Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein.

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.     

Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3' end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5' phosphates in the target DNA (strand transfer). Previous investigators have shown that purified IN requires Mn2+ or Mg2+ to promote strand transfer in vitro, although Mg2+ is the likely metal cofactor in vivo. IN activity in the presence of Mg2+ in vitro requires high IN concentrations and low concentrations of salt. Here, we show that the viral nucleocapsid protein NCp7 allows efficient IN-mediated strand transfer in the presence of Mg2+ at low enzyme concentrations. This potentiating effect appears to be unique to NCp7, as other small DNA-binding proteins, while capable of stimulating integration in the presence of Mn2+, all failed to stimulate strand transfer in the presence of Mg2+.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.     

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.     

Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions.

Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration. The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group. This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA. We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions. Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining. However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions. The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity. A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position. Single-stranded DNA served as an in vitro integration target for HIV IN. The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated.     

Activities of the feline immunodeficiency virus integrase protein produced in Escherichia coli.

Retroviral DNA integration requires the activity of at least one viral protein, the integrase (IN) protein. We cloned and expressed the integrase gene of feline immunodeficiency virus (FIV) in Escherichia coli as a fusion to the malE gene and purified the IN fusion protein by affinity chromatography. The protein is active in site-specific cleavage of the viral DNA ends, DNA strand transfer, and disintegration. FIV IN has a relaxed viral DNA substrate requirement: it cleaves and integrates FIV DNA termini, human immunodeficiency virus DNA ends, and Moloney murine leukemia virus DNA ends with high efficiencies. In the cleavage reaction, IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack. In vitro, either H2O, glycerol, or the 3' OH group of the viral DNA terminus can serve as nucleophile in this reaction. We found that FIV IN preferentially uses the 3' OH ends of the viral DNA as nucleophile, whereas HIV IN protein preferentially uses H2O and glycerol as nucleophiles.     

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.     

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.     

In vitro activities of purified visna virus integrase.

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.     

Changes in the mechanism of DNA integration in vitro induced by base substitutions in the HIV-1 U5 and U3 terminal sequences.

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration with specially designed mini-donor DNA, a supercoiled plasmid acceptor, purified bacterial-derived HIV-1 integrase (IN), and host HMG-I(Y) protein (Hindmarsh, P., Ridky, T., Reeves, R., Andrake, M., Skalka, A. M., and Leis, J. (1999) J. Virol. 73, 2994-3003). Integration in this system is dependent upon the mini donor DNA having IN recognition sequences at both ends and the reaction products have all of the features associated with integration of viral DNA in vivo. Using this system, we explored the relationship between the HIV-1 U3 and U5 IN recognition sequences by analyzing substrates that contain either two U3 or two U5 terminal sequences. Both substrates caused severe defects to integration but with different effects on the mechanism indicating that the U3 and the U5 sequences are both required for concerted DNA integration. We have also used the reconstituted system to compare the mechanism of integration catalyzed by HIV-1 to that of avian sarcoma virus by analyzing the effect of defined mutations introduced into U3 or U5 ends of the respective wild type DNA substrates. Despite sequence differences between avian sarcoma virus and HIV-1 IN and their recognition sequences, the consequences of analogous base pair substitutions at the same relative positions of the respective IN recognition sequences were very similar. This highlights the common mechanism of integration shared by these two different viruses.     

DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein.

The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.     

Characterization of a replication-defective human immunodeficiency virus type 1 att site mutant that is blocked after the 3' processing step of retroviral integration

Two activities of retroviral integrase, 3' processing and DNA strand transfer, are required to integrate viral cDNA into a host cell chromosome. Integrase activity has been analyzed in vitro using purified protein and recombinant DNA substrates that model the U3 and U5 ends of viral cDNA or by using viral preintegration complexes (PICs) that form during virus infection. Numerous studies have investigated changes in integrase or viral DNA for effects on both 3' processing and DNA strand transfer activities using purified protein, but similar analyses have not been carried out using PICs. Here, we analyzed PICs from human immunodeficiency virus type 1 (HIV-1) strain 604del, an integration-defective mutant lacking 26 bp of U5, and revE1, a revertant of 604del containing an additional 19-bp deletion, for levels of 3' processing activity that occurred in infected cells and for levels of in vitro DNA strand transfer activity. Whereas revE1 supported one-third to one-half of the level of wild-type DNA strand transfer activity, the level of 604del DNA strand transfer activity was undetectable. Surprisingly, integrase similarly processed the 3' ends of 604del and revE1 in vivo. We therefore conclude that 604del is blocked in its ability to replicate in cells after the 3' processing step of retroviral integration. Whereas Western blotting showed that wild-type, revE1, and 604del PICs contained similar levels of integrase protein, Mu-mediated PCR footprinting revealed only minimal protein-DNA complex formation at the ends of 604del cDNA. We propose that 604del is replication defective because proteins important for DNA strand transfer activity do not stably associate with this cDNA after in vivo 3' processing by integrase.     

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.     

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.     

Substrate specificity of Ty1 integrase.

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 requires the element-encoded integrase (IN) protein, which is a component of cytoplasmic virus-like particles (VLPs). Using purified recombinant Ty1 IN and an oligonucleotide integration assay based on Ty1 long terminal repeat sequences, we have compared IN activity on substrates having either wild-type or altered donor ends. IN showed a marked preference for blunt-end substrates terminating in an A:T pair over substrates ending in a G:C pair or a 3' dideoxyadenosine. VLP activity on representative substrates also showed preference for donor strands which have an adenosine terminus. Staggered-end substrates showed little activity when nucleotides were removed from the end of the wild-type donor strand, but removal of one nucleotide from the complementary strand did not significantly diminish activity. Removal of additional nucleotides from the complementary strand reduced activity to minimal detection levels. These results suggest that the sequence specificity of Ty1 IN is not stringent in vitro. The absence of Ty1 IN-mediated 3' dinucleotide cleavage, a characteristic of retroviral integrases, was demonstrated by using selected substrates. In addition to the forward reaction, both recombinant IN and VLP-associated IN carry out the reverse disintegration reaction with long terminal repeat-based dumbbell substrates. Disintegration activity exhibits sequence preferences similar to those observed for the forward reaction.     

The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO(4) was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3'-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19-42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ~32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN-DNA complex in the absence or presence of strand transfer inhibitors.     

DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2.

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.     

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.