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(The FASEB Journal. 2000;14:1389-1399.)
© 2000 FASEB

High-level expression of active HIV-1 integrase from a synthetic gene in human cells

PETER CHEREPANOV1, WIM PLUYMERS1, ANJE CLAEYS, PAUL PROOST, ERIK DE CLERCQ and ZEGER DEBYSER2

Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium

2Correspondence: Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: zeger.debyser{at}uz.kuleuven.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A synthetic gene encoding for HIV-1 integrase was designed to circumvent the intrinsic instability and the repressor elements present in the wild-type gene. High-level expression of HIV-1 integrase was obtained in various human cell lines independently of viral accessory proteins. A human 293T cell line was selected that stably expresses HIV-1 integrase and has growth kinetics comparable to the parental cell line. The enzyme was localized in the nucleus and remained stably associated with the chromosomes during mitosis. Lentiviral vector particles carrying the inactivating D64V mutation in the integrase gene were capable of stably transducing 293T cells when complemented in the producer cells with integrase expressed from the synthetic gene. When the cell line that stably expresses integrase was infected with the defective viral particles, complementation of integrase activity was detected as well. Expression of active HIV-1 integrase in human cells will facilitate the study of the interplay between host and viral factors during integration.—Cherepanov, P., Pluymers, W., Claeys, A., Proost, P., De Clercq, E., Debyser, Z. High-level expression of active HIV-1 integrase from a synthetic gene in human cells.


Key Words: complementation • expression • human immunodeficiency virus type 1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AS A RETROVIRUS, the human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), has a replication cycle characterized by the reverse transcription of its genomic RNA into a double-stranded DNA copy flanked by long terminal repeats (LTRs) and by the integration of this viral DNA copy into the host chromosome. The only viral enzyme required for integration is integrase (IN), encoded by the 3' fragment of the retroviral pol gene. Since integration is an essential step in the replication cycle of HIV (1 , 2) and no human counterpart is known to exist, there is great interest in developing inhibitors of HIV integrase (3 , 4) .

Retroviral integrases recognize specific sequences in the long terminal repeat elements of the viral cDNA (For a review, see ref 5 ). The terminal 15 bp of the LTR are necessary and sufficient for site-specific cleavage and integration. A highly conserved CA dinucleotide immediately upstream of the cleavage site is critical for enzymatic activity. 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) (6 , 7) . This reaction occurs in the cytoplasm within a nucleoprotein complex, referred to as the preintegration complex (PIC) (8) . After entering the nucleus, the processed viral dsDNA is joined to host DNA. The joining reaction involves a coordinated 4–6 bp staggered cleavage of the host DNA and the ligation of the processed CA-3' OH viral DNA ends to the 5' phosphate ends of the target DNA. Repair of the remaining gaps is probably accomplished by host-cell DNA repair systems, although reverse transcriptase and integrase may play a role. Staggered strand transfer and gap repair result in the duplication of host cell sequences immediately flanking the inserted provirus. The duplication size is virus-specific and, in the case of HIV, consists of a 5 bp direct repeat (9) .

HIV-1 integrase is a protein of 32 kDa composed of three functional domains. The amino-terminal region (residues 1–50) is characterized by an ‘HHCC’ zinc finger-like motif (10) . Evidence suggests that binding of zinc promotes the self-assembly of IN into an active multimeric complex (11) . The central core domain (residues 50–212) contains a highly conserved triad of acidic residues D64, D116, and E152 (DDE motif) that are involved in catalysis. Mutation of any of these acidic residues abolishes catalytic activity of the enzyme (12 , 13) . The less conserved carboxyl-terminal domain (residues 212–288) harbors nonspecific DNA binding activity (14 , 15) . Although the overall conformation of functional integrase remains unknown, complementation observed between proteins containing mutations in distinct domains suggests the existence of an oligomeric state (16 , 17) .

The lentiviral PIC is recognized by the cellular nuclear import machinery and actively transported through the nucleopore (18 , 19) . Purified PICs contain HIV cDNA, IN, viral matrix (MA), nucleocapsid, reverse transcriptase, Vpr proteins, and cellular proteins. MA and Vpr are believed to facilitate nuclear transport in nondividing cells (19 20 21) . Tagged HIV-1 integrase, injected intracytoplasmatically, localized in the nucleus (22) . An atypical bipartite nuclear localization signal in the carboxyl-terminal region of IN was shown to interact with the importin/karyopherin pathway. The nuclear localization of integrase fusion proteins expressed in mammalian cells corroborates the karyophilic properties of HIV-1 integrase (23 , 24) .

An active search for cellular host factors that participate in HIV integration is ongoing. Three cellular proteins have been put forward. The integrase interacting protein 1 binds tightly to HIV-1 IN in vitro and stimulates its activity. This nuclear factor has been proposed to promote integration and targeting of viral DNA to actively transcribed genes (25) . Recently, the high mobility group protein HMG I(Y) was identified as a cellular host factor essential for PIC activity in vitro (26) . HMG I(Y) is a known nonhistone chromosomal protein involved in transcriptional control and nuclear architecture. It has been postulated that HMG I(Y) binds to HIV cDNA and juxtaposes both LTR ends prior to concerted integration. A third candidate host protein of ~10 kDa, first isolated from Moloney murine leukemia virus-infected cells, acts as a barrier to autointegration factor (BAF) (27) . It has been suggested that BAF acts by creating a meshlike structure through formation of intramolecular bridges in the viral cDNA. An essential role during HIV-1 replication for any of the proposed host factors awaits confirmation.

An understanding of the interplay of the HIV integration machinery with cellular proteins would be facilitated by the development of an efficient expression system for HIV-1 integrase in human cells. The integrase of HIV-1 has been expressed in Escherichia coli (E. coli) (6) and insect cells using baculovirus (7) and Saccharomyces cerevisiae (28) . High-level expression of HIV-1 integrase in mammalian cells has remained elusive, in large part because expression of HIV-1 Gag and Pol proteins in general is Rev dependent (29) . In the absence of Rev, multiple instability sequences (INS) or cis-acting repressor elements in the mRNA of the integrase gene interfere with protein expression (30 , 31) . In human cells, low-level Rev-dependent expression of HIV-1 IN has been reported previously (32) . Rev-independent expression was obtained when IN was fused to ß-galactosidase or green fluorescent protein (GFP) (23 , 33) or when detection was made more sensitive using a highly antigenic epitope tag (24) . Low-level expression of the integrase of avian sarcoma virus has also been reported (34 , 35) . Herein we describe construction of a synthetic gene coding for HIV-1 IN that enabled efficient expression of integrase in human cell lines. The ability of the enzyme to complement defective integrase carried by HIV-1-derived vector particles provides full proof for its enzymatic activity in the cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Assembly of the synthetic gene
The restriction sites NheI, PstI, BamHI, NaeI, and NarI divide the sequence of the synthetic gene into six fragments, each ~150 bp long, that correspond to the sequences 1–149, 144–306, 301–456, 451–623, 618–776, and 771–930 (see Fig. 3 ). Each of these fragments was constructed separately by annealing and extending two partially complementary oligonucleotides (85–95 nt long, PAGE-purified and 5'-phosphorylated, synthesized by Gibco BRL, Merelbeke, Belgium) using Sequenase (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (see Fig. 3 ). Each fragment was cloned into the EcoRV site of the vector pBluescript KS(+) (Stratagene, La Jolla, Calif.). The sequence errors found in the resulting clones were repaired using either the QuickChange procedure for site-directed mutagenesis (Stratagene) with Pyrococcus furiosus (Pfu) polymerase (for a base substitution within the fragment 451–623) or polymerase chain reaction (PCR) (for deletions in the terminal regions of the fragment 1–149). The full 930 bp sequence was built by stepwise assembly of the fragments. Choice of the cloning vector (pBluescript KS or SK) at each step was dictated by toxicity of the IN coding DNA. Finally, the two halves of the IN gene (1–451 and 452–930) were ligated together and cloned into pBluescript KS(+) resulting in pINs. The plasmid pINs(D64V) carrying the D64V-inactivating mutation in the INs gene was obtained from pINs using the QuickChange procedure (Stratagene) for site-directed mutagenesis with the oligonucleotides 5'-GCATCTGGCAGCTCGTCTGTACTCACCTGGAGGG and 5'-CCCTCCAGGTGAGTACAGACGAGCTGCCAGATGC.



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  		Figure 3. Sequence and structure of the synthetic gene. A) Sequence of the synthetic DNA coding for pNL4–3 HIV-1 integrase. The amino acid sequence is shown in the single letter code. The restriction sites used in construction are indicated. The translation initiation site is underlined. B) A schematic representation of the structure of the synthetic gene. The following regions are indicated: the 5'- and 3'- untranslated regions (UTR) derived from ß-globin mRNA, the Met-Gly dipeptide, and the integrase open reading frame (ORF). The three domains of the integrase protein are shown.

Expression constructs
The plasmid pCEP-INs was obtained by subcloning the 1 kb EcoRI/XhoI fragment of pINs carrying the integrase synthetic gene between the PvuII and XhoI sites of pCEP4 (Invitrogen, Leek, The Netherlands). The EcoRI end of the fragment was filled in using T4 DNA polymerase. The plasmid pCEP-INs(D64V) was constructed in a similar way, using pINs(D64V) as the source of the mutant integrase gene. To obtain pRSV-INs, the EcoRI/XhoI fragment of pINs was inserted between the NheI and XhoI sites of pBK-RSV (Stratagene).

The wild-type IN open reading frame was PCR amplified from the HIV-1 clone pNL4–3 using Pfu DNA polymerase with the primers 5'-CCCCCAAGCTTGCCAGCCATGTTTTTAGATGGAATAGATAAGG and 5'-CCCGCTCGAGCTTTCCTTGAAATATACATATGGTG, and subcloned into pCEP4 to obtain pCEP-INwt1. The CTE sequence (from the plasmid pS12; 36 ) was cloned into pCEP4 in the sense orientation, followed by insertion of the integrase gene upstream of the CTE giving pCEP-INwt1-CTE. To obtain pCEP-INwt2, the IN coding region from the pNL4–3 HIV-1 clone was amplified in two consecutive PCRs with the primers 5'-ATCACTAGCAACCTCAAACAGACACCATGGGAT + 5'-CCCAGTTTAGTAGTTGGACTTAATCCTCATCCTGTCTACT and 5'-AAACAGACACCATGGGATTTTTAGATGGAATAGATAAGG + 5'-TATCACTCGAGATCATAATATCCCCCAGT TTAGTAG, and cloned into the BamHI/PvuII sites of pCEP4. The expression construct for the GFP-IN fusion was described before (23) .

Cell culture
HeLa cells were from American Type Culture Collection (Rockville, Md.). Human embryonic kidney cells 293T, expressing SV40 large T antigen, were obtained from Dr. O. Danos (Evry, France). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 20 µg/ml gentamicin at 37°C in 5% CO2 humidified atmosphere. HeLa cells were transfected by electroporation. 293T cells were transfected using polyethylenimine (PEI) (37) or lipofectamine (Gibco BRL). To establish stable cell lines, cells transfected with pBK-INs or pCEP-INs or pCEP-INs(D64V), were cultured in the presence of 500 µg/ml geneticin (G418) or 200 µg/ml hygromycin B (both from Gibco BRL), respectively.

Western blotting and indirect immunofluorescence
For Western blotting and indirect immunofluorescence, we used rabbit polyclonal antibodies raised against recombinant HIV-1 integrase. Western blotting was performed using the ECL+ chemiluminescent detection system (Amersham Pharmacia Biotech) with biotinylated goat anti-rabbit antibodies (Dako A/S, Glostrup, Denmark) and streptavidin-horseradish peroxidase (Amersham Pharmacia Biotech). Detection limit was ~10 pg of recombinant IN.

For detection of IN expression in situ by indirect immunofluorescence microscopy, cells were grown on glass slides (HeLa cells) or in permanox chamber slides (Gibco BRL) (293T cells). Cells were fixed in 100% methanol and blocked with 10% FCS in phosphate-buffered saline (PBS). Incubations with antibodies were carried out at 37°C in the blocking solution. The primary antibody (rabbit anti-IN) was diluted 1:20 to 1:80; the secondary FITC-conjugated swine anti-rabbit antibody (Dako) was used at the dilution of 1:40. Nuclear staining was performed with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Bornem, Belgium) in methanol. Fluorescence microscopy was performed with a Leitz microscope (Wetzlar, Germany) using filter blocks I2 (for FITC) or A (for DAPI).

Amino-terminal sequence analysis of integrase expressed in 293T cells
Integrase was purified from 293T cells transiently transfected with pCEP-INs using in-house-produced rabbit polyclonal antibodies coupled to CNBr-activated Sepharose (Amersham Pharmacia). The protein was eluted in 50 mM glycine buffer, pH 2. Amino-terminal microsequencing was performed by Edman degradation on a 477A/120A protein sequencer (Perkin-Elmer, Norwalk, Conn.).

Lentiviral vector production
HIV-1-derived vector particles, pseudotyped with the envelope of vesicular stomatitis virus, were produced by transfecting 293T cells with a packaging plasmid containing the HIV-1 gag and pol genes (pCMV{Delta}R8.2), a plasmid expressing the envelope of vesicular stomatitis virus (pMDG), and a plasmid containing a reporter gene flanked by two LTRs (pHR'-CMVLacZ) (38) . The first generation packaging plasmid, containing all HIV-1 genes except for env, and the transfer vector were a kind gift from Dr. O. Danos (Généthon, France). For transfection of a 10 cm dish of 293T cells, a 700 µl mixture of three plasmids was made in 150 mM NaCl: 20 µg of vector plasmid, 10 µg of packaging construct, and 5 µg of envelope plasmid. To this DNA solution, 700 µl of a PEI solution (110 µl of a 10 mM stock solution in 150 mM NaCl) was added slowly. After 15 min at room temperature, the DNA-PEI complex was added dropwise to the 293T cells in DMEM medium with 1% FCS. After overnight incubation, medium was replaced with medium containing 10% FCS. Supernatants were collected from day 2 to 5 five post-transfection. The vector particles were sedimented by ultracentrifugation in a swinging-bucket rotor (SW27 Beckman, Palo alto, Calif.) at 25,000 rpm for 2 h at 4°C. Pellets were redissolved in PBS. Different viral stocks were normalized based on p24 antigen content (HIV-1 p24 Core Profile ELISA, DuPont, Dreieich, Germany) for use in complementation assays.

Complementation experiments
Integrase-defective virus particles were produced using pCMV{Delta}R8.2IN(D64V), obtained from Dr. D. Trono (Geneva, Switzerland), as a packaging plasmid (38) . Complemented vectors were produced by expressing integrase from pCEP-INS in 293T cells after quadruple transient transfection. Vector preparations were normalized for p24 antigen count. Vector was added to target cells in the presence of 2 µg/ml polybrene and left overnight. After removal of vector, cells were incubated for an additional 36 h. Cells were washed with PBS, fixed with 0.75% formaldehyde/0.05% glutaraldehyde in PBS, and stained with freshly prepared X-gal substrate (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, and 100 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) (Biotech Trade & Service Gmbh, St. Leon-Rot, Germany) in PBS at 37°C overnight. Each transduction experiment was done in duplicate in a 96-well plate. Transduction efficiency was determined by counting the number of blue cells 48 h after infection in one of the wells, whereas the cells in the duplicate well were split 1:2. Half of the sample remained in the well and was stained at confluency (passage 1) whereas the other half was cultured in a 24-well plate. At confluency, these cells were again split 1:2 (passage 2). Finally, cells were brought in a 6-well plate and grown to confluency (passage 3). It was estimated that by this time each cell had to undergo eight subsequent divisions. After staining, the efficiency of stable transduction was measured by counting ß-galactosidase-positive colonies (>=10 blue cells).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HIV-1 integrase from the wild-type gene
We made expression constructs using the wild-type HIV-1 IN gene. The basic construct, pCEP-INwt1, contains the pNL4–3 IN gene under the control of the immediate early human cytomegalovirus (hCMV) promoter/enhancer. In human 293T or HeLa cells transiently transfected with this plasmid, no expression of IN was detected by either immunofluorescence microscopy or Western blotting (Fig. 1 , lanes 3 and 9). It is known that the constitutive transport elements (CTE) of the type-D retroviruses can substitute for the HIV RRE-Rev system (39) . To circumvent the intrinsic instability of the IN mRNA, we introduced the CTE of the simian retrovirus type 1 (SRV-1) downstream of the IN coding region. The CTE-containing construct, pCEP-INwt1-CTE, resulted in low but detectable expression of IN (Fig. 1 , lanes 4 and 10).



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  		Figure 1. Western blot analysis of transient expression of HIV-1 IN in 293T cells using different expression strategies. 293T cells were transiently transfected with various expression vectors. At 48 h post transfection, cell extracts representing 104 (lanes 2–7) or 105 cells (lanes 8–11) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto PVDF membranes. Integrase was detected by Western blotting using polyclonal antibodies against HIV-1 integrase and the ECL+ system. Transfections were done with the following plasmids: lanes 2 and 8, pCEP4; lanes 3 and 9, pCEP-INwt1; lanes 4 and 10, pCEP-INwt1-CTE; lanes 5 and 11, pCEP-INwt2; lanes 6 and 7, pCEP-INS. Lane 1 contains 0.1 ng of recombinant HIV-1 integrase.

Previously we reported expression of the carboxyl-terminal fusion of IN to the green fluorescent protein (GFP-IN) (23) . No transport/stabilization element was required, in accord with Kukolj et al. (33) , who expressed integrase as a carboxyl-terminal fusion with ß-galactosidase. The effect of the instability elements in the IN gene on protein expression levels is illustrated in Fig. 2 . Presence of the IN coding sequence in the GFP-IN expression construct results in an approximately 10-fold decrease in expression levels compared to the parental GFP expression vector. Based on these results, we decided to design a synthetic gene for HIV-1 integrase with an increased intrinsic mRNA stability.



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  		Figure 2. Effect of the integrase sequence on the expression level of GFP in 293T cells. HeLa cells were transfected with pGFP or pGFP-IN. After 48 h, the fluorescence of transfected cells relative to mock-transfected cells was measured. Average values ± SD for duplicate experiments are shown.

Design and construction of the synthetic integrase gene
To achieve high-level expression of HIV-1 integrase in human cell lines, we recreated the gene maintaining the amino acid sequence of IN from the pNL4–3 clone of HIV-1, but adapting its GC content and codon usage to those of constitutively and highly expressed human genes. A first version of an artificial IN reading frame (not shown) was based on random choice between alternative codons at each position, biasing in favor of preferential triplets as found in the human ß-globin, {alpha}-, {gamma}-actin, and EF2 genes. Next, the DNA sequence was substantially edited to remove potential splice sites and to reduce the number of CpG methylation sites, but keeping the overall GC content and codon usage close to optimal. The final version of the synthetic gene (INs) (Fig. 3 ) contains fragments of the 5'- and 3'-untranslated regions (UTRs) from the ß-globin mRNA. This gene encodes for wild-type HIV-1 integrase with addition of the amino-terminal Met-Gly dipeptide. The extra glycine codon completes the Kozak’s consensus sequence (ANNATGG) required for efficient initiation of translation (40) . In the synthetic gene, the overall GC content is 59% as compared to 40% in the wild type. The gene was constructed from six synthetic DNA fragments, each ~150 bp long, by stepwise cloning. A variant synthetic gene carrying the inactivating D64V mutation, INS(D64V) was made by site-directed mutagenesis.

Transient and stable expression of HIV-1 integrase in 293T and HeLa cells
The synthetic gene was cloned into the expression vectors pCEP4 and pBK-RSV under control of the hCMV and Rous sarcoma virus (RSV) promoters, respectively. Transient expression of IN was obtained in both 293T and HeLa cell lines, as verified by immunoblotting (Fig. 1 , lanes 6 and 7) and indirect immunofluorescence (see Fig. 5 ). In 293T cells, expression levels of IN varied between 1 and 50 µg per 10 x 106 cells in different transfection experiments.



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  		Figure 5. Visualization of IN expression by indirect immunofluorescence. HIV-1 integrase expression was visualized by indirect immunofluorescence using polyclonal antibodies directed against integrase and FITC-conjugated secondary antibodies. A) Stable expression of IN in the 293T-INS cell line. B) 293T cells as negative control. C) Same field as in panel A, but with filter block A in order to visualize nuclei stained with DAPI. D) Transient transfection of HeLa cells with pCEP-INS. Original magnification was 500x (A–C) and 200x (D).

Transfection of 293T cells with the episomal expression vector pCEP-INS, followed by selection with hygromycin, resulted in a stable cell line, referred to as 293T-INS. Indirect immunofluorescence staining revealed that 80–90% of selected cells produce integrase at detectable levels (see Fig. 5A ). A cell line stably expressing D64V integrase was established as well. The level of integrase expression in both cell lines was ~1 µg of integrase per 10 x 106 cells (2x106 copies of IN per cell) (Fig. 4 ). The cell growth kinetics of 293T-INS and 293T-INS(D64V) were similar to those of the parental 293T cells (doubling time of 22 h). After 2 months of cell culture, IN expression levels remained stable as verified both by Western blotting and immunofluorescence.



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  		Figure 4. Stable expression of HIV-1 IN from the synthetic gene in 293T cells. Cell extracts (10 µg, or ~105 cells) were separated in 15% SDS-PAGE; integrase was detected by Western blotting using polyclonal antibodies and the ECL+ detection system. Lane 1: 2.5 ng of recombinant His-tagged HIV-1 integrase; lane 2: extract of 293T cells; lane 3: 293T cells stably transfected with the vector pCEP4; lane 4: 293T cells stably expressing IN (293T-INS); lane 5: 293T cells stably expressing D64V IN (293T-INS(D64V)).

In HeLa cells, integrase was found exclusively in the nuclei (Fig. 5D ). In 293T cells, transient transfections typically gave rise to an irregular, granular cytoplasmatic distribution of IN, probably due to precipitation of the protein (data not shown). In a subpopulation of those cells expressing lower amounts of IN, the protein was localized in the nuclei. In the 293T cell line selected to stably express IN, nuclear localization of IN was evident (Fig. 5A, C ). During the metaphase and anaphase steps of mitosis, IN remained stably associated with chromosomes (Fig. 6 ).



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  		Figure 6. Integrase is bound to chromosomes during mitosis. The indirect immunofluorescence microscopy was performed as in Fig. 5 . The 293T- INS cells were stained with antibody against HIV-1 integrase (A, C) or DAPI (B, D).

Solid phase amino-terminal sequencing of integrase purified from transiently transfected 293T cells revealed the amino terminus Gly-Phe-Leu-Asp-Gly-Ile-Asp-Lys, the starting methionine being removed post-translationally.

It was of interest to see whether the wild-type integrase gene could be expressed when put in the same context as the synthetic gene in pCEP-INs. Therefore, we constructed pCEP-INwt2, which contains the wild-type integrase gene with the same 5'- and 3'-untranslated regions as the synthetic gene as well as the Met-Gly dipeptide at its NH2 terminus. No significant IN expression was obtained when transfecting this plasmid in 293T cells (Fig. 1 , lanes 5 and 11).

Complementation of IN-defective vector particles
To verify whether integrase protein expressed from the synthetic gene in human cells is enzymatically active, we tested its ability to complement integrase-defective HIV-1-derived lentiviral vectors. We used the lentiviral vector system developed by Naldini et al. (38) . Pseudotyped vector particles were produced by transfecting 293T cells with a packaging plasmid containing HIV-1 gag and pol genes, a plasmid expressing the envelope G protein of the vesicular stomatitis virus, and a transfer vector containing the ß-galactosidase reporter gene, the HIV-1 genomic RNA packaging signal, and the long terminal repeats. The packaging plasmid pCMV{Delta}R8.2 containing the functional integrase gene was used to produce the ‘wild-type’ vector (WT vector). Integrase-defective virus particles (D64V vector) were produced using pCMV{Delta}R8.2IN(D64V), a packaging construct carrying the D64V mutation in the integrase gene (38) . This mutation is known to abolish integrase activity without affecting any other steps of infection (41) . All viral preparations were diluted so that an equal amount of p24 antigen was used in each experiment. The transducing titer of the D64V vector in 293T cells was 20-fold lower than the titer of the WT vector (Table 1 , transient transduction), in agreement with previously reported results (38) . The ß-galactosidase expression observed 2 days postinfection with the D64V vector is predominantly due to transcription from nonintegrated circularized viral DNA and is drastically reduced on passaging the cells (Table 1 , stable transduction). Nevertheless, in some experiments stable ß-galactosidase-positive clones were observed (0.5–1% of WT) (Table 1) . A residual transducing activity of the D64V virus has been described before (42) . It has been suggested that it occurs through an integrase-independent mechanism.


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  		Table 1. Complementation of integrase-defective lentiviral vector particles

Complemented vector (D64V+INS) was prepared by cotransfecting the 293T producer cells with the mutant packaging plasmid pCMV{Delta}R8.2IN(D64V) and the integrase expression construct pCEP-INS. Transducing activity of virus from the D64V+INS preparations was partially restored (Table 1) . The relative transducing titer of the D64V+INS vector amounted to 30% of WT (Table 1) , varying between 6 and 30% for different batches of D64+INS and WT vectors. The complementation was due to stable integration of the transfer vector, since an equal proportion of ß-galactosidase-positive colonies was counted after multiple passages of the transduced cells. In our protocol, transduced cells undergo ~8 subsequent cell divisions before final counting. Integration sites of the complemented D64V+INS vector in the chromosomal DNA of 293T cells were cloned using LTR-Alu PCR (43) . Sequencing of 23 clones revealed that integration was preceded by specific 3' processing, thus confirming HIV-1 integrase-mediated integration (data not shown).

Moreover, evidence for trans-complementing activity of integrase expressed from the synthetic gene in target cells was obtained (Table 1) . Transduction of IN-expressing 293T-INS cells with IN-defective virus particles, resulted in a higher transduction efficiency than with the parental 293T cells. After passaging the transduced cells, the difference became even more pronounced. This points to a direct or indirect interaction of integrase present in the receptor cell with the preintegration complex of the incoming vector. The WT and the complemented D64V+INS vectors also showed increased transduction efficiencies in the 293T-INS cells, which may suggest that the amount of active integrase present in the viral particle is dose-limiting or that integrase present in the target cell traps inhibitory or competing host factors.

The authenticity of the complementation was verified by repeating the experiments, using a synthetic gene carrying the D64V mutation. New WT, D64V, D64V+INS vectors were produced in parallel with a D64V+INS(D64V) vector. No complementation was obtained with the D64V vector when supplemented with INS-D64V in the producer cells or when the D64V vector was used to infect 293T-INS(D64V) cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Expression of HIV-1 proteins from partially spliced and unspliced mRNAs is dependent on Rev-RRE interaction (29) . When expressed in the absence of Rev protein or RRE, these mRNAs are shown to be unstable and, according to other reports, trapped in the nuclei or not translated efficiently (30 , 31 , 44 , 45) . A number of regulatory elements within Rev-dependent viral mRNAs have been identified. Apart from RRE, the cis-acting repressor sequences or instability elements are widespread throughout the viral genome and occur in the regions coding for Gag (30 , 44) , Pol (30 , 31) , and Env (45) . It is firmly established that they function post-transcriptionally and do not depend on the occurrence of splice sites. In their extensive study, Pavlakis and co-workers (30) identified a number of inhibitory sequences. The two major instability elements (INS-1 and INS-2) within the Gag coding region were shown to be responsible for a 100-fold reduction in the expression of the Gag protein in the absence of Rev/RRE (30) . They were found to decrease steady-state levels of gag mRNA by reducing its half-life. There is no sequence homology or specific secondary structure associated with various instability elements in gag and pol genes. Nevertheless these regions have one common feature: high AU content. Directed mutagenesis of the AU-rich regions present in gag and pol genes (including the 5'-portion of the integrase locus) resulted in Rev-independent expression of viral particles (30) .

Inhibitory/instability determinants have been found in 5'- and 3'- untranslated regions as well as in coding regions of different cellular mRNAs, especially in fast decaying mRNAs of proto-oncogenes and cytokines (for reviews, see refs 46 , 47 ). The AU-rich elements (AREs) have been identified in the 3'-UTRs of most short-lived mRNAs and have a minimal core pentanucleotide sequence AUUUA. Regulation of mRNA stability via a common regulatory element in the 3'-UTR would preclude differential regulation of retroviral gene expression. Instead, lentiviruses adopted the strategy of mRNA destabilization via interspersed INS, which can be suppressed by trans-acting factors. Unlike AREs, instability elements within coding regions of eukaryotic mRNAs lack a consensus sequence, likely as a result of their coding function. An interesting example is the rare-codon/AU-rich INS within the MATalpha1 yeast mRNA (48) . Whereas this element is contained within a 65 nt region, the HIV Gag-Pol mRNA bears multiple, independent INS. An example of a cellular mRNA with several INS is the c-fos mRNA where different, nonoverlapping fragments of the coding region were found to confer instability to chimeric mRNAs (49) . Apparently, the diverse cellular and viral mRNA inhibitory/instability elements represent binding sites for trans-regulatory factors responsible for degrading mRNA or sequestering it from efficient transport and translation.

Expression of a wide variety of genes is regulated at the level of mRNA stability. Since no clear consensus for INS exists, a rational prediction of cryptic INS in a particular mRNA is not feasible. Redesigning the gene by randomization of codon usage with a bias for preferential triplet choice will circumvent this problem. Our synthetic gene for HIV-1 integrase provides a proof for this principle.

The wild-type HIV-1 IN coding region has a GC content of 40%, whereas in highly expressed human genes it is around 60%. By taking advantage of the degenerative nature of the genetic code, we increased the GC content of the integrase gene to 59%. The other characteristics of our synthetic gene design are 1) removal of potential splice sites, 2) reduction of the number of CpG methylation sites, 3) introduction of 5'- and 3'-UTRs of a stable mammalian mRNA (in our case, from human ß-globin), and 4) addition of an extra amino-terminal peptide (in our case Met-Gly) for efficient initiation of translation. As a consequence of these modifications, we obtained very efficient expression of HIV-1 integrase in various human cell lines independently of the viral regulatory factors (Rev or Tat). Thus, transient expression of IN from pCEP-INS is ~100-fold higher than CTE-dependent expression from the wild-type gene in pCEP-INwt1-CTE. When the wild-type integrase gene was supplied with the same UTRs and the Kozak’s consensus sequence as the synthetic gene (pCEP-INwt2), no detectable expression of IN was observed in transient transfections of 293T cells. It follows that the design of the synthetic coding region is the major determinant for high-level expression. A variant of the synthetic gene with the Ala codon (GCC) instead of the penultimate Gly was constructed as well. The properties of the protein expressed from this gene are similar to the original reported here. Data on this and other modifications of the integrase gene will be presented elsewhere.

Codon optimization is classically carried out to improve expression of eukaryotic genes in bacteria. In contrast, few examples of synthetic genes for expression in mammalian cells exist. Haas et al. (50) achieved Rev-independent expression of a synthetic HIV-1 envelope glycoprotein (gp120) in human cells. The relative contribution of optimized codon usage and removal of INS for efficient expression of HIV-1 IN or gp120 from synthetic genes is not clear. Although codon usage may play a role, it is not a major one. If tRNA availability were an important factor, it would be difficult to explain high-level Rev-dependent Gag and Env expression during HIV replication.

HIV-1 integrase, expressed transiently or stably from the synthetic gene, clearly localizes in the nucleus (Fig. 5) . This finding corroborates previous results pointing to the karyophilic properties of HIV-1 integrase (22 23 24) . An interesting result is that during mitosis integrase remains stably bound to the chromosomes, as has been visualized in metaphase and anaphase (Fig. 6) . This is a strong indication of the direct interaction of integrase with the cellular DNA. Conspicuously, HIV-1 integrase is known to possess a low level of unspecific DNA nicking/cleavage activity (5) . It is therefore remarkable that the high levels of integrase expression were compatible with the replication of the cell, as evidenced by the establishment of cell lines (293T and HeLa) that stably express integrase with growth kinetics comparable to that of the parental cells lines. Moreover, we could not detect considerable chromosomal nicking in the 293T-INS cells compared to the 293T or 293T-INS(D64V) using TUNEL or COMET assays (data not shown) (51 , 52) . On the other hand, since toxicity of integrase would be detrimental to HIV replication, integrase may have no unspecific nuclease activity in vivo, or cellular inhibitors or compensatory factors may be induced. This is the focus of ongoing studies.

Integrase expressed from the synthetic gene was capable of complementing the inactivating D64V mutation in the integrase gene of an HIV-1 vector packaging construct. It is known that integrase molecules can functionally exchange domains (16 , 17) . Our results point to the enzymatic activity of (at least) the catalytic core of the enzyme expressed from the synthetic gene. The relative transduction efficiency of the complemented vectors amounted up to 30% of the transduction efficiency achieved with the wild-type vector. The principle of trans-complementation of IN-defective virus was shown previously using VPR-IN fusion expression constructs, with VPR targeting integrase into the virion (53) . The transducing activity of catalytic domain mutants of IN was restored for 20% by trans-complementation with VPR-IN. However, in the absence of VPR, the expression construct for wild-type integrase, achieved only 0.04% complementation efficiency (53) . In the absence of VPR, our synthetic gene results in a complementation activity that is 750-fold more pronounced. This is likely due to the high level of protein expression. It is possible that the integrase expressed from the synthetic gene is packaged into the virion by direct association with the gag-pol protein precursor.

Complementation of D64V HIV-1 vectors also occurred by integrase produced in the target cells. Apparently, expression of HIV-1 integrase in human cells also increases the efficiency of transduction by wild-type lentiviral vectors (Table 1) . These data may suggest that endogenous integrase can interact and exchange functional domains with the integrase in the preintegration complex. This may provide a tool for studying the actual organization of the PIC in the infected cell. Alternatively, integrase expression might activate cellular DNA recombination pathways that could promote lentiviral integration. Unfortunately, the interplay between retroviral integration and cellular DNA repair and recombination is not understood at all at this time, although early evidence has recently been put forward (54) .

The integration process is the keystone of retroviral replication. Once integrated into the chromosome, the provirus will remain stable throughout the life span of the target cell. It is of a great importance to study expression of active integrase in the living cell. This model system enables study of the interaction of this viral protein with cellular processes. Apparently between 50 and 100 molecules of integrase are present in the HIV virion. We can speculate that a fraction thereof is directly involved in LTR processing and integration and that the rest of the integrase pool has a structural or regulatory role. Extra integrase may be necessary to recruit a cellular factor to the PIC being formed or to trap an inhibitory host factor. Investigations on the catalytic function of IN within the cell can now be initiated in the context of all the proteins and cofactors that are potentially involved in the formation and function of the PIC. The intriguing question remains as to whether it is possible to uncouple HIV integration and reverse transcription in vivo. Is it possible to have correct and efficient integration with IN as the only viral protein within the cell? The interaction of HIV-1 integrase with mini-HIV type substrates (55) in the human cell can now be studied, which may eventually lead to the development of an intracellular integrase test for the evaluation of integrase inhibitors. Expression of HIV-1 integrase at high levels in mammalian cells will facilitate the identification of interacting host factors, the role of which is much debated for the moment.

We believe that the synthetic gene technology presented here will be useful for the further development of lentiviral vectors for gene therapy. First, Tat/Rev-independent gag-pol gene expression may yield higher viral titers (using packaging constructs without HIV LTR promoter). Second, the design of the synthetic packaging constructs will decrease the likelihood of recombination between producer constructs or with endogenous retroviral elements, thus improving the biosafety of the lentiviral vectors.


   ACKNOWLEDGMENTS
 
We appreciate the help from Dr. J. Neyts (Rega Institute) with antibody production. We thank Drs. O. Danos (Genethon, France), D. Trono (University of Geneva, Switzerland), and I. Verma (Salk Institute, San Diego) for providing us with the HIV lentiviral vector constructs and 293T cells. Work was supported in part by the Biomedical research Program of the European Commission. Z.D. is a postdoctoral fellow of the Flemish Research Foundation. W.P. has a grant from the Flemish Institute supporting Scientific-Technological Research in Industry (IWT). P.C. designed and constructed the synthetic gene and performed IF studies; W.P. analyzed expression, constructed the D64V mutant, and established the stable cell lines; Z.D. and A.C. designed and performed the complementation experiments. P.P. performed protein sequence analysis. The paper was written by Z.D., P.C., and E.D.C.


   FOOTNOTES
 
1 These authors contributed equally to the work.

Received for publication July 20, 1999. Revision received February 4, 2000.
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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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