.

5 NUCLEAR LOCALIZATION OF HIV-1 INTEGRASE 331 The expression of the gag, pol, and env genes of HIV-1 in mammalian cells depends on the presence of the viral Rev protein (Smith et al., 1990; Cochrane et al., 1991; Myers et al., 1992). Expression of HIV-1 IN is hampered by the presence of inhibitory or instability sequences throughout the IN gene (Schwartz et al., 1992; Schneider et al., 1997). The presence of the Rev responsive element (RRE) downstream of the IN gene and Rev is believed to circumvent this instability to some extent (Cochrane et al., 1991; Faust et al., 1995; Schneider et al., 1997). When IN was expressed as a fusion protein with Vpr, to transcomplement IN in virions lacking IN, it was also necessary to introduce the RRE and to coexpress Rev (Fletcher et al., 1997; Liu et al., 1997; Wu et al., 1997). In contrast to these observations, in our hands expression of IN as a fusion protein did not require Rev coexpression (data not shown). This is in accordance with Kukolj et al. (1997), who expressed IN as a C-terminal fusion protein with -galactosidase in the absence of Rev. Creation of the GFP-IN fusion was in our hands a successful approach to express detectable levels of wild-type HIV-1 IN in mammalian cells. Moreover, the presence of GFP enables quantitative analysis by flow cytometry and opens the possibility to perform biophysical studies in vivo on the interaction of HIV-1 IN with DNA. The expression of GFP-IN can also be useful to develop a cellular integration assay and to study the enzymatic activity of IN in vivo; such an assay should be helpful in the identification of small-molecular-weight inhibitors of HIV integration or nuclear transport. Recombinant plasmids MATERIALS AND METHODS The open reading frame of the HXB2 HIV-1 IN was PCR amplified using the Pfu DNA polymerase (Stratagene) with the primers IN-LEFT (5 -CCCCCAAGCTTGCCAGC- CATGTTTTTAGATGGAATAGATAAGG-3 ) and IN-RIGHT (5 -CCCGCTCGAGCTTTCCTTGAAATATACATATGGTG-3 ) and subcloned in pcep4 (InVitrogen), resulting in pcep- IN. The 1-kb HindIII XhoI fragment of pcep-in containing the IN gene was cloned in the vector pegfp-c2 (Clontech) to obtain pegfp-in. This construct contains the gene of the EGFP-IN chimera (EGFP fused to the N-terminus of the HIV-1 IN, which will be further denoted GFP-IN) under the control of the human immediate-early cytomegalovirus enhancer/promoter. The full IN open reading frame was sequenced to confirm the absence of mutations. No frameshift mutation or stop codon was introduced behind the EGFP gene, as verified by DNA sequence analysis of the fusion part of both genes. Cell culture and transfections The 293T cells (obtained from O. Danos, Evry, France) were grown in DMEM (GIBCO BRL) with glutamax supplemented with 10% FCS, 500 U/ml penicillin, and 500 g/ml streptomycin solution. Cells were seeded onto 6-well plates at a density of 100,000 cells/well 24 h before transfection. On the day of transfection, 5 g of DNA was diluted in 100 l of 150 mm sterile NaCl. Then, 13.5 l of EXGEN 500 (polyethyleneimine) (Euromedex, Strasbourg) was diluted in 75 l of 150 mm NaCl. Both solutions were mixed and incubated for 15 min at room temperature. During this incubation, the cells were washed once with DMEM (GIBCO BRL) plus glutamax and 1% FCS, and 600 l of this medium was used to cover the cells. After incubation, the DNA polycation complexes were added dropwise to the cells, and the plates were put in a 5% CO 2 incubator. The day after transfection, the medium was changed with complete DMEM with 25 mm HEPES. HeLa cells were grown in DMEM (GIBCO BRL) supplemented with 10% FCS, 0.12% (v/w) sodium bicarbonate (GIBCO BRL), 2 mm glutamine (GIBCO BRL), and 20 g/ml gentamicin (GIBCO BRL). For electroporation, the cells were trypsinized at 80% confluence and were pelleted by low-speed centrifugation. The cells were then resuspended at a density of cells/ml in growth medium; 0.5 ml of this solution was aliquoted into 4-mm cuvettes (Eurogentec, Seraing, Belgium), and 20 g of DNA was added to the cell suspension. After the electric pulse (10 F, 250 V), the cells were allowed to rest for 10 min at room temperature before plating. The human osteosarcoma cell line OST/TK (ATCC CRL-8303) was grown in MEM (GIBCO BRL) supplemented with 10% FCS, 0.12% (v/w) sodium bicarbonate, 2 mm glutamine, and 20 g/ml gentamicin. The day before transfection, the cells were seeded onto a 35-mm dish ( cells/dish). The transfection procedure was identical to that of 293T cells using the appropriate medium for this cell line. Cell cycle arrest with aphidicholin Cells were grown on 6-well plates as described above. At a confluence of 40%, aphidicholin (Sigma) was added to the medium at a final concentration of 15 g/ml. At 24 h after this treatment, cells were transfected as described above. Growth arrest by aphidicholin was verified in separate control experiments by cell counting. Western blot analysis At 48 h post-transfection, cells were lysed in SDS PAGE loading buffer (25 mm Tris HCl, ph 6.8, 50 mm DTT, 1% SDS, 0.05% bromophenol blue, and 5% glycerol) at 3000 cells/ l. After the samples were boiled for 5 min, 8 l was loaded onto a 15% SDS-polyacrylamide gel. Detection was performed on PVDF membranes (Bio-Rad) using the ECL chemiluminescent system (Amersham- Pharmacia) with rabbit polyclonal anti-hiv-1 IN (produced in house) or anti-gfp (Clontech) antibodies.

6 332 PLUYMERS ET AL. Fluorescence microscopy At 48 h post-transfection, cells were washed twice with PBS, covered with a coverslip, and analyzed using the Nikon fluorescence microscope, with the filter set at B2 (DM510; Nikon), to visualize the expression of EGFP and EGFP-IN. FACS analysis At 48 h post-transfection, cells were washed twice with PBS and subsequently fixed with 5% paraformaldehyde (Sigma) in PBS. Cells were analyzed with an FAC- Scan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Data were acquired and analyzed with CellQuest software (Becton Dickinson). ACKNOWLEDGMENTS Wim Pluymers is funded by a grant from the Flemish Institute supporting Scientific-Technological Research in Industry (IWT). Zeger Debyser has a postdoctoral fellowship from the Flemish Fund for Scientific Research (FWO). We thank Kristel Van Laethem and Anne-Mieke Vandamme for help with PCR and sequencing work and Bart Degreve for helpful discussions. 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