©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Essential Interaction between Distinct Domains of HIV-1 Integrase Mediates Assembly of the Active Multimer (*)

(Received for publication, August 8, 1994; and in revised form, November 18, 1994)

Viola Ellison (1) Jennifer Gerton (1) (2) Karen A. Vincent (1)(§) (3) Patrick O. Brown (1) (3)(¶)

From the  (1)Department of Biochemistry, the (2)Department of Microbiology and Immunology, and the (3)Howard Hughes Medical Institute, Stanford University, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3`-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved ``DD35E'' motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.


INTRODUCTION

Integration is a critical step in the retroviral life cycle (for a review of the retroviral life cycle, see (1) ). From genetic analyses, one viral protein, integrase, and sequences located at the ends of the viral genome are known to be required for integration (2, 3, 4) . The reaction is accomplished in vivo by a large viral nucleoprotein complex(5, 6) . Three steps in the reaction have been deduced from the analysis of reaction intermediates(6, 7, 8) : (i) removal of the terminal nucleotides from each viral DNA end distal to the highly conserved CA dinucleotide by a reaction termed 3`-end processing; (ii) joining of the two recessed 3`-ends of viral DNA to 5` staggered sites in the target DNA by a concerted cleavage and ligation reaction; and (iii) repair of the small gaps in the target sequence flanking the viral genome and joining of the 5`-ends of viral DNA to host DNA. The resulting provirus is colinear with unintegrated DNA, except for the loss of the two terminal base pairs from each end of the unintegrated viral DNA precursor.

The 3`-end processing and DNA joining steps of the reaction can be reproduced in vitro with purified integrase and model viral DNA substrates(9, 10, 11) . Like the in vivo reaction, integration in vitro is mediated by a stable complex between integrase and the viral DNA end(12) , which processively catalyzes the 3`-end processing and DNA joining steps in the reaction. Neither step requires a high energy cofactor, and both proceed by a one-step transesterification reaction(13) . Integrase is an enzyme, as demonstrated by its ability to catalyze a reversal of the DNA joining reaction, termed disintegration(14) , and to turn over catalytically in both the integration and disintegration reactions(15, 16) .

Mutational analyses have defined two functionally distinct structural features of HIV (^1)integrase(17, 18, 19, 20, 21, 22) characterized by amino acid sequence motifs that are highly conserved among all retroviruses and retrotransposons. The first, commonly called the DD35E region, lies within a protease-resistant core of the enzyme and contains the motif DX39-58DX35E. Mutations in the evolutionarily conserved aspartic or glutamic acid residues result in loss of all catalytic activities, suggesting a role for these residues in active site function(17, 18, 19) . The second feature, located near the N terminus, is the zinc-finger-like motif HX3-7HX23-32CX2C, termed the HHCC domain. The predicted secondary structure of this domain, as determined by spectroscopic studies, is similar to that of the zinc finger domain of TFIIIA(23) . Mutations in the conserved cysteine or histidine residues impair only 3`-end processing and DNA joining without eliminating disintegration activity(17, 18, 19, 20, 21) . These mutations also compromise the ability of the enzyme to bind zinc ions(24) .

The disintegration activity of the HHCC mutants shows an altered pattern of substrate specificity that suggests that the HHCC region participates in interactions with viral DNA at sites internal to the phylogenetically conserved, subterminal CA/TG dinucleotide pair(21) . Nevertheless, mutants lacking the HHCC domain retain the ability to recognize the features of the viral DNA end (like the conserved subterminal CA/TG) that are absolutely required for the 3`-end processing and DNA joining reactions(21) . Paradoxically, although mutations in the HHCC domain abolish integration of conventional model substrates, these mutations do not block the ``reintegration'' of viral DNA ends released by the mutant enzyme from the disintegration substrate(21) . Thus, the activity of the HHCC domain appears to be required at an early step in the integration pathway in which a productive interaction with viral DNA is established. Once this interaction is established, integration can proceed in the absence of a functional HHCC domain. In this report, we further characterize the properties of the HHCC mutants of HIV integrase. Our results support a model in which the HHCC domain participates in a divalent cation-dependent asymmetric interaction between integrase protomers that plays a critical role in assembly of a stable complex between integrase and viral DNA.


EXPERIMENTAL PROCEDURES

Oligonucleotides and Preparation of Substrates

Oligonucleotides were purchased from Operon Technologies Inc. and purified by electrophoresis through a 15% denaturing polyacrylamide gel. The standard integration substrate consists of the last 21 base pairs from the U5 end of HIV-1 DNA and was prepared using the oligonucleotides C220 (5` ATGTG GAAAA TCTCT AGCAGT 3`) and C120 (5` ACTGC TAGAG ATTTT CCACAT 3`). C220 was labeled with T4 Polynucleotide Kinase (New England Biolabs) and [ P]ATP (ICN) to a specific activity of 700,000 cpm/pmol and annealed to a 1.5-fold excess of C120 in 50 mM NaCl by heating first to 90 °C for 5 min., 37 °C for 15 min., and then room temperature for 10 min. The unincorporated nucleotide was removed by centrifugation through a 1-ml bed volume Sephadex G-15 column, equilibrated in 10 mM Tris, pH 7.5, 50 mM NaCl. A pre-processed version of the wild-type integration substrate corresponding to the product of the 3`-end processing reaction was made by annealing B2-1 (5` ATGTG GAAAA TCTCT AGCA 3`) to C120. The preparation of the disintegration Y-oligomer substrate was as described previously(14) .

Standard Integration and Disintegration Assays

Wild-type HIV integrase and two mutant versions with alterations in the HHCC domain, H12A (containing amino acid changes at positions 12 and 13) and DeltaHHCC (containing a deletion of the HHCC region), were expressed in Escherichia coli using the T7 expression system and purified as described(21) . Integrase proteins with alterations in the phylogenetically conserved aspartic acid residues at positions 64 and 116 or the glutamic acid residue at position 152, which constitute the DD35E motif, were obtained from two sources. D116N, which contains a substitution of asparagine for aspartic acid at position 116, and E152Q, which contains a substitution of glutamine for glutamic acid at position 152(17) , were kindly provided by A. Engelman and R. Craigie (National Institutes of Health). D64V (an aspartic acid to valine substitution), E152N (a glutamic acid to asparagine substitution), D116I (an aspartic acid to isoleucine substitution), and R199C (an integrase variant with a deletion of amino acids 200 to 288) (18) , were all kindly provided by A. Leavitt and H. Varmus (University of California, San Francisco). Typically, 100 nM integrase protomer was incubated at 37 °C with 10 nM viral DNA substrate in a 25-µl reaction containing 20 mM Hepes, pH 7.5, 1 mM DTT, 0.05% Nonidet P-40, 0.5 mM CHAPS, 20 mM NaCl, and 5 mM MnCl(2) (Fig. 1). In complementation experiments, pairs of mutant integrases were used at a 1:1 stoichiometry with a total concentration of 100 nM integrase protomer. All incubations were performed at 37 °C by prewarming tubes containing the reaction buffer for 5-10 min, then adding all DNA substrates and, when indicated, competitor substrates, and finally by adding integrase. Reactions were stopped by adding an equal volume of gel loading buffer (95% formamide, 30 mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue) and analyzed on a denaturing 15% polyacrylamide gel. Time courses were performed by beginning with a 50-µl reaction volume and taking 5-µl aliquots at indicated time points. Reaction products were quantitated using a Molecular Dynamics PhosphorImager. The disintegration assay was performed under the same experimental conditions, except the Y-oligomer substrate was used at a concentration of 5 nM (Fig. 1).


Figure 1: In vitro assays for integrase. In the integration assay, an oligonucleotide mimicking one end of viral DNA is used as a substrate, and the reaction proceeds in two steps. In the 3`-end processing reaction, integrase catalyzes the removal of the terminal nucleotides distal to the phylogenetically conserved CA dinucleotide. In the second step, integrase catalyzes a concerted cleavage and ligation reaction in which the recessed 3`-end of viral DNA is joined to target DNA. In disintegration, a reversal of the DNA joining reaction, a substrate that mimics the integration intermediate (the Y-oligomer) is resolved by integrase into an intact target DNA molecule and a processed viral DNA end.



Prebinding Protocol For Stable Integrase-DNA Complexes

The experimental design for the assay is described in detail elsewhere (12) . Briefly, to measure stable complex formation during disintegration, all integrase proteins (100 nM) were incubated with 5 nM Y-oligomer for 15 s at 37 °C, after which 100 ng of heparin (Sigma) was added to inhibit further substrate binding. The reaction was then incubated for an additional 30 min.

NEM Modification Experiments

A 50 mM solution of N-ethylmaleimide (NEM) (Sigma) was prepared in distilled water within 24 h of use. Integrase (400 nM) was incubated with 5 mM NEM in Buffer N (20 mM Hepes pH 7.5, 0.05% Nonidet P-40, 20 mM NaCl) for 30 s at room temperature. The reaction was then quenched by the addition of 10 mM DTT and incubation on ice for 5 min. The modified enzyme was then assayed under standard assay conditions (described above) for retention of both integration and disintegration activities. For the protection experiments, the NEM modification was performed as described above, except integrase was first preincubated in Buffer N containing 5 mM MnCl(2) for 5 min at room temperature. 5 mM NEM was then added, and the reaction was incubated for 30 s, quenched with 10 mM DTT, and assayed. For the protection experiments with complementing pairs of mutant integrase subunits, both mutant proteins were present at a concentration of 200 nM and treated as described above. NEM modification of individual mutant integrases (200 nM) was performed as described for the wild-type protein. After quenching the reaction with DTT, an equimolar quantity of the complementing subunit was added, and the protein mixture was assayed for integration activity.

Aggregation/Pelleting Experiments

Integrase (100 nM) was incubated in 20 mM Hepes, pH 7.5, 1 mM DTT, 0.05% Nonidet P-40, 0.5 mM CHAPS, and 50 mM NaCl for 5 min at room temperature in the presence or absence of 5 mM MnCl(2). The reaction (20 µl) was then layered on top of a 60-µl solution of the same reaction buffer containing 20% glycerol, in a 500-µl siliconized Eppendorf microcentrifuge tube, and centrifuged at 10,000 times g for 10 min at 4 °C. The supernatant was removed, and the pellet was resuspended in 80 µl of reaction buffer without divalent cation by pipetting the solution in and out of the tube 20 times. The protein content of each fraction was determined by SDS-PAGE and Western blotting using polyclonal human antiserum (Scripps) and the ECL detection system (Amersham Corp.).


RESULTS

NEM-modified HIV Integrase Has a Phenotype Similar to That of an HHCC Mutant

The biochemical properties of HIV integrase are altered by exposure to the alkylating agent NEM (Fig. 2). Modification of the enzyme with NEM, in the absence of a divalent cation, abolished both 3`-end processing and DNA joining activity ( Fig. 2left panel, compare lane1 versus lane3). However, the ability of the enzyme to catalyze the disintegration reaction was not affected ( Fig. 2right panel, lane3). Thus, the phenotype of NEM-modified integrase is similar to that of an HHCC mutant.


Figure 2: Activities of NEM-modified integrase. Integrase was modified by incubating the protein with 5 mM NEM for 30 s and then quenching the reaction by incubating with 10 mM DTT. The modified enzyme was assayed for disintegration and integration activity, as shown in the left and rightpanels, respectively. Wild-type integrase was assayed before exposure to NEM (lane1), after treatment first with 10 mM DTT and then 5 mM NEM (lane2), and after treatment with 5 mM NEM (lane3). In the lanes labeled -, integrase was omitted from the reaction.



A Divalent Cation-dependent Structural Change in Integrase Is Associated with Integration

Under conditions permissive for integration, integrase undergoes a structural change in the presence of a divalent cation (Mn is typically used, Zn and Mg will also suffice), which results in aggregation of the enzyme at a low protein concentration (3 µg/ml) (Fig. 3). Aggregation was manifested as a change in sedimentation behavior ( Fig. 3panelA, compare lanes 2p and 3p) and the precipitated enzyme was active for both 3`-end processing and DNA joining (data not shown). Protein aggregation in vitro can be a manifestation of specific, functionally-important protein-protein interactions (e.g. DnaA(25) , RecA(26) , Zeste(27) ). To investigate the possibility of a functional relationship between integration activity and integrase aggregation, we tested NEM-modified, HHCC mutant, and DD35E mutant variants of integrase for divalent cation-induced aggregation. Although the Mn-dependent aggregration of the DD35E mutant was similar to that of wild-type integrase (data not shown), aggregation of the HHCC mutant and the NEM-modified protein was markedly diminished (Fig. 3, B and C, compare lanes2p and 3p in each panel). Thus, mutations in the HHCC region or modification of integrase with NEM abolish both the metal ion-induced aggregation and integration activity.


Figure 3: Mn-dependent aggregation of wild-type and variant integrases. PanelsA-C show analyses of wild-type, DeltaHHCC, and NEM-modified integrase, respectively. Integrase was incubated either in the presence (lanes3, s and p) or absence (lanes2, s and p) of 5 mM MnCl(2), and the samples were then subjected to centrifugation for 10 min at 10,000 times g. The sample analyzed in lane1 was not subjected to centrifugation. s represents the supernatant fraction, and p represents the pelleted fraction.



The divalent cation-dependent structural change also resulted in protection against NEM modification (Fig. 4). When the enzyme was incubated with 5 mM Mn before and during NEM treatment, integrase retained approximately 35% of initial levels of 3`-end processing and DNA joining activities (compare lane3 with lanes4 and 5). The same NEM treatment in the absence of a divalent cation completely abolished both activities. This protection from NEM inactivation was also observed when integrase was pre-incubated with Ca or Mg. The ability of integrase to aggregate was also protected against inactivation by NEM when the enzyme was pre-incubated with a divalent metal ion before exposure to NEM (data not shown). Thus, the divalent cation-dependent structural change that leads to aggregation also appears to protect the NEM-sensitive site (NSS) from NEM modification so that both aggregation and integration activity were partially retained.


Figure 4: Protection of integrase against NEM inactivation by preincubation with a divalent cation. Integrase was preincubated for 5 min in the presence or absence of 5 mM MnCl(2), after which NEM was added to a final concentration of 5 mM. After a 30-s incubation with NEM, all reactions were quenched with 10 mM DTT, and the samples were then assayed for integration activity. Reaction conditions were as follows: integrase was not exposed to NEM (lane1); integrase was incubated first with DTT for 30 s and then with NEM (lane2); integrase was preincubated in the absence of MnCl(2) and then incubated with NEM (lane3); integrase was preincubated in the presence of either 5 mM MnCl2 (lane4) or 5 mM MgCl(2) (lane5), and then incubated with NEM. In the lane labeled -, integrase was omitted from the reaction.



What is this divalent cation-dependent structural change and how are the HHCC domain and NSS of the protein involved? We hypothesized that formation of a stable complex between integrase and viral DNA, an initial step in the integration pathway, requires multimerization of integrase and that multimerization involves interactions between the NSS of one integrase subunit and the HHCC domain of another subunit (Fig. 5). The interaction with the HHCC domain protects the NSS against modification, and propagation of this interaction leads to aggregation. This model predicts that: (i) integrase functions as a multimer, (ii) the HHCC domain and NSS on different subunits of the multimer interact and therefore function in trans, and (iii) both features are required for integrase to form a stable active complex with viral DNA. These predictions were tested.


Figure 5: A model for integrase multimerization. Wild-type integrase is depicted in the center, with the triangular protrusion on the right side of the molecule representing the HHCC domain, and the indentation on the left representing the NSS. Modification of integrase with NEM or deletion of the HHCC domain results in the integrase variants shown on the left and right side of wild-type, respectively. Although depicted here as a single molecule, wild-type integrase is predominantly a dimer in the absence of a divalent cation, and dimerization of integrase appears not to be disrupted by alterations in the NSS or HHCC domains. The important features of this model are: (i) the active form of integrase is a multimer, (ii) multimerization is mediated by interactions between the HHCC domain of one subunit and the NSS of another subunit and is promoted by a divalent cation, (iii) propagation of the HHCC domain-NSS interaction leads to aggregation of the enzyme, (iv) alterations in the HHCC domain and NSS of the enzyme impair both integration and aggregation, (v) integrase multimerization is critical for formation of a stable integrase-viral DNA complex. After assembly, integrase processively executes the 3`-end processing and DNA joining steps in the integration pathway.



The HHCC-, DD35E-, and NEM-sensitive Features Can Be Distributed among at Least Two Separate Protomers in an Active Integrase Multimer

The functional organization of the enzyme was investigated by testing for reconstitution of integration activity by mixing pairs of defective proteins (Fig. 6, data summarized in Fig. 7). When incubated independently, each mutant had virtually undetectable integration activity (Fig. 6A: wild-type integrase, lane1; NEM-modified integrase, lane2; HHCC mutants, lanes3-5; DD35E mutants, lanes6-9). However, both 3`-end processing and DNA joining activity were partially restored when specific combinations of mutants were mixed. No complementation was observed between pairs of HHCC mutants or between pairs of DD35E mutants (data not shown). Reconstitution of enzyme activity was greatest when an HHCC mutant was combined with either a DD35E mutant (Fig. 6B, lanes1-8) or a C-terminal truncation mutant (Delta amino acids 200-288, data not shown). Weak complementation was observed between the C-terminal truncation and catalytic core mutants (data not shown). Thus, the HHCC region functions in trans to the catalytic core and amino acids 200-288 are not required in cis to the HHCC domain. Since only weak complementation was detected between the DD35E mutants and the C-terminal truncation mutant, a feature in the C terminus most likely functions primarily in cis to the catalytic core.


Figure 6: The NEM-sensitive, HHCC, and DD35E regions define functional modules of integrase. Various integrase mutants were assayed for integration activity either separately (panelA) or in combinations (panelsB-D). In panelA, assays were performed with wild-type integrase (lane1); NEM-modified integrase (lane2); H12A (lane3); DeltaHHCC (lane4); C43A (lane5); D116N (lane6); D64V (lane7); E152N (lane8); E152Q (lane9). Note that the D64V (lane7) and E152N (lane8) protein preparations contain a contaminating nuclease activity, which does not significantly affect the conclusions from the experiment. In panelB, combinations of mutants were premixed in reaction buffer without the divalent cation, and then assayed for integration activity. In the assays in lanes1-4, H12A was mixed with either D64V (lane1), E152N (lane2), D116N (lane3), or E152Q (lane4). In the reactions shown in lanes5-8, DeltaHHCC was mixed with either D64V (lane5), E152N (lane6), D116N (lane7), or E152Q (lane8). In panelC, H12A (lanes1-3) and DeltaHHCC (lanes5-7) were treated with NEM for 30 s in the absence of a divalent cation. After quenching the reactions with DTT, the NEM-treated proteins were mixed with either D64V (lanes1 and 5), D116N (lanes2 and 6), or E152Q (lanes3 and 7) and then assayed for integration. In lanes4 and 8, H12A (lane4) and DeltaHHCC (lane8) were preincubated with 5 mM MnCl(2) for 5 min and then treated with NEM for 30 s. After quenching the reactions with DTT, each protein was mixed with D116N and then assayed for integration. In panelD, D64V (lanes1 and 3), D116N (lanes2 and 5), and E152Q (lanes3 and 6) were incubated with NEM in the absence of a divalent cation for 30 s. After quenching the reaction with DTT, each protein was mixed with either H12A (lanes1-3) or DeltaHHCC (lanes4-6), and then assayed for integration activity. In the lane labeled -, integrase was omitted from the reaction. The results from the complementation analysis are summarized in Fig. 7.




Figure 7: Summary of results of in vitro complementation experiments. The results are consistent with a model in which subunit interactions that involve the NSS of one subunit and the HHCC domain of a second subunit are required for assembly of the active multimer. These two features are related asymmetrically to the putative active site: HHCC domain functions effectively in trans to the intact DD35E motif, but the NSS does not.



Complementation between the HHCC mutant and the NEM-modified protein suggested that these two regions may function in trans. We tested this hypothesis by treating either the HHCC or DD35E mutant with NEM prior to mixing the two mutant proteins. Complementation was moderately compromised when the DD35E mutant was NEM-modified (panelD, lanes1-6) but virtually abolished when the HHCC mutant was modified with NEM (panelC, lanes1-8). Therefore, in the active integrase multimer, the NEM-sensitive region functions primarily in trans to the HHCC domain.

There are 6 cysteines in integrase: 2 in the HHCC domain, at positions 40 and 43; 3 in the protease-resistant core of the enzyme, at positions 56,65, and 130; and 1 near the C terminus, at position 280. (Cys-40, Cys-43, and Cys-280 could be excluded by the complementation analysis). To determine which cysteine was sensitive to NEM modification, alanine was substituted for cysteine at either position 56, 65, or 130. If the NEM-sensitive cysteine itself were necessary for NSS function, then replacement of the cysteine would be predicted to yield an enzyme with a phenotype mimicking NEM-modified integrase. However, if the NEM-sensitive cysteine were dispensible, replacement would be predicted to have no effect on enzyme function, yet would render integrase resistant to NEM modification. As shown in Fig. 8, the latter phenotype was observed only for the mutant with alanine substituted for Cys-56, implicating this cysteine as the target of the inhibitory NEM modification. This cysteine, and the corresponding NEM-sensitive functional site, are probably contained in the protease-resistant core domain that includes the enzyme's active site (17, 22) .


Figure 8: The cysteine at position 56 in HIV-1 integrase confers sensitivity to NEM modification. A synthetic gene encoding HIV-1 integrase (J. Gerton, unpublished results) was modified to direct incorporation of alanine instead of cysteine at position 56, and the integrase variant was purified by a modified version of the protocol described in(24) . Wild-type integrase and the integrase variant were assayed for integration activity as described under ``Experimental Procedures,'' except DTT was used at 12.5 mM. Reactions were performed with the following integrase proteins: C56A (lane1), or wild-type integrase (lane2), not exposed to NEM; C56A (lane3) or wild-type integrase (lane4), first incubated with 5 mM NEM for 30 s and then adjusted to 10 mM DTT. Integrase was omitted from the reaction analyzed in lane5.



Substitution of alanine for the single cysteine outside of its HHCC domain did not impair the in vitro enzymatic activity of the Moloney murine leukemia virus (MLV) integrase, which has also been shown to be sensitive to NEM(28) . The NEM sensitivity of this mutant form of the Moloney murine leukemia virus (MLV) integrase was not tested.

An Intermolecular Interaction Involving the HHCC and NSS Mediates the Divalent Cation-dependent Aggregation and Protection from NEM Modification

To test the hypothesis that an intermolecular interaction between the HHCC domain and NSS mediates both aggregation and protection from NEM inactivation, we tested complementing pairs of mutant integrases in both the NEM protection and divalent cation-induced precipitation assays.

Because the previous experiment had shown that the NSS and HHCC domain function preferentially or exclusively in trans, we tested whether a DD35E-defective mutant integrase subunit with an intact HHCC domain could protect an HHCC-defective mutant subunit against NEM modification. An enzyme reconstituted by mixing a DeltaHHCC mutant with a D116N mutant retained both 3`-end processing and DNA joining activities after exposure to NEM, and the protection against NEM depended on preincubating the mutant proteins together with 5 mM MnCl(2) (Fig. 9A, compare lanes3 and 4). The divalent cation alone did not confer protection of the DeltaHHCC mutant from NEM inactivation (Fig. 9Alane2, also Fig. 5, panelC, lanes4 and 8). Therefore, a feature of the D116N mutant that is not provided by the DeltaHHCC mutant itself confers protection of the NEM-sensitive site of the DeltaHHCC mutant. This feature is most likely in the HHCC domain. Thus, the HHCC domain and NSS mediate an interaction between subunits of the active multimeric form of integrase, and this interaction requires a divalent cation.


Figure 9: A, NEM protection requires a functional HHCC domain in trans. DeltaHHCC and D116N, either separately or in combination, were treated with NEM in the presence or absence of a divalent cation and then assayed for integration activity. Lane1, DeltaHHCC and D116N were mixed (no NEM treatment); lane2, DeltaHHCC was preincubated with 5 mM MnCl(2) for 5 min, after which the protein was incubated with 5 mM NEM for 30 s, and then DTT was added at 10 mM, before assaying integration activity. Lanes3 and 4, same as lane2, except that DeltaHHCC and D116N were mixed and preincubated for 5 min in the presence (lane3) or absence (lane4) of 5 mM MnCl(2), prior to NEM treatment. B, mixing the DeltaHHCC variant and NEM-modified integrase subunits does not restore aggregation. DeltaHHCC and NEM-modified wild-type integrase were mixed in the absence (lanes2, s and p) or the presence of 5 mM MnCl(2) (lanes3, s and p) and then subjected to centrifugation 10,000 times g for 10 min. The sample in lane1 was not subjected to centrifugation. The supernatant and pellet were analyzed for protein content by SDS-PAGE and Western blotting. The s represents the supernatant fraction, and p represents the pelleted fraction. DeltaHHCC migrates slightly faster than wild-type integrase.



Complementing pairs of mutants were also used to investigate the potential role of the interaction between the NSS and the HHCC domain in integrase aggregation. If aggregation was the result of a decrease in solubility accompanying a conformational change to the active form of the enzyme, then we would expect to reconstitute aggregation along with integration activity. On the other hand, if aggregation was due to serial propagation of protein-protein interactions involving the NSS and HHCC domain of integrase, then the active enzyme reconstituted by mixing an HHCC mutant with an NEM-modified integrase would not be expected to precipitate, since propagation of the pairwise subunit interactions would require the subunits to have both an intact NSS and an intact HHCC domain. As shown in Fig. 9B, neither defective subunit precipitated when the reconstituted enzyme was incubated with the divalent cation. Therefore, a conformational change resulting in a less soluble active state of the enzyme cannot simply account for the precipitation of wild-type integrase. These data support the oligomerization model illustrated in Fig. 5, but other models have not been excluded. We are currently conducting further experiments to test specific features of the model.

Role for the NEM-sensitive Feature and the HHCC Domain in Assembly of a Stable Integrase-DNA Complex

A divalent cation has been shown to be required for the formation of a functional, stable complex between integrase and viral DNA molecules in vitro(12) . After formation of this initial stable complex, integrase processively catalyzes the 3`-end processing and DNA joining steps of the reaction. Stable complex formation is also an initial step in the disintegration reaction. (^2)The requirement for a divalent cation in both the NSS-HHCC domain-mediated subunit interaction and stable complex assembly suggested that this subunit interaction might play a role in assembly of a stable integrase-viral DNA complex.

Since the HHCC mutant and NEM-modified integrase are active for disintegration, we tested their ability to form active stable complexes with a disintegration substrate. After a very brief pre-incubation of integrase with the disintegration substrate, heparin was added to block further binding of integrase to the substrate, and stable complexes formed during the preincubation were assayed for disintegration. Wild-type integrase formed a heparin-resistant complex with the disintegration substrate (Fig. 10A, lane4). Formation of this complex depended on preincubating integrase with the substrate (Fig. 10, compare panel A, lane 4, and panel D, lane 1) in the presence of a divalent cation (Fig. 10A, compare lanes3 and 4) before the addition of the heparin. Modification of integrase with NEM or deletion of the HHCC domain (Fig. 10, lane4 of panelsC and B, respectively) greatly impaired the stability of the complex formed between integrase and the disintegration substrate. Therefore, both the HHCC domain and the NEM-sensitive feature of integrase appear to be critical for the formation of a stable integrase-DNA complex.


Figure 10: Effect of NEM-modification or HHCC mutation on assembly of stable complexes between integrase and DNA substrates. Disintegration assays were performed with wild-type integrase (panelA), DeltaHHCC (panelB), and NEM-modified integrase (panelC). To detect stable complex formation during disintegration, integrase proteins were preincubated for 15 s at 37 °C with the disintegration substrate in the presence or absence of 5 mM MnCl(2). Heparin was then added to a final concentration of 5 µg/ml, and the divalent cation was added to the reactions from which it was omitted during the preincubation. The samples were then incubated for an additional 30 min. In panels A-C, standard disintegration assays were performed for 15 s (lane1) and 30 min (lane2). Integrase was preincubated with the disintegration substrate in the absence (lane3) or in the presence (lane4) of 5 mM MnCl(2). Heparin was then added (and 5 mM MnCl(2) was added to the reactions in lanes3), and the reactions were incubated an additional 30 min. Control reactions, panel D, in which heparin was added simultaneously with the disintegration substrate were also performed with wild-type integrase (lane1), H12A (lane2), DeltaHHCC (lane3), and NEM-modified integrase (lane4). In the lane labeled -, integrase was omitted from the reaction.




DISCUSSION

We have confirmed that the active form of HIV integrase is a multimer and that the NSS, HHCC domain, and DD35E motif, all critical for integration, can be provided by different subunits of the active multimer. Similar observations regarding the HHCC domain and DD35E region have been reported(29, 30) . Rather than defining a separate structural domain, the NSS, although it plays a distinct functional role, is most likely a feature of the protease-resistant core domain, which also contains the essential DD35E motif.

Our results suggest a model for the role of the NSS and HHCC domain in the integration pathway (Fig. 5). In this model, an NEM-sensitive site on one integrase protomer and an HHCC domain on another are required for assembly of an active multimeric form of integrase. This assembly is a prerequisite for formation of an initial stable complex between integrase and viral DNA. After assembly of the stable complex, integrase processively executes the 3`-end processing and DNA joining steps in the reaction(12) . Several predictions of the model have been confirmed: (i) integrase functions as a multimer within which the NSS and HHCC region are required in trans; (ii) alteration of either domain disrupts the inter-subunit interactions that lead to aggregation; (iii) the HHCC domain protects the NEM-sensitive site in trans; and (iv) defects in either domain impair the ability of integrase to form a stable, active complex with viral DNA substrates.

Like the HHCC-domain-dependent subunit interaction that confers protection against NEM, integrase aggregation required a divalent cation. The loss of either HHCC or NSS function abolished aggregation, indicating that a protein-protein interaction involving these two domains is a prerequisite for integrase aggregation. Moreover, unlike integration, extensive aggregation was not reconstituted by mixing HHCC mutant integrase with NEM-treated integrase, suggesting that propagation of the HHCC-NSS-dependent interaction is necessary for extensive aggregation of the enzyme. Direct physical measurements indicate that in the absence of a divalent cation, integrase is predominantly a dimer. The subunit interaction that depends upon the HHCC domain and the NEM-sensitive site is not required for dimerization since (i) it requires a divalent cation and (ii) the HHCC mutant proteins retain the ability to dimerize. (^3)This interaction therefore presumably promotes higher order multimerization of the constitutive integrase dimers. Thus, we propose that the active form of integrase contains a minimum of four protomers. We are investigating this aspect of the model.

Mutations in the HHCC-domain appear to render integrase defective in exploiting binding interactions with the viral DNA substrate, except for those at the very distal end of the DNA(21, 24, 28) . This defect makes the disintegration activity of HHCC-defective mutant integrases hyperdependent upon the remaining interactions with the terminal bases of the viral DNA end, and with the target DNA portion of the disintegration substrate. Thus, while HHCC mutant enzymes are indifferent to truncation of the viral DNA portion of the substrate, they cannot tolerate truncation or elimination of the target DNA portion or substitutions for the conserved CA dinucleotide at the 3`-end of the viral DNA component(21, 24, 28) . This evidence that the HHCC domain contributes to interactions between integrase and subterminal sites on the viral DNA end suggests that the HHCC domain-dependent multimerization may serve to extend the enzyme's binding sites for viral DNA inward from the tip of the viral DNA molecule.

Formation of a stable integrase-viral DNA complex is a critical step in the integration reaction (12) that precedes the 3`-end processing and DNA joining steps. The HHCC mutant and NEM-modified form of integrase have properties compatible with a primary defect in stable complex assembly. All of the HHCC mutants characterized thus far, as well as NEM-modified integrase, are capable of remaining bound to the processed viral DNA end released by disintegration, and can subsequently catalyze the reintegration of that DNA molecule, albeit at a reduced level. Thus, these defective enzymes retain the ability to form a functional complex with viral DNA by an apparently distinct alternative mechanism. The existence of alternative routes to stable complex assembly, and the distinct requirements for assembly and function of an enzyme-DNA complex has parallels in the bacteriophage Mu transposition reaction (31) .

A higher order protein-DNA complex is also an essential intermediate in the recombination reaction catalyzed by resolvase(32) . The formation of the resolvosome, a stable complex in which res DNA is wrapped around a tetramer of resolvase, is facilitated by cooperative interactions between resolvase dimers bound to distant res sites. This interaction between the dimeric units of resolvase is required for recombination, since resolvase mutants that are defective for interdimer interactions are incapable of forming the correct higher order protein-DNA architecture necessary for recombination. Thus, such mutants are incompetent for recombination. Similarly, specific interactions between HIV integrase protomers appear to be required for the assembly of a stable complex with viral DNA, and this process is perturbed by mutations in the HHCC domain.

Although the zinc binding domain(s) of numerous proteins have been shown to be important for interactions with nucleic acids(33) , several have also been reported to play a role in protein-protein interactions. The single zinc finger motif in the SV40 large T antigen(34) , the last three finger motifs of transcription factor IIIA(35) , and the zinc binding domain of the regulatory chain of aspartate transcarbamoylase (36) , have either been implicated in or directly shown to mediate protein-protein interactions critical for the assembly of an active stable protein complex. The HHCC domain of HIV-1 integrase, which has been demonstrated to bind zinc (24) and is predicted to form a structure similar to the prototypical zinc finger of TFIIIA(23) , appears to perform an analogous role in HIV integrase.

The assembly of a specialized nucleoprotein complex is a critical step in many processes involving enzymatic manipulation of nucleic acids (37) . HIV integration in vivo(38, 39, 40) and in vitro(12) , proceeds through a stable complex between integrase and viral DNA. Future analyses of the protein and DNA requirements for assembly of this key intermediate should provide valuable insights into the mechanism and regulation of retroviral integration.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI27205 (to P. O. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Genzyme Corp., Framington, MA 01701.

Assistant investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 415-723-0039; Fax: 415-723-1399.

(^1)
The abbreviations used are: HIV, human immunodeficiency virus; DTT, dithiothreitol; NEM, N-ethylmaleimide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; NSS, NEM-sensitive site.

(^2)
V. Ellison and P. O. Brown, unpublished results.

(^3)
V. Ellison, J. Gerton, and P. O. Brown, unpublished observations.


ACKNOWLEDGEMENTS

We thank all members of the Brown laboratory, Harold Varmus, Andrew Leavitt, and Karl Reich for stimulating and enlightening discussions; Andrew Leavitt, Harold Varmus, Allan Engelman, and Robert Craigie for providing integrase mutants; and Allan Engelman and Robert Craigie for communicating results prior to publication.


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