Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells

Retrovirus preintegration complexes (PIC) in virus-infected cells contain the linear viral DNA genome (approximately 10 kbp), viral proteins including integrase (IN), and cellular proteins. After transport of the PIC into the nucleus, IN catalyzes the concerted insertion of the two viral DNA ends into the host chromosome. This successful insertion process is termed "full-site integration." Reconstitution of nucleoprotein complexes using recombinant human immunodeficiency virus type 1 (HIV-1) IN and model viral DNA donor substrates (approximately 0.30 to 0.48 kbp in length) that are capable of catalyzing efficient full-site integration has proven difficult. Many of the products are half-site integration reactions where either IN inserts only one end of the viral donor substrate into a circular DNA target or into other donors. In this report, we have purified recombinant HIV-1 IN at pH 6.8 in the presence of MgSO4 that performed full-site integration nearly as efficiently as HIV-1 PIC. The size of the viral DNA substrate was significantly increased to 4.1 kbp, thus allowing for the number of viral DNA ends and the concentrations of IN in the reaction mixtures to be decreased by a factor of approximately 10. In a typical reaction at 37 degrees C, recombinant HIV-1 IN at 5 to 10 nM incorporated 30 to 40% of the input DNA donor into full-site integration products. The synthesis of full-site products continued up to approximately 2 h, comparable to incubation times used with HIV-1 PIC. Approximately 5% of the input donor was incorporated into the circular target producing half-site products with no significant quantities of other integration products produced. DNA sequence analysis of the viral DNA-target junctions derived from wild-type U3 and U5 coupled reactions showed an approximately 70% fidelity for the HIV-1 5-bp host site duplications. Recombinant HIV-1 IN successfully utilized a mutant U5 end containing additional nucleotide extensions for full-site integration demonstrating that IN worked properly under nonideal active substrate conditions. The fidelity of the 5-bp host site duplications was also high with these coupled mutant U5 and wild-type U3 donor ends. These studies suggest that recombinant HIV-1 IN is at least as capable as native IN in virus particles and approaching that observed with HIV-1 PIC for catalyzing full-site integration.     

Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.     

DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro

Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN-4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ~20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.     

Avian retrovirus DNA internal attachment site requirements for full-site integration in vitro

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.     

Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.

The in vitro assembly process for forming nucleoprotein complexes containing linear retrovirus-like DNA and integrase (IN) was investigated. Solution conditions that allowed avian myeloblastosis virus IN to efficiently pair two separate linear DNA fragments (each 487 bp in length) containing 3' OH recessed long terminal repeat termini were established. Pairing of the viral termini by IN during preincubation on ice permitted these nucleoprotein complexes to catalyze the concerted insertion of the two termini into a circular DNA target (full-site reaction), mimicking the in vivo reaction. The three major solution determinants were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethylene glycol. The aprotic solvent dioxane (15%) was significantly better (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half-site integration events. Half-site reactions by IN involved the insertion of a single donor terminus into circular pGEM. Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent half-site reactions from occurring at high NaCl concentrations. Under optimal solution conditions, the concerted integration reaction was directly proportional to a sixfold range of IN. The complexes appeared not to turn over, and few half-site donor-donor molecules were produced. In the presence of 0.15 or 0.35 M NaCl, dioxane prevented efficient 3' OH trimming of a blunt-ended donor by IN, suggesting that the complexes formed by IN with blunt-ended donors were different from those formed with donors containing 3' OH recessed termini for strand transfer. The results suggest that IN alone was capable of protein-protein and protein-DNA interactions that efficiently promote the in vitro concerted integration reaction.     

Biochemical and biophysical analyses of concerted (U5/U3) integration

Retrovirus integrase (IN) integrates the viral linear DNA genome (10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.     

Molecular and genetic determinants of rous sarcoma virus integrase for concerted DNA integration

Site-directed mutagenesis of recombinant Rous sarcoma virus (RSV) integrase (IN) allowed us to gain insights into the protein-protein and protein-DNA interactions involved in reconstituted IN-viral DNA complexes capable of efficient concerted DNA integration (termed full-site). At 4 nM IN, wild-type (wt) RSV IN incorporates approximately 30% of the input donor into full-site integration products after 10 min of incubation at 37 degrees C, which is equivalent to isolated retrovirus preintegration complexes for full-site integration activity. DNase I protection analysis demonstrated that wt IN was able to protect the viral DNA ends, mapping approximately 20 bp from the end. We had previously mapped the replication capabilities of several RSV IN mutants (A48P and P115S) which appeared to affect viral DNA integration in vivo. Surprisingly, recombinant RSV A48P IN retained wt IN properties even though the virus carrying this mutation had significantly reduced integrated viral DNA in comparison to wt viral DNA in virus-infected cells. Recombinant RSV P115S IN also displayed all of the properties of wt RSV IN. Upon heating of dimeric P115S IN in solution at 57 degrees C, it became apparent that the mutation in the catalytic core of RSV IN exhibited the same thermolabile properties for 3' OH processing and strand transfer (half-site and full-site integration) activities consistent with the observed temperature-sensitive defect for integration in vivo. The average half-life for inactivation of the three activities were similar, ranging from 1.6 to 1.9 min independent of the IN concentrations in the assay mixtures. Wt IN was stable under the same heat treatment. DNase I protection analysis of several conservative and nonconservative substitutions at W233 (a highly conserved residue of the retrovirus C-terminal domain) suggests that this region is involved in protein-DNA interactions at the viral DNA attachment site. Our data suggest that the use of recombinant RSV IN to investigate efficient full-site integration in vitro with reference to integration in vivo is promising.     

Inhibition of human immunodeficiency virus type 1 concerted integration by strand transfer inhibitors which recognize a transient structural intermediate

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (~15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3'-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3'-OH processing. It follows that the IN-viral DNA complex is "trapped" by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.     

HIV-1 integrase crosslinked oligomers are active in vitro

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.     

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.     

Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted Integration

A macromolecular nucleoprotein complex in retrovirus-infected cells, termed the preintegration complex, is responsible for the concerted integration of linear viral DNA genome into host chromosomes. Isolation of sufficient quantities of the cytoplasmic preintegration complexes for biochemical and biophysical analysis is difficult. We investigated the architecture of HIV-1 nucleoprotein complexes involved in the concerted integration pathway in vitro. HIV-1 integrase (IN) non-covalently juxtaposes two viral DNA termini forming the synaptic complex, a transient intermediate in the integration pathway, and shares properties associated with the preintegration complex. IN slowly processes two nucleotides from the 3′ OH ends and performs the concerted insertion of two viral DNA ends into target DNA. IN remains associated with the concerted integration product, termed the strand transfer complex. The synaptic complex and strand transfer complex can be isolated by native agarose gel electrophoresis. In-gel fluorescence resonance energy transfer measurements demonstrated that the energy transfer efficiencies between the juxtaposed Cy3 and Cy5 5′-end labeled viral DNA ends in the synaptic complex (0.68 ± 0.09) was significantly different from that observed in the strand transfer complex (0.07 ± 0.02). The calculated distances were 46 ± 3 Å and 83 ± 5 Å, respectively. DNaseI footprint analysis of the complexes revealed that IN protects U5 and U3 DNA sequences up to ∼ 32 bp from the end, suggesting two IN dimers were bound per terminus. Enhanced DNaseI cleavages were observed at nucleotide positions 6 and 9 from the terminus on U3 but not on U5, suggesting independent assembly events. Protein-protein cross-linking of IN within these complexes revealed the presence of dimers, tetramers, and a larger multimer (> 120 kDa). Our results suggest a new model where two IN dimers individually assemble on U3 and U5 ends before the non-covalent juxtaposition of two viral DNA ends, producing the synaptic complex.     

Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.

The integration of linear retrovirus DNA by the viral integrase (IN) into the host chromosome occurs by a concerted mechanism (full-site reaction). IN purified from avian myeloblastosis virus and using retrovirus-like DNA restriction fragments (487 bp in length) as donors and circular DNA (pGEM-3) as the target can efficiently catalyze that reaction. Nonionic detergent lysates of purified human immunodeficiency virus type 1 (HIV-1) virions were also capable of catalyzing the concerted integration reaction. The donor substrates were restriction fragments (469 bp) containing either U3-U5 (H-2 donor) or U5-U5 (H-5 donor) long terminal repeat sequences at their ends. As was shown previously with bacterially expressed HIV-1 IN, the U5 terminus of H-2 was preferred over the U3 terminus by virion-associated IN. The reactions involving two donors per circular target by HIV-1 IN preferred Mg2+ over Mn2+. Both metal ions were equally effective for the circular half-site reaction involving only one donor molecule. The linear 3.8-kbp recombinant products produced from two donor insertions into pGEM were genetically selected, and the donor-target junctions of individual recombinants were sequenced. A total of 55% of the 87 sequenced recombinants had host site duplications of between 5 and 7 bp, with the HIV-1 5-bp-specific duplication predominating. The other recombinants that migrated at the linear 3.8-kbp position were mainly small deletions that were grouped into four sets of 17, 27, 40, and 47 bp, each having a periodicity mimicking a turn of the DNA helix. Aprotic solvents (dimethyl sulfoxide and 1,4-dioxane) enhanced both the half-site and the linear 3.8-kbp strand transfer reactions which favored low-salt conditions (30 mM NaCl). The order of addition of the donor and target during preincubation with HIV-1 IN on ice did not affect the quantity of linear 3.8-kbp recombinants relative to that of the circular half-site products that were produced; only the quantity of donor-donor versus donor-target recombinants was affected. The presence of Mg2+ in the preincubation mixtures containing donor and target substrates was not necessary for the stability of preintegration complexes on ice or at 22 degrees C. Comparisons of the avian and HIV-1 concerted integration reactions are discussed.     

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.     

Structural organization of avian retrovirus integrase in assembled intasomes mediating full-site integration

Retrovirus preintegration complexes (PIC) purified from virus-infected cells are competent for efficient concerted integration of the linear viral DNA ends by integrase (IN) into target DNA (full-site integration). In this report, we have shown that the assembled complexes (intasomes) formed in vitro with linear 3.6-kbp DNA donors possessing 3'-OH-recessed attachment (att) site sequences and avian myeloblastosis virus IN (4 nm) were as competent for full-site integration as isolated retrovirus PIC. The att sites on DNA with 3'-OH-recessed ends were protected by IN in assembled intasomes from DNase I digestion up to approximately 20 bp from the terminus. Several DNA donors containing either normal blunt-ended att sites or different end mutations did not allow assembly of complexes that exhibit the approximately 20-bp DNase I footprint at 14 degrees C. At 50 and 100 mm NaCl, the approximately 20-bp DNase I footprints were produced with wild type (wt) U3 and gain-of-function att site donors for full-site integration as previously observed at 320 mm NaCl. Although the wt U5 att site donors were fully competent for full-site integration at 37 degrees C, the approximately 20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 degrees C. Under suboptimal assembly conditions for intasomes using U3 att DNA, DNase I probing demonstrated an enhanced cleavage site 9 bp from the end of U3 suggesting that a transient structural intasome intermediate was identified. Using a single nucleotide change at position 7 from the end and a series of small size deletions of wt U3 att site sequences, we determined that sequences upstream of the 11th nucleotide position were not required by IN to produce the approximately 20-bp DNase I footprint and full-site integration. The results suggest the structural organization of IN at the att sites in reconstituted intasomes was similar to that observed in PIC.     

HIV-1 integrase interaction with U3 and U5 terminal sequences in vitro defined using substrates with random sequences

Successful integration of viral genome into a host chromosome depends on interaction between viral integrase and its recognition sequences. We have used a reconstituted concerted human immunodeficiency virus, type 1 (HIV-1), integration system to analyze the role of integrase (IN) recognition sequences in formation of the IN-viral DNA complex capable of concerted integration. HIV-1 integrase was presented with substrates that contained all 4 bases at 8 mismatched positions that define the inverted repeat relationship between U3 and U5 long terminal repeats (LTR) termini and at positions 17-19, which are conserved in the termini. Evidence presented indicates that positions 17-20 of the IN recognition sequences are needed for a concerted DNA integration mechanism. All 4 bases were found at each randomized position in sequenced concerted DNA integrants, although in some instances there were preferences for specific bases. These results indicate that integrase tolerates a significant amount of plasticity as to what constitutes an IN recognition sequence. By having several positions randomized, the concerted integrants were examined for statistically significant relationships between selections of bases at different positions. The results of this analysis show not only relationships between different positions within the same LTR end but also between different positions belonging to opposite DNA termini.     

Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.     

Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration.     

FRET analysis reveals distinct conformations of IN tetramers in the presence of viral DNA or LEDGF/p75

A tetramer of HIV-1 integrase (IN) stably associates with the viral DNA ends to form a fully functional concerted integration intermediate. LEDGF/p75, a key cellular binding partner of the lentiviral enzyme, also stabilizes a tetrameric form of IN. However, functional assays have indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for productive concerted integration. Here, we employed Förster Resonance Energy Transfer (FRET) to monitor assembly of individual IN subunits into tetramers in the presence of viral DNA and LEDGF/p75. The IN–viral DNA and IN–LEDGF/p75 complexes yielded significantly different FRET values suggesting two distinct IN conformations in these complexes. Furthermore, the order of addition experiments indicated that FRET for the preformed IN–viral DNA complex remained unchanged upon its subsequent binding to LEDGF/p75, whereas pre-incubation of LEDGF/p75 and IN followed by addition of viral DNA yielded FRET very similar to the IN–LEDGF/p75 complex. These findings provide new insights into the structural organization of IN subunits in functional concerted integration intermediates and suggest that differential multimerization of IN in the presence of various ligands could be exploited as a plausible therapeutic target for development of allosteric inhibitors.