Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners

Jorge Vera1, Vincent Parissi2,3, Andrea García1, Roberto Zúñiga1, Marie-Line Andreola2,3, Anne Caumont-Sarcos2,3, Laura Tarrago-Litvak2,3 and Oscar Leon1

1 Programa de Virologia, ICBM, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile
2 CNRS UMR 5097, Bordeaux, F-33000 France; Université Victor Segalen Bordeaux 2, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France
3 Bordeaux, F-33000 France; IFR 66 ‘Pathologies Infectieuses et Cancers’, Bordeaux, F-33000 France. 146 rue Léo Saignat, 33076 Bordeaux cedex, France

Correspondence
Oscar Leon
oleon{at}med.uchile.cl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in Escherichia coli led the authors to explore the use of Saccharomyces cerevisiae for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel–NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from E. coli and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the in vitro assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, S. cerevisiae was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that S. cerevisiae could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Integration of viral DNA into the host genome is an essential step for retrovirus replication. This process is mediated by a large nucleoprotein complex known as preintegration complex (PIC). The exact composition of PICs for different retroviruses has not been defined, but in addition to viral DNA and integrase (IN), it comprises several other viral and cellular proteins (Turlure et al., 2004). Within the complex, IN removes two deoxynucleotides from both 3' ends of the long terminal repeat (LTR) termini (3' processing) to expose a subterminal 5'-CA-3' dinucleotide, conserved among all retroviruses and retrotransposons [Reviewed by Craigie (2001)]. Subsequently, the PIC migrates to the host nucleus and integrates the viral DNA into chromosomal DNA in a concerted transesterification reaction (Craigie et al., 1990; Engelman et al., 1991). Repair of the gaps on the integration intermediate results in a short direct duplication of host DNA sequences. Processing and strand-transfer reactions have been measured in vitro by using synthetic oligonucleotides corresponding to the LTR ends. In addition, IN catalyses another in vitro reaction called disintegration, a reversal of the strand-transfer reaction (Chow, 1992).

Although the general process of integration is similar among retroviruses, there are several aspects that are not yet completely understood, especially those dealing with virus import into the nucleus. The role of viral and cellular proteins in the pathway for PIC migration to the nucleus, entry and targeting is under intense research, not only to determine possible approaches to inhibit virus replication but also to understand the mechanisms involved. For Moloney murine leukemia virus (M-MuLV), the viral proteins capsid, reverse transcriptase and IN have been reported to be PIC components (Fassati & Goff, 1999). Recently, the cellular proteins barrier-to-autointegration (BAF) and lamina-associated polypeptide 2{alpha} (LAP2{alpha}) have been described as collaborating to organize the M-MuLV PIC. For human immunodeficiency virus type 1 (HIV-1), the cellular proteins BAF (Chen & Engelman, 1998), HMGA1 (Farnet & Bushman, 1997) and more recently LEGF/p75 (Maertens et al., 2004) have been shown to be important in the integration process.

Retroviral INs have essentially three functional domains [Reviewed by Chiu & Davies (2004)]. The HisHis/CysCys (HHCC) motif is highly conserved among all INs. For M-MuLV, this domain is essential for integration (Jonsson et al., 1996) and it is also involved in protein multimerization (Yang et al., 1999; Leon & Roth, 2000). Some basic residues of this domain have been identified as critical for 3' processing and strand transfer (Yang et al., 2001). In addition, the importance of the HHCC domain to the efficiency of coordinated two-end integration has been described (Yang & Roth, 2001). The core domain or catalytic site of INs is characterized by a D-D(35)-E motif that is highly conserved among retroviruses and retrotransposons. In the case of the M-MuLV, the conserved DDE motif comprises Asp125, Asp184 and Glu220. Substitutions of any of these acidic residues in HIV-1 and Rous sarcoma virus INs result in loss of all catalytic activities (Drelich et al., 1992; Engelman & Craigie, 1992; Kulkosky et al., 1992; Leavitt et al., 1993). The C-terminal domain of INs is less conserved. This domain contributes non-specific DNA-binding activity and participates in oligomerization (Engelman et al., 1994). IN seems to exist in a dynamic equilibrium of monomers, dimers, tetramers and high-order oligomers (Bao et al., 2003). In vivo and in vitro complementation studies suggest that the active M-MuLV IN is a multimer (Jonsson et al., 1996; Yang et al., 1999; Yang & Roth, 2001). On the basis of its amino acid sequence, the monomeric M-MuLV IN has an estimated 46 kDa molecular mass.

Most in vitro studies have been carried out with the enzyme produced in Escherichia coli. Given the high similarity between yeast and higher eukaryotes regarding protein folding, membrane trafficking and other cellular mechanisms, yeast has been widely used to uncover and establish basic aspects of fundamental biology. As a first step toward defining the cellular protein factors able to interact with IN, we used the yeast Saccharomyces cerevisiae.

In this work, we present the results of the expression and purification of M-MuLV IN in yeast. We also describe the results of the expression of M-MuLV IN in yeast strains that are deficient in RAD52 and SNF5.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strains, culture media and growth conditions.
Yeast strains used in this work are listed in Table 1. The culture media were: yeast complete medium (YEPD) (1·0 % yeast extract, 2·0 % bactopeptone, 2·0 % glucose); yeast selective medium [YNB lacking uracil and leucine (0·67 % yeast nitrogen base without amino acids, 0·1 % glucose)]; or YCAD lacking uracil (YNB supplemented with 0·5 % Casamino acids, 0·01 % adenine, 2·0 % glucose, 0·2 % tryptophan). Amino acids and bases (20–30 mg l–1) were added as required. Liquid cultures were performed in 2 l Erlenmeyer flasks filled to one fifth of their capacity and shaken in an orbital incubator. Solid media were obtained by supplementing liquid media with 2·5 % bacto-agar. Yeast strains were incubated at 28 °C.


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Table 1. Yeast strains

 
Bacterial strains, culture media, transformation procedure, growth conditions, DNA preparation and DNA analysis.
E. coli strain DH5{alpha} was used for plasmid amplification. Bacterial transformation was performed as described elsewhere (Hanahan, 1983). Bacteria were grown on Luria–Bertani medium containing ampicillin (50 or 200 µg ml–1). Plasmids were extracted from bacteria using the Miniprep Express Matrix (BIO 101 Systems) and the plasmid SV miniprep kit (Promega). DNA was purified from gels using the QIAquick Gel Extraction system (Qiagen) and the PCR SV gel (Promega). DNA restriction endonucleases and DNA ligase (Promega) were used under conditions recommended by the suppliers. DNA fragments were analysed by gel electrophoresis on 1 % agarose in the presence of 0·5 mg ethidium bromide ml–1 under UV light.

Construction of the M-MuLV IN expression vector in yeast.
The yeast–E. coli shuttle expression vectors used in this work were those previously described by Caumont et al. (1996). These vectors are self-replicating recombinant plasmids that contain the 2 µ replication-origin sequence, the URA3 yeast selection markers and the leu2-d genes (Erhart & Hollenberg, 1983). These plasmids were used to transform yeast strains mutated in their LEU2 and URA3 genes. The expression vector pJV M-MuLV IN was constructed in two steps. First, the histidine-tagged M-MuLV IN expression vector pETINH1 (Jonsson et al., 1993) was used as a template to amplify the IN coding sequence. A NcoI site at the start codon and a SalI site at the 3' terminus of the IN coding sequence were incorporated using the oligonucleotides 5'-CGCGCCATGGTAGAAAATTCATCACCCTACA-3' and 5'-CGCGGTCGACCTATAACCTTATTTTTAAGGGGTT-3', respectively. The PCR amplification was carried out using ELONGASE Enzyme Mix (Gibco-BRL). The PCR product was digested with SalI and partially digested with NcoI to obtain a 1200 bp DNA fragment.

The gel-isolated product was ligated to pBS100 digested with NcoI and SalI to directionally insert the IN gene under the control of the alcohol dehydrogenase 2/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAPDH)-inducible hybrid promoter (Cousens et al., 1987) to obtain the construct pBS100/M-MuLV IN. Positive clones were selected by the release of a 787 bp fragment by total digestion with NcoI. PBS100/M-MuLV IN was digested with BamHI and SalI to release the expected 2420 bp fragment corresponding to the ADH2/GAPDH promoter and IN, gel-isolated and ligated to a BamHI/SalI-digested pBS24.1 vector (Fig. 1). The mutant D184A IN was constructed by overlapped PCR amplification using the mutagenic oligonucleotides 5'-TTGGGAACTGGCAATGGGCCTG-3' and 5'-CAGGCCCATTGCCAGTTCCCAA-3'. The flanking primers for amplification were the same as used for the wild-type IN. The procedure used to ligate the mutated IN to the plasmid PBS24.1 was similar to that described for wild-type IN.



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Fig. 1. Expression vector pJV-M-MuLV IN.

 
To facilitate the purification of IN, a hexahistidine tag was incorporated at the C terminus of both WT and D184A IN by PCR amplification of the constructs pJV-M-MuLV IN (wild-type) and pJV-M-MuLVD184A IN using the Advantage 2 kit (Clontech) and the oligonucleotides 5'-CGGAAGCGAGAAGAATCATAA-3' and 5'-TTTCGGTCGACCTAGTGATGGTGATGGTGATGTAACCTTATTTTTAA-3'. The fragments were cleaved with BamHI and SalI, and ligated to a BamHI/SalI-digested pBS24.1 vector. The sequences of the wild-type and D184A INs were confirmed by DNA sequencing. Plasmid maintenance was selected on a medium lacking either uracil or both uracil and leucine.

Yeast transformation.
Yeast transformations were performed by the one-step procedure (Chen et al., 1992). Transformed cells were inoculated on YCAD solid medium lacking uracil, supplemented with 2·0 % glucose. The plates were incubated at 28 °C for 3–5 days.

Yeast lethality test.
The effect of M-MuLV IN expression on yeast growth on solid media was determined using a ‘drop test’ (Caumont et al., 1996). Briefly, 3 µl droplets of plasmid-containing yeast standard suspensions (about 20 000 URA+ yeast cells) were dropped on YNB solid medium lacking uracil and leucine. In addition, the medium contained 0·1 % glucose, which allows high IN expression. The plates were incubated for 5–7 days at 28 °C and the phenotypes were observed. All lethality tests were done with IN without the His tag.

Yeast growth.
To evaluate the lethal effect of M-MuLV IN expression on yeast as a function of incubation time, liquid cultures of yeast expressing IN in a selective medium were performed. Cell growth was monitored by measuring OD600. Yeast suspensions expressing M-MuLV IN (1 ml) were prepared under the conditions described above and were inoculated in 50 ml YNB liquid medium lacking uracil and leucine. The OD600 was measured between 0 and 24 h incubation at 28 °C.

M-MuLV integrase expression and purification.
In order to analyse the expression level of M-MuLV IN, the yeast strain JSC-310 containing the expression plasmid pJV M-MuLV IN-HT was grown in 250 ml YEPD complete medium, as described by Caumont et al. (1996). After 72 h culture, yeast pellets were lysed with glass beads, as reported by Pichuantes et al. (1990), in a solution containing 50 mM HEPES (pH 7·4), 0·5 M NaCl, 10 mM CHAPS, 1 mM EDTA and 10 mM 2-mercaptoethanol (buffer A). Just before lysis, 1 mM PMSF, 1 mM benzamidine and 2 mg leupeptin ml–1 were added. The supernatant was mixed with 0·5 ml nickel–NTA agarose equilibrated with buffer A containing 15 mM imidazole. The mixture was left overnight at 4 °C in a rotator. The resin was placed in a small column and washed with 5 ml buffer A containing 20 mM imidazole. The protein was eluted with 300 mM imidazole in buffer A and 0·5 ml fractions were collected. Glycerol (0·5 ml) was added to the fractions, which were stored at –80 °C. The fractions were analysed by SDS-PAGE in 15 % polyacrylamide gels and by Western blotting using a rabbit polyclonal anti-M-MuLV IN antibody, kindly supplied by Dr M. Roth, University of Medicine and Dentistry of New Jersey. Protein was estimated by the Bio-Rad assay.

In vitro assays of M-MuLV integrase.
The 3'-processing and strand-transfer reactions contained 1 pmol labelled substrate, and the reaction buffer contained 20 mM MES (pH 6·2) 100 mM KCl, 10 mM MnCl2, 10 mM dithiothreitol and 10 % (v/v) glycerol in a final volume of 15 µl. The reaction was started by adding 1–5 µl IN. The reaction mixture was incubated at 37 °C for 1 h and then stopped by adding 15 µl loading buffer (95 % formamide, 20 mM EDTA, 0·05 % bromophenol blue) and heating at 90 °C for 5 min. Disintegration activity was determined in 20 mM PIPES (pH 6·4). The reaction products were analysed by electrophoresis on 12 % polyacrylamide gels with 7 M urea in Tris/borate/EDTA (TBE) buffer (pH 7·6) and autoradiographed. Substrates were labelled at the 5' end with T4 polynucleotide kinase and annealed to the complementary unlabelled strand at a ratio of 1 : 2 in 100 mM NaCl. The oligonucleotides were annealed for 3 min at 95 °C and then cooled to 25 °C. The 3'-processing and strand-transfer reactions were performed using the 5'-labelled oligodeoxynucleotides (ODNs) 5'-GATCCGACTACCCGTCAGCGGGGGTCTTTCATT-3' and 5'-GATCCGACTACCCGTCAGCGGGGGTCTTTCA-3', respectively, annealed to 5'-AATGAAAGACCCCCGCTGACGGGTAGTCGGATC-3' (Invitrogen). Disintegration assays were performed using a 5'-end radiolabelled dumb-bell substrate, as described previously (Donzella et al., 1998). The reaction products were run on 17·5 % acrylamide sequencing gels, dried, and exposed on a phosphorImager (Bio-Rad). Images were processed using Adobe Acrobat 6.0 Professional.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and expression of M-MuLV integrase
M-MuLV IN has been expressed in E. coli and purified under denaturing (Jonsson et al., 1993) and native conditions (Villanueva et al., 2003). In this work, we chose to express M-MuLV IN in the yeast S. cerevisiae, since direct expression of eukaryotic proteins in yeast has been shown to be efficient and offers many advantages, as its biosynthetic pathways resemble those of higher eukaryotic cells in many respects. A construct for expression of M-MuLV IN in yeast was generated by inserting the IN coding sequence carrying a hexahistidine tag at the C terminus in the vector pBS24.1, as described in Methods (Fig. 1).

The protease-deficient yeast strain JSC-310 was used for expression of M-MuLV IN. The strain containing the plasmid harbouring the IN coding sequences was grown in YEPD complete medium, as indicated in Methods. The addition of a His tag at the C terminus of IN allowed its purification after a single chromatographic step. In parallel experiments we constructed a mutated IN to obtain an inactive protein. Mutation D184A was introduced into M-MuLV IN, since residue D184 has been postulated to be one of the amino acids of the putative catalytic triad of M-MuLV IN (Kulkosky et al., 1992). Both wild-type and D184A His-tagged INs were purified by chromatography on nickel–NTA agarose. The purified proteins were obtained with a yield of 1–2 mg (l yeast culture)–1.

Fig. 2(a) shows a SDS-PAGE analysis of the protein fractions. A protein band migrating near the 45 kDa marker was observed in the yeast strains that were transformed with the plasmids containing either WT IN or the mutant D184A IN (lanes 3 and 4). This protein band was not observed in a cell lysate of the JSC-310 yeast strain that was not transformed with plasmid pJV M-MuLV IN (lane 2). After elution with 300 mM imidazole, a main protein band was present for both the wild-type (lane 5) and D184A (lane 6) IN. The presence of IN in the fractions was confirmed by Western blotting (Fig. 2b, lanes 5 and 6). In Fig. 2(b), lanes 2 and 3 show the proteins stained with Ponceau S (Sigma) before blotting. These results indicate that M-MuLV IN was efficiently expressed in yeast, and that the D184A mutation did not affect protein expression.



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Fig. 2. (a) Analysis of M-MuLV integrase expression. Several fractions were subjected to SDS-PAGE in 15 % acrylamide. Lane 1, molecular mass standards (Winkler SA); lane 2, soluble fraction of JSC310 yeast, 80 µg; lane 3, soluble fraction of JSC310 yeast cultures expressing wild-type M-MuLV IN, 160 µg; lane 4, soluble fraction of yeast JSC310 expressing the mutant D184A M-MuLV IN, 88 µg; lane 5, wild-type M-MuLV IN purified by nickel–NTA agarose, 3 µg; lane 6, mutant D184A M-MuLV IN purified by nickel–NTA agarose, 4 µg. (b) Western blot of purified IN. Lanes 1 and 4, molecular mass protein standards (Winkler SA); lanes 2 and 5, purified wild-type M-MuLV IN (3 µg); lanes 3 and 6, D184A M-MuLV IN (3 µg). The purified proteins were stained with Ponceau S prior to blotting (lanes 2 and 3). Blotted IN proteins are shown in lanes 5 and 6.

 
The in vitro 3'-processing, strand-transfer and disintegration reactions were assayed under the optimal conditions described in Methods. The 3'-processing reaction was assayed in the presence of the 5'-end radiolabelled duplex ODN. As shown in Fig. 3(a), lane 2, the wild-type M-MuLV IN expressed in yeast showed a robust 3'-processing activity with production of the (–2) nt processed product. This was not the case with the mutated D184A IN. The latter enzyme presented no activity, showing that D184 is absolutely required for this activity (lane 3).



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Fig. 3. 3'-Processing (a), strand-transfer (b) and disintegration (c) activities of the purified M-MuLV IN. Lane 1, no enzyme; lane 2, WT M-MuLV IN (4·5 pmol); lane 3, mutated D184A M-MuLV IN(4·5 pmol). The reaction products are indicated by arrows. The structure of the dumb-bell substrate and of the disintegration product are also shown.

 
Each purified protein was then tested for strand-transfer activity on the specific radiolabelled duplex substrate. Strand-transfer activity was detected for the enzyme carrying the wild-type sequence (Fig. 3b, lane 2), whereas no strand-transfer activity was observed for the mutated IN protein (lane 3).

Regardless of its relevance in vivo, the disintegration reaction has been very useful in the characterization of several mutants, particularly those encoding substitutions in the core domain. We therefore assayed the disintegration activity in the presence of the ‘dumb-bell-oligomer’ disintegration substrate (Fig. 3c). Only wild-type IN was able to catalyse the standard disintegration reaction (lane 2). Mutated IN showed no activity (lane 3). In contrast to human and avian retroviral INs, in which it has been experimentally shown that the DDE triad is esential for IN activity, this is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN.

Many if not all M-MuLV INs have been expressed and purified using the bacterial system. It thus seemed interesting to compare both systems, the yeast- and the E. coli-expressed INs. Fig. 4 shows a comparison of the disintegration and strand-transfer activities of the wild-type INs prepared from S. cerevisiae and E. coli. The enzyme purifed from yeast was about twofold more active than the enzyme from E. coli. This result is in agreement with the hypothesis stated above that expression of eukaryotic proteins in yeast can be more efficient.



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Fig. 4. Strand-transfer and disintegration activities of integrase from S. cerevisiae and E. coli. The activities were determined in triplicate, as described in Methods, using 5 pmol of each enzyme. The products of the reactions were separated by electrophoresis and quantified by scanning the dried gel in a Bio-Rad phosphorimager.

 
Expression of M-MuLV integrase induces a lethal phenotype
When assaying different requirements for expression of IN we observed that under certain conditions of growth, some yeast strains, such as the rad52 haploid W839-5C (deficient in DNA repair) became sensitive to M-MuLV IN expression. We hypothesized that this could be due to DNA damage induced by the enzyme nuclease activity.

The effect of M-MuLV IN expression on yeast growth was determined on selective solid medium lacking leucine under conditions that allowed high levels of IN expression (0·1 % glucose). The diploid yeast strain AB2 (d.RAD52+) and the rad52 haploid yeast strain W839-5C were transformed with the plasmid pJV M-MuLV IN. The results shown in Fig. 5(a, b), lanes 3 and 4, revealed that under these conditions expression of M-MuLV IN produced a lethal phenotype in both yeast strains. In contrast, after 5 days incubation, no lethal phenotype was observed when the yeasts were transformed with the control plasmid lacking the M-MuLV IN gene (Fig. 5a, b, lane 1). No lethality was observed also when the yeast strains AB2 or W839-5C were transformed with the plasmid containing the inactive D184A M-MuLV IN coding sequence (Fig. 5a, b, lane 5). Another way to detect the lethal effect produced by IN on yeast was obtained by measuring the yeast growth as a function of time. The experiment reported in Fig. 5(c) shows that in liquid selective medium the lethal effect can also be observed when using the active M-MuLV IN.



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Fig. 5. Lethal effect of M-MuLV IN expression. Yeast strains were grown on selective solid medium lacking leucine in the presence of 0·1 % glucose. (a) W839-5C strain; (b) AB2 strain. Lane 1, yeast transformed with the plasmid without the IN insert; lane 2, non-transformed yeast; lanes 3 and 4, yeast transformed with the pJV-M-MuLV IN vector; lane 5, yeast transformed with the pJV-M- MuLV D184A IN (corresponding to an inactive integrase). (c) Effect of expressing M-MuLV IN on yeast growth. Liquid yeast cultures were incubated for 24 h in 0·1 % glucose. {bullet}, W839-5C yeast strain transformed with pBS24.1 without the IN gene; {square}, W839-5C yeast strain transformed with pJV-M-MuLV IN; {blacksquare}, AB2 yeast strain transformed with pJV-M-MuLV IN.

 
The lethal effect of M-MuLV integrase depends on an active SNF5
In an attempt to reveal possible cellular partners of IN, we first studied the possible interactions between M-MuLV IN and the yeast SNF5 factor. This transcription factor (Ini1/ySNF5) has been well characterized in human and yeast cells. The human Ini1 factor has been shown to interact with HIV-1 IN (Kalpana et al., 1994). We thus studied the role of yeast SNF5 on the lethal effect produced by IN to determine whether this interaction was functionally important in vivo. For that purpose, we analysed the IN activity in a yeast strain sensitive to IN-induced lethality and in the corresponding strain disrupted for the gene encoding SNF5 (W839-5C snf5 : : ura3). The two yeast strains W839-5C and W839-5C snf5 : : URA were grown in the presence of 0·1 % glucose (Fig. 6). In our control experiment, the lethal phenotype was observed in the W839-5C yeast strain expressing M-MuLV IN (Fig. 6, lane 2), while the control plasmid lacking the M-MuLV IN gene gave no such effect (Fig. 6, lane 1). Then we analysed the effect of disrupting the SNF5 gene on yeast. Disruption of SNF5 did not affect the viability of this strain in the presence of 0·1 % glucose (data not shown), indicating that this factor is not essential under those conditions.



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Fig. 6. Effect of SNF5 gene disruption on integrase-induced lethality in yeast. Yeast cells were grown in YNB containing 0·1 % glucose. 1, W839-5C transformed with the PBS24.1 vector without the IN gene (no insert); 2, W839-5C transformed with pJV-M-MuLV IN; 3, W839-5C snf5 : : ura3 transformed with PBS24.1 (no insert); 4, W839-5C snf5 : : ura3 transformed with pJV-M-MuLV IN.

 
To determine the effect of SNF5 on the expression of a lethal phenotype by M-MuLV IN, the yeast strain lacking SNF5 was transformed with the plasmid pJV M-MuLV IN. Interestingly, no lethal phenotype was observed in SNF5-disrupted yeast strains expressing active M-MuLV IN (Fig. 6, lane 4). Fig. 6, lane 3 shows a normal growth of the {Delta}SNF5 mutant yeast transformed with the vector without the M-MuLV IN gene. The need of SNF5 for lethality suggested that this factor may interact with IN or it may favour the targeting of the chromosomal DNA.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Retroviruses and yeast retrotransposons share several features in their life cycle. These include reverse transcription and integration (Wilhelm & Wilhelm, 2001). In order to determine the possible cellular partners involved in integration we studied here the expression of M-MuLV IN in yeast and used it as a system for screening cellular proteins that interact with IN to further identify possible homologous proteins from murine cells. Yeast cells are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce ‘authentic’ and bioactive mammalian proteins. In addition, the phenotypes conferred on yeast cells by the production of certain proteins offer important insights into fundamental aspects of their biology.

Under defined conditions, the overexpression of M-MuLV IN produced a lethal phenotype in yeast. A similar observation has been previously described for HIV-1 IN (Caumont et al., 1996). The yeast lethal phenotype suggested that HIV-1 IN could enter into the yeast nucleus and act as a nuclease, probably cleaving chromosomal DNA (Parissi et al., 2000a, 2003). The mechanism of entry of HIV-1 IN into the yeast nucleus has not been completely elucidated. Maertens et al. (2004) have reported that LEDGF/p75 is essential for nuclear localization of HIV-1 IN. Previous studies have shown that in murine cells, mitosis is required for integration of M-MuLV (Roe et al., 1993). Since the nuclear membrane of the budding yeast is not disrupted during mitosis it is possible that M-MuLV and HIV-1 INs pass through the nuclear membrane pores to produce DNA cleavage (Barnes & Rine, 1985).

Several lines of evidence indicate that cellular factors could affect the selection of integration sites, since host proteins could enhance or decrease integration at nearby sites (reviewed by Engelman, 2003). Yeast proteins may direct retroviral PIC to integration sites in Ty1, Ty3 (Chalker & Sandmeyer, 1992) and Ty5 (Zou & Voytas, 1997) retrotransposons. The requirement of the transcription factor ySNF5 (homologous to human Ini1) by both HIV-1 and M-MuLV INs to induce a lethal phenotype revealed an analogous interaction. ySNF5 is a component of the SWI/SNF chromatin-remodelling system. It is possible that ySNF5 could direct both INs to active regions of chromatin (Kalpana et al., 1994). M-MuLV IN does not contain nuclear-localization signals and its entry into the nucleus may be determined by interaction with yeast proteins. It will be of great interest to identify those proteins.

Our results constitute the second item of evidence for lethality caused by expression of a retroviral IN in S. cerevisiae. In addition, this expression system could provide important information to search for counterparts of the natural host to define the role of cellular proteins in the import of PICs and in target-site selection. Finally, the lethal effect could also be used as a selection marker to select IN mutants, as in the case of HIV-1 (Parissi et al., 2000b).


   ACKNOWLEDGEMENTS
 
This work was funded in part by ECOS-SUD-C0B01, Fondecyt 1040409, Fondecyt 2000143 (J. V.) and the French National Research Agency against AIDS (ANRS), the CNRS and the University of Bordeaux 2. V. P. benefitted from a post-doctoral fellowship from ‘Ensemble contre le SIDA, SIDACTION’.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 1 March 2005; accepted 7 June 2005.



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