Activity of recombinant HIV-1 integrase on mini-HIV DNA

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.     

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.     

Functional nucleotides of U5 LTR determining substrate specificity of prototype foamy virus integrase

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.     

Interactions of HIV-1 DNA heterocyclic bases with viral DNA

Human immunodeficiency virus type 1 integrase is one of three viral enzymes, and it realizes a key process of the viral replication cycle, i.e. viral DNA integration into infected cell genome. Integrase recognizes nucleotide sequences located at the ends of the viral DNA U3 and U5 LTRs and catalyzes 3'-processing and strand transfer reactions. To study the interactions between integrase and viral DNA at present work, we used modified integrase substrates mimicking the terminal U5 LTR sequence and containing non-nucleoside insertions in one or/and both strands. It is shown that the substrate modifications have no influence on the integrase binding rate, while the heterocyclic bases removal in the 5th and 6th substrate positions and in the 3rd position of the substrate processed strand distinctly inhibits the integrase catalytic activity. This fact demonstrates these bases significance for the active enzyme/substrate complex formation. On the contrary, modification of the 3rd position within substrate non-processed strand stimulates 3'-processing. Since heterocyclic base elimination results in disruption of the DNA complementary and staking interactions, this result shows that DNA double helix destabilization close to the cleaved bond promotes the 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.     

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.     

Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication.     

Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.     

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.     

Presteady-state analysis of avian sarcoma virus integrase. II. Reverse-polarity substrates identify preferential processing of the U3-U5 pair

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556-2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5'-5' phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089-12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s(-1) for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.     

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.     

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.     

A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration.

We have probed the nucleoprotein organization of Moloney murine leukemia virus (MLV) pre-integration complexes using a novel footprinting technique that utilizes a simplified in vitro phage Mu transposition system. We find that several hundred base pairs at each end of the viral DNA are organized in a large nucleoprotein complex, which we call the intasome. This structure is not formed when pre-integration complexes are made by infecting cells with integrase-minus virus, demonstrating a requirement for integrase. In contrast, footprinting of internal regions of the viral DNA did not reveal significant differences between pre-integration complexes with and without integrase. Treatment with high salt disrupts the intasome in parallel with loss of intermolecular integration activity. We show that a cellular factor is required for reconstitution of the intasome. Finally, we demonstrate that DNA-protein interactions involving extensive regions at the ends of the viral DNA are functionally important for retroviral DNA integration activity. Current in vitro integration systems utilizing purified integrase lack the full fidelity of the in vivo reaction. Our results indicate that both host factors and long viral DNA substrates may be required to reconstitute an in vitro system with all the hallmarks of DNA integration in vivo.     

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916.

The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases.     

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.     

Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases.

A comparison of the extents of site-specific cleavage of U5 and U3 viral DNA termini by the integrases of human immunodeficiency virus type 1 and visna virus guided the quantitative testing of oligonucleotide substrates containing specific base substitutions. The simultaneous exchange of positions 5 and 6 between U3 substrates switched the patterns of differential susceptibility to the two integrases. The activity of visna virus integrase was more dependent on the identity of position 5 adjacent to the invariant CA bases than on position 6, whereas human immunodeficiency virus type 1 integrase appeared to interact even more critically with position 6. Although the paired natural substrates of most lentiviral integrases match at positions 7 and 8, these bases were not important for susceptibility of U5 substrates. In fact, the final six U5 positions contained all of the sequence information necessary for susceptibility. These results suggest that constraints other than integration influence the terminal inverted repeats of retroviral DNA.     

Nuclease mechanism of the avian retrovirus pp32 endonuclease.

In vivo, the inferred circular retrovirus DNA precursor to the provirus contains two long terminal repeats (LTRs) in tandem. We studied the site-specific nicking of supercoiled DNA that contains tandem copies of avian retrovirus LTR DNA in vitro by using purified avian myeloblastosis virus pp32 endonuclease, Mg2+, and viral DNA substrates containing different LTR circle junction sequences. The results confirmed our previous observation that the pp32 protein generates two nicks, one in either viral DNA strand, each 2 nucleotides from the circle junction site. The specificity of nicking by pp32 was unchanged over an eight-fold range of protein concentration and with different avian retrovirus LTR circle junction substrates. These data are consistent with models which propose a role for the endonuclease in removal of two nucleotides from the LTR termini on integration of viral DNA in vivo.