A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles
- Olivier Delelis†1,
- Caroline Petit†1Email author,
- Herve Leh2,
- Gladys Mbemba3,
- Jean-François Mouscadet3 and
- Pierre Sonigo1Email author
© Delelis et al; licensee BioMed Central Ltd. 2005
Received: 20 April 2005
Accepted: 18 May 2005
Published: 18 May 2005
Retroviral integration is central to viral persistence and pathogenesis, cancer as well as host genome evolution. However, it is unclear why integration appears essential for retrovirus production, especially given the abundance and transcriptional potential of non-integrated viral genomes. The involvement of retroviral endonuclease, also called integrase (IN), in replication steps apart from integration has been proposed, but is usually considered to be accessory. We observe here that integration of a retrovirus from the spumavirus family depends mainly on the quantity of viral DNA produced. Moreover, we found that IN directly participates to linear DNA production from 2-LTR circles by specifically cleaving the conserved palindromic sequence found at LTR-LTR junctions. These results challenge the prevailing view that integrase essential function is to catalyze retroviral DNA integration. Integrase activity upstream of this step, by controlling linear DNA production, is sufficient to explain the absolute requirement for this enzyme.
The novel role of IN over 2-LTR circle junctions accounts for the pleiotropic effects observed in cells infected with IN mutants. It may explain why 1) 2-LTR circles accumulate in vivo in mutants carrying a defective IN while their linear and integrated DNA pools decrease; 2) why both LTRs are processed in a concerted manner. It also resolves the original puzzle concerning the integration of spumaretroviruses. More generally, it suggests to reassess 2-LTR circles as functional intermediates in the retrovirus cycle and to reconsider the idea that formation of the integrated provirus is an essential step of retrovirus production.
Keywordsspumaretrovirus integrase substrate palindrome at LTR-LTR junctions 2-LTR circles DNA
Integration of viral genomes into host cell DNA is a key element of the life cycle and pathogenesis of many viruses. DNA viruses integrate by relying solely on cell machinery. In contrast, retroviruses possess a specialized endonuclease, also designated integrase (IN), which is essential for their replication (for a review, see ). After entering a target cell, reverse transcriptase (RT) converts genomic RNA into linear double-stranded cDNA with a copy of the viral long terminal repeat (LTR) at each end. Such linear genomic cDNA included in a preintegration complex (PIC) [2–9] can be used as a template for integration in vivo. Consequently, circular viral genomes that are detected in infected cells were considered until now as «dead-end» molecules, without essential function in the integration process and the viral cycle in general .
Integration mediated by the retrovirus IN occurs in two catalytic steps, referred to as 3'-processing and strand transfer (or joining), respectively. Interestingly, the two steps appeared on distinct reactions catalyzed by virus IN in two different compartments in the infected cells. The strand transfer reaction joins viral DNA to cellular DNA in the cell nucleus. The viral cDNA ends are used to cut the target DNA in a staggered manner, which covalently links the viral 3' ends to the 5' phosphates of the cut (for reviews see [10, 11]. The 3' hydroxyl groups at the LTR termini are the nucleophiles that promote DNA strand transfer . Efficient strand transfer requires previous endonucleolysis of DNA that produces recessed 3'hydroxyl ends [3, 5]. This occurs in the cytoplasm very soon after reverse transcription is completed [13–16], as viral genomes with blunt ends are extremely rare in the infected cytoplasm. Following these reactions, host cell enzymes likely repair the gap remaining between host and provirus DNA [17, 18].
IN recognizes and acts on short sequences (12 to 20 bp) called attachment (att) sites that are located at the LTRs . Att site includes the invariant CA dinucleotides, which are conserved in all retroviruses whereas the other nucleotides of the att site, while not conserved in sequence, form an (imperfect) inverted repeat (IR) in all retroviruses, that has to be maintained intact for viral replication. Att mutagenesis experiments showed that mutation in one LTR precludes the processing of the other, demonstrating that activity of IN is concerted onto the two viral LTRs that are simultaneously cleaved in vivo . The structural basis of such concerted processing of both extremities is unknown. More surprisingly, in the case of spumaretroviruses, a subfamily of retroviruses that share some features of DNA viruses [21–23], the IN may process only one of the two LTRs, although the att sites are present at the two LTRs. Based on the sequences of both 2-LTR DNA and integrated proviruses, an asymmetric processing of att sites has been proposed, in which IN may cleave the right, U5 end and may leave the left, U3 end intact [24, 25]. As the human spumaretrovirus (PFV) IN presents the usual features of other IN and carries out in vitro an endonucleolytic activity, as well as strand transfer and disintegrase activities [26, 27], the reason for this unusual mechanics is not understood at present.
The att recognition site of IN is present at least one time on all forms of viral DNA. In addition to linear and integrated forms, viral DNA is found in the infected cells as covalently closed DNA circles containing either one or two copies of the LTR, referred to as 1-LTR and 2-LTR circles, respectively . Interestingly in the 2-LTR circles, the att sites are in a closed configuration due to the juxtaposition of the two LTRs and are included within a palindromic motif formed by the inverted repeat sequences in all retroviruses [28–31]. These 2-LTR circles are believed to result from a direct covalent joining of LTR ends at the so-called circle junction [32, 33]. Circularization is thought to occur by blunt-end ligation of the ends of linear proviral DNA, even no direct evidence has been provided until now to support this hypothesis. 2-LTR could be formed in part by the non-homologous end-joining (NHEJ) pathway of DNA recombination . The two-LTR circle forms could, theoretically, serve as a potential precursor for the integrated provirus . In spleen necrosis virus (SNV), Rous sarcoma virus (RSV), avian sarcoma virus (ASV) and avian leukosis virus (ALV), closed circular forms were initially proposed to act as substrates templates for integration [31, 32, 35], although these reports have not been substantiated. Although they are currently described in a productive infection as "dead end" molecules, precisely because of their incapacity to be directly integrated , intriguing observations invite some to reconsider their place. First, 2-LTR molecules were shown to be used as functional templates for the transcription machinery in HIV infected cells [36–39]. Second, 2-LTR viral DNA were detected in the cytoplasm of MLV and PFV infected cells at a very early time post infection, suggesting that they are not formed in the nucleus by an alternative fate to the integration way [40, 41]. In this context, we asked whether 2-LTR circles, rather than being substrate for integration nor "dead end" molecules, would be used as substrates for a preintegrative endonucleolytic activity of PFV IN.
Such interrogation comes within the scope of the more global questioning concerning the pleiotropic actions of IN. Indeed, the mechanisms underlying the essential requirement for integration are still unclear in the retrovirus cycle. Why is integration critical for viral production when unintegrated DNA is abundant and competent for transcription [36–39, 42–45]? Is it possible that preintegrative function of IN explain its essential requirement rather than integration per se? Indeed, in addition to its roles in the establishment of the proviral integrated state, IN participates to other critical steps, such as reverse transcription [23, 46–52], nuclear import of HIV-1 preintegration complex (PICs) (for a review, see ), and the postintegration step of viral particle assembly (reviewed in ). Among the PIC constituents, IN is a logical and probable candidate for facilitating the efficient nuclear import of cDNA, since it has karyophilic properties [55–61]. Reflecting the pleiotropic activities of IN, non-replicative IN mutants of HIV were divided in two phenotypic classes depending on their defects . The properties of IN mutants of PFV are less extensively described, and we suspected that PFV IN could play a key role in early preintegrative steps.
In an attempt to better characterize the properties and substrates of the original IN of PFV, we analyzed both its in vivo properties and in vitro activity. We observed that the 2-LTR circles could serve as templates for the 3' processing reaction of the IN. This allows spumaretrovirus to follow a symmetrical mechanism of integration and leads to reexamine the role of 2-LTR molecules and the importance of preintegrative function of IN.
Results and discussion
The mutations inPFV IN do not alter its karyophilic property
Retroviral INs from oncoviruses [62, 63], lentiviruses [55, 59, 64, 65] and spumavirus  are karyophilic proteins, since they localize to cell nuclei in the absence of any other viral protein. Nuclear accumulation of INs may be a general feature of retroviruses. The intrinsic karyophilic property of retrovirus INs could be of high importance for the import of preintegration complex containing viral genomes in the nucleus (for a review, see ), where the transcription step occurs.
PFV harboring mutant IN genes are impaired in their replication at an early step
PFV-1 replication defective IN mutants display an abnormal pattern of viral DNA synthesis with an accumulation of 2-LTR circles
To further document the early steps at which the replication of defective mutant IN viruses is impaired, detailed kinetic analyzes of the different viral DNA forms were conducted in infected cells. The importance of IN in the virus replication might be very early since it participates to reverse transcription [23, 46–52], and may be even in close contact with the viral DNA all along its synthesis since it was shown to directly interact with the RT [46, 47].
Various DNA extracts were then analyzed for their content in molecules carrying 2-LTR junctions. As previously shown , viral DNA containing a LTR-LTR junction could be detected as early as 3 hours post-infection, and it continuously increased during viral replication (Figure 3B). The kinetics of production of 2-LTR species for IN mutant viruses paralleled that of the wild-type virus, indicating that their reverse transcription products were quite compatible with the formation of viral DNA containing LTR-LTR junctions. Using these quantitative data, we calculated the ratio of 2-LTR versus gag containing DNA in the same extracts. As for other retroviruses [77, 78], viral DNA species with an LTR-LTR junction represented a minority of the total viral DNA, from 0.6% early in the replicative cycle to a maximum of 9% 24-hour post-infection, in the case of wild-type virus (Figure 3C).
Interestingly, for all IN-mutant viruses, we noticed a marked increase in the proportion of 2-LTR species as compared to the wild-type virus. The over-representation of 2-LTR molecules increased all along infection, reaching a remarkable 35% of total viral DNA in the case of the M8 mutant (Figure 3C). 2-LTR PCR does not allow to distinguish between 2-LTR circles and other molecules containing a LTR-LTR junction such as concatemeric linear or circular genomes. As the later molecules were not described, we assume that the 2-LTR junctions we quantified are indeed carried by circular genomes as in other retroviruses. However, such circles were difficult to detect during spumavirus infection by Southern blot , and further studies will be required to precisely answer this question.
Our kinetic analyses revealed that the impaired global production of viral DNA due to inactivation of IN was associated with an abnormal accumulation of 2-LTR DNA species. Importantly, this overaccumulation of 2-LTR species has also been associated with IN-defective HIV viruses [50, 80–82]. To explain this observation, it is currently assumed that linear HIV DNA, representing the precursor of integration [3, 5], accumulates because it cannot be integrated and is rerouted into the circularization pathway producing 2-LTR molecules in the nucleus [29, 83–85]. However, 2-LTR circles are also detected in WT infected cells. In this case, 2-LTR formation was suggested to result from aberrant att sequences preventing their recognition by IN . Moreover, since 2-LTR molecules have been detected both in the cytoplasm and the nucleus of PFV WT infected cells , as well as at very early time-points in cytoplasm of MLV infected cells , overproduction of 2-LTR DNA cannot simply be explained by such a rerouting of non-integrated viral DNA. Alternatively, PFV-1 IN might be directly involved in the processing and/or turnover of viral DNA containing LTR-LTR junctions explaining their accumulation when IN is defective. To address this hypothesis, we tested whether PFV-1 IN might use LTR-LTR circle as a substrate in vitro.
PFV IN can specifically cleave the conserved palindromic sequence found at LTR-LTR junctions to generate 3'-end processed LTRs
Since inactivation of PFV IN led to the accumulation of 2-LTR viral DNA containing a palindrome reminiscent of enzymatic restriction sites, we tested whether this palindrome was a possible substrate for the endonuclease activity of IN, as proposed for avian retroviruses . Recombinant PFV IN was produced in E. coli and purified on nickel column. The purified IN, able to catalyze integration in vitro, was incubated with a double stranded 32P-labeled oligonucleotide containing the palindrome. Reaction products were analyzed by electrophoresis in a polyacrylamide sequencing gel. A cleavage product appeared in the presence of IN confirming that IN harbors endonuclease activity. Moreover, the digestion fragment was found to be unique (Figure 4B and 4C, lanes 2 and 6) and corresponded to a cut between the two consecutive adenines in the middle of the palindrome, as determined by comigration of the sequencing reaction (Figure 4B, lane (G+A)). This digestion was dependent on IN activity as only the initial oligonucleotide was detected when IN was inactivated by EDTA treatment (Figure 4B and 4C, lanes 1 and 5). Moreover, this activity of PFV-1 IN was highly dependent on the target sequence since oligonucleotides carrying mutations that disrupt the palindromic character of the LTR-LTR junction (Figure 4C lane 10 and Figure 4D), and an irrelevant scrambled oligonucleotide (Figure 4D) did not undergo specific cleavage. Finally, PFV-1 IN did not cleave palindromes that are found at HIV-1 and MLV retroviral LTR-LTR junctions (Figure 4D). These data demonstrated that IN double-stranded DNA cleavage activity is restricted to the palindrome at the LTR-LTR junction found in corresponding infected cells and thus carries the same sequence specificity as already documented for the 3'processing of LTR extremities . Detailed analysis indicated that the digestion had operated on the two strands (U5- and U3-end labeling) of the oligonucleotide substrate generating cohesive ends with a 5'-protuding AT (compare lanes 2 and 3, or 6 and 7, Figure 4C).
Altogether, these data reveal a new substrate for IN endonuclease activity. This endonucleolytic activity is able to cleave specifically the palindromic sequence generated at the LTR-LTR junctions of viral DNA. The cleavage of 2-LTR circles into linear genomes justifies revisiting them as functional intermediates in the retroviral cycle. This is reinforced by recent observations showing their stability and contribution to the viral transcription [36, 37, 77, 78]. Interestingly, many DNA viruses replicate by using circular intermediates resembling the retroviral 2-LTR circles, and require the activity of a virally encoded endonuclease reminiscent of the IN. Identification of new IN activity should improve our understanding of the early steps of the retroviral replication cycle, allow screening of anti-retroviral drugs as well as design of new non-integrating retroviral vectors.
That IN operates on 2-LTR molecules to produce linear DNA with each LTR end 3'-processed avoids the need for asymmetrical integration in spumavirus
In light of our observation that 2-LTR molecules are possible substrates for PFV-1 IN (Figure 4), the 3'-processing of both ends of the linear DNA might be generated in a single reaction that produces the two 3'-processed ends simultaneously (Figure 5B). Such concerted processing might explain the influence of one LTR on the processing of the other, as observed for HIV-1 . The subsequent integration of such processed extremities would eliminate the two nucleotides that are lost between the LTR-LTR junction and the integrated provirus. No asymmetric integration is required to account for the previous observations [24, 25]. This mechanic, when generalized to other retroviruses carrying a different palindrome at the LTR-LTR junction, would result during integration in the loss of the number of nucleotides comprised between the conserved CA.
In support of our symmetrical integration model, Pahl and Flügel  previously reported an efficient 3'-processing activity of PFV IN on LTR containing the two additional nucleotides AT. The substrate of concerted processing corresponds to the extended substrate they tested. We confirmed the 3'-processing cleavage of the extended U3 LTR carrying an additional AT (Figure 4C), as well as the fact that the 3'-processing does not occur onto the shorter U3 LTR lacking these nucleotides (not shown).
Integration depends on preintegrative IN activity
Role of IN in PFV retrovirus replication cycle
We conclude from these experiments that PFV IN displays a specific activity on the 2-LTR circles, which may constitute a substrate for the 3'processing reaction in vivo. This action of IN generates linear DNA that might be then integrated in the cell genome following a classical symmetrical integration process. The fact that early actions of IN may influence later steps of replication, including integration, certainly participates in the pleiotropic effects of IN mutations. Finally, IN seems to be essential not because of its participation to the integration per se but for its upstream activities able to influence integration efficacy.
Although the consensus sequences in the C ter region of IN may differ between the lentiviruses and the nonlentiviruses, the carboxyterminal region of IN is well conserved in all retroviruses , and further studies are now required to evaluate whether the revised replication model we propose here, applies to all retroviruses. The fact that the typical phenotype associated with a defective IN, either due to mutations or inhibitors, resulting in reduced DNA synthesis but a persistence of integration and an accumulation of 2-LTR molecules, is commonly observed among retroviruses [73, 82, 90], argues in favour of a conserved IN function. Such an early participation of IN sheds new light on reports showing both that viral transcription occurs from nonintegrated HIV DNA [38, 44, 45, 91], and that the most prevalent form of HIV DNA during the asymptomatic phase of infection is full-length unintegrated DNA [42, 92]. Whereas IN activity is clearly required, formation of integrated provirus as an obligate step of retroviral replication now needs to be reconsidered. On the other hand, early preintegrative activities of IN are of capital importance. This provides new answers to the puzzling question of why is integration essential to retrovirus replication, when many authors have shown that unintegrated genomes are abundant and expressed [36–39, 42–45, 93]. Our proposal is simply: integrase is essential, integration is not; and IN is required given its critical preintegrative influence on genomic DNA production in vivo, as we precisely measured here.
Given the above, retroviruses better fit the classical schemes of distinct lytic and lysogenic phases exemplified by the lambda phage: integration (lysogeny) contributes to viral persistence and pathogenesis, but it is not essential for acute viral production (lytic cycle). Finally, a fascinating evolutionary conservation appears between retroviruses and DNA viruses (such as poxviruses). All use circular DNA intermediates and a specialized endonuclease activity for genome production.
Cells, virus infections and reagents
BHK-21, FAB, HeLa and U373-MG cells were cultivated in DMEM with 10% foetal calf serum, 1 μg per ml of streptomycine-streptavidine. For FAB indicator cells, 1 μg per ml of G418 (Sigma) was added.
PFV-1 virus stocks were prepared by transfecting BHK-21 cells with the PFV-1 molecular WT and mutant clones using the calcium phosphate method. Cells were infected by WT and mutant viruses with same amounts of viral particles, as evaluated by a reverse transcription assay. The culture medium was changed two hours post-infection with fresh medium.
Cell free virus stocks were titrated on FAB cells . In some experiments, infected cells were treated with 3'-azido-3'-deoxythymidine (AZT, Sigma) at 100 μM.
DNA quantifications by real time PCR
Total DNAs were extracted from 106 cells using the DNA Blood Mini kit (Qiagen) in a final volume of 200 μl and analysed by real time PCR as described previously . Integrated viral DNA was also quantified by two rounds of PCR . The first one amplifies integrated DNA using primers ALU1 (5'-CCT CAG CCT CCC GAG TAG CTG GGA-3'), ALU2 (5'-CTG TAA TCC CAG CAC TTT GGG AGG C-3'), and λ TSPA (5'-ATG CCA CGT AAG CGA AAC TTA GTA TAA TCA TTT CCG CTT TCG-3'). Sequence in bold represents a sequence in the lambda phage, which is unknown in all mammals' databanks. The other part of the sequence of λ TSPA primer can hybridize in PFV LTR. Amplification was performed in a 20 μl reaction volume containing 1X Light Cycler Fast Start DNA Hybridation probes, 3.5 mM MgCL2, 300 nM of primer ALU1, ALU2 and 10 nM of primer λ TSPA. The same mix, containing only primer λ TSPA, was prepared. DNA from U373-MG chronically infected cells was used as a standard for integrated copies. All reactions were further diluted in a final volume of 200 μl of water. 2 μl over 200 μl was used for the second PCR. This amplification was performed with 300 nM of each primers Nested R (5'-GAA ACT AGG GAA AAC TAG G-3'), lambdaT (5'-ATG CCA CGT AAG CGA AAC T-3') and 100 nM of each hybridation probes SpuFL (5'-CAC TCT CGA CGC AGC GAG TAG TGA A X-3') and SpuLC (5'-GCC TCC CGT ACA ATC TAG AAA CTA TCC T p-3'). This assay is quite specific of integrated provirus only, as attested by performing the following control reactions: – a carry-over control in which all primers were omitted in the first PCR, data obtained indicated always that the second-round amplification of nonpreamplified viral DNA is efficiently prevented; -a parallel reaction with the Alu primers in the first-round PCR, in order to calculate the linear amplifications resulting from all the viral DNA species. The copy number due to the linear amplification was systematically subtracted from the signal obtained in the presence of Alu primer. We evaluated that this interfering amplification never exceeded 6.7 % of the global amplification.
Quantifications were performed with the LightCycler software Version 3.5 according to manufacturer's instructions.
Virion-associated RT assays
48 hours post transfection viral supernatants were collected. 10 μl of viral supernatant was incubated with 20 μl of reaction buffer (Tris pH 8 50 mM – KCl 75 mM – Dithiotreitol 2 mM – rA/dT 25 μg/ml – NP40 0,05% – MnCl2 5 mM – dTTP α-32P 20 μCi/ml). The reaction mixtures were incubated at 37°C for 90 min. 10 μl of the reaction was spotted onto DE81 filter and allowed to dry. The filters were washed four times with 2xSSC (1xSSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min each, followed by two washes with 95% ethanol. The filters were then dried and counting by scintillation fluid.
Construction of Flag-PFV IN mutants and their cell localisation by immunofluorescence staining
To express the INs in the absence of other viral products, we used the pFlag expression vector ; in which we inserted the PFV-1 IN sequence under the control of the simian virus 40 promoter. The IN fragment was amplified by PCR with the following primers, which created a BamH1 and an XhoI restriction site at the 5' and 3' ends, respectively, of the IN sequence: 5'-GGA TCC TAC ATA TTT TTT AGA AGA TGG C-3'; and 5'-CTC GAG TTA TTC ATT TTT TTC CAA TGA TCC-3'. The resulting PCR fragment was digested with BamHI and XhoI and ligated into the corresponding cloning sites of pSG-Flag , in the plasmid called pSG-FlagIN PFV. The pSG-FlagIN PFV expression vector was used for the mutagenesis, with the Quick Change mutagenesis kit (Stratagene), and the primers: 5'-CAA TTT GGC TCT CAC AGG ACG TGA AGC C-3' and 5'-GGC TTC ACG TCC TGT GAG AGC CAA ATT G-3' for the M5 mutant; 5'-ATT CAC TCT GGT CAA GGT GCA GC-3' and 5'-GCT GCA CCT TGA CCA GAG TGA AT-3' for the M8 mutant; and 5'-GGC AAA GGG CCA GTA TAG TCA AT-3' and 5'-ATT GAC TAT ACT GGC CCT TTG CC-3' for the M9 mutant.
HeLa cells (2 × 105) were spread on glass coverslips in 24-well plates, transfected with 1 μg of the corresponding plasmids, and stained for immunofluorescence 36 hours later. Cells were fixed in 3.7% formaldehyde-PBS for 20 min, washed three times in PBS, and incubated for 10 min in 50 mM NH4Cl to quench free aldehydes. Cells were washed three times in PBS and incubated in a permeabilization buffer (0.05% saponin, 0.01% Triton X-100, 2% bovine serum albumin, PBS) for 15 min and incubated 1 h with the first MAb (M2 anti-Flag MAb at 7.5 μg/ml) in permeabilization buffer. Cells were washed three times in permeabilization buffer and incubated with Cy3-conjugated anti-mouse MAbs (Amersham) at a final dilution of 1:200. Cells were washed three times in permeabilization buffer and once in PBS and mounted in 133 mg of Mowiol (Hoechst) per ml-33% glycerol-133 mM Tris HCl (pH 8.5). Confocal microscopy was performed and optical sections were recorded. One representative medial section was mounted by using Adobe Photoshop software.
Construction of PFV proviruses
We inserted a DNA fragment containing the PFV-1 IN sequence into a Litmus 38 plasmid, in which a PacI site had been added. The viral fragment was amplified by PCR with the following primers: 5'-GGA TCC TAC ATA TTT TTT AGA AGA TGG C-3' and 5'-CTC GAG TTA TTC ATT TTT TTC CAA TGA TCC-3', and cloned after a BspEI-PacI digestion into the modified Litmus. This plasmid containing the WT IN was used for the mutagenesis, with the Quick Change mutagenesis kit and the primers used above for the expression IN vector mutagenesis. After the mutagenesis, the PacI-BspEI digestion fragments from the mutated Litmus vectors were substituted for the corresponding sequence of the PFV-1 full-length clone. All constructions were confirmed by DNA sequencing of the entire PCR-amplified fragment.
2 LTR junction sequence analysis
Total DNA from acutely BHK-21 infected cells of two independent infections were extracted and analyzed by a PCR amplification specific for the LTR-LTR junction from the 2-LTR circles, using the following primers: R, 5'-TAC GAG ACT CTC CAG GTT TG-3'; and U3, 5'-CGA CGC AGC GAG TAG TGA AG-3' and the Pfu polymerase (Stratagene) . PCR products were cloned in a pSK+ plasmid (PCR-Script cloning kit, Stratagene). 50 independent cloned were sequenced.
Construction and purification of PFV recombinant IN
Histidine-tagged PFV-1 IN, corresponding to aminoacids 752-1143 of the Pol polyprotein, was expressed and purified by nickel affinity. The preparation and purification of recombinant PFV-1 IN protein were performed as described for HIV IN . To obtain wild type IN protein, plasmid pET15b (Novagen) was digested with NdeI and BamHI. The DNA fragment containing the PFV IN was obtained from pHSRV clone C55 by PCR using the Pfu DNA polymerase (Stratagene). The sequence of the primers used to amplify the fragment were 5'-ACA TAT GTG TAA TAC CAA AAA ACC AAA CCT GG-3' and 5'-AGG ATC CTT ACT CGA GTT CAT TTT TTT C-3'. PCR amplifications were done at 92°C for 1 min, 55°C for 45 s, and at 72°C for 90 s; the cycle was repeated 28 times. The resulting PCR fragment were digested with NdeI and BamHI and ligated into the corresponding cloning sites of pET15b. Plasmid pET15bIN was used to express the His-tagged IN in E. coli BL21 (DE3) cells. 500 ml of BL21 (DE3) pET15bIN cells was grown at 37°C in LB medium (supplemented with 50 mg/ml ampicilin) to an A600 of 0.6–0.8. To induce IN protein expression, isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 1 mM; bacteria were grown for another 4 hours and harvested by low speed centrifugation. The pellet was resuspended in 24 ml of 50 mM Tris-HCl, pH8, 1 M NaCl, 4 mM β-mercaptoethanol (buffer A). Cells were lysed with French Press and centrifugated at 14,000 rpm and 4°C for 30 min to remove cells debris
The supernatant was filtered (0.45 μm) and incubated over night with Ni-NTA agarose beads (Qiagen). The beads were washed with 10 volumes of buffer A. Then, IN was purified under native conditions according to manufacturer's instructions using batch procedure. His-tagged IN was eluted with buffer A supplemented with 50 μM ZnSO4 and 1 M imidazole. The IN concentration was adjusted to 0.1 mg/ml in buffer A and dialysed over night against 20 mM Tris-HCl, pH 8, 1 M NaCl, and 4 mM β-mercaptoethanol. Fractions were aliquoted and rapidly frozen at -80°C.
Nucleic acid substrates
All oligonuleotides U5B (5'-CCT TAG GAT AAT CAA TAT ACA AAA TTC CAT GAC AAT-3'), (U5A 5'-ATT GTC ATG GAA TTT TGT ATA TTG ATT ATC CTA AGG-3'), U3 B (5'-ATT GTG GTG GAA TGC CAC TAG AAA T-3'), U3A (5'-ATT TCT AGT GGC ATT CCA CCA CAA T-3'), LTR-LTRB (5'-CCT TAG GAT AAT CAA TAT ACA AAA TTC CAT GAC AAT TGT GGT GGA ATG CCA CTA GAA AT-3') and LTR-LTRA (5'-ATT TCT AGT GGC ATT CCA CCA CAA TTG TCA TGG AAT TTT GTA TAT TGA TTA TCC TAA GG-3') were purchased from Eurogentec and further purified on an 15% denaturing acrylamide/urea gel. 100 pmol of U5 B, U3 B and LTR-LTR B were radiolabeled using T4 polynucleotide kinase and 50 μCi of [γ-32P]ATP (3000 Ci/mmol) during 2 hours at 37°C. The T4 kinase was heat inactivated, and unincorporated nucleotides were removed using a Sephadex G-10 column (Pharmacia). NaCl was added to a final concentration of 100 mM and complementary unlabeled strand was added to either U5 B, U3 B or LTR-LTR B. The mixture was heated to 90°C for 3 min, and the DNA was annealed by slow cooling.
LTR processing, LTR-LTR junction cleavage
Processing and LTR-LTR cleavage were performed in buffer containing 50 mM Hepes, 5 mM DTT and 10 mM MgCl2. 150 nM of PFV-1 IN was used for reaction. The reaction was initiated by addition of substrate DNA, and the mixture was incubated 2 hours at 37°C and stopped by phenol/chloroform extraction. DNA products were precipitated with ethanol, dissolved in TE containing 7 M urea and electrophoresed on a 15% denaturing acrylamide/urea gel. Gels were analysed using a STORM Molecular Dynamics phosphorimager.
List of abbreviations
- Att :
human immunodeficiency virus
long terminal repeat
primate foamy virus
We warmly acknowledge Olivier Neyrolles, Sebastien Petit and the OCU for stimulating remarks and daily help. We are grateful to William Jacques Speare and Alexandre Matet for their corrections and for continued enthusiastic discussion regarding this research. We also thank Marc Alizon, Olivier Danos and Olivier Schwartz for stimulating and thoughtful comments, and constructive criticisms on the manuscript. We finally thank Naomi Taylor and Marc Sitbon for insightful discussions concerning the retrovirus replication models, as well as for their meticulous reading of our original manuscript.
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