Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

Replication of retroviruses requires integration of the linearviral DNA genome into the host chromosomes. Integration requiresthe viral integrase (IN), located in high-molecular-weight nucleoproteincomplexes termed preintegration complexes (PIC). The PIC insertsthe two viral DNA termini in a concerted manner into chromosomesin vivo as well as exogenous target DNA in vitro. We reconstitutednucleoprotein complexes capable of efficient concerted (full-site)integration using recombinant wild-type human immunodeficiencyvirus type I (HIV-1) IN with linear retrovirus-like donor DNA(480 bp). In addition, no cellular or viral protein cofactorsare necessary for purified bacterial recombinant HIV-1 IN tomediate efficient full-site integration of two donor terminiinto supercoiled target DNA. At $~$ 30 nM IN (20 min at 37°C),approximately 15 and 8% of the input donor is incorporated intotarget DNA, producing half-site (insertion of one viral DNAend per target) and full-site integration products, respectively.Sequencing the donor-target junctions of full-site recombinantsconfirms that 5-bp host site duplications have occurred witha fidelity of $~$ 70%, similar to the fidelity when using IN derivedfrom nonionic detergent lysates of HIV-1 virions. A key factorallowing recombinant wild-type HIV-1 IN to mediate full-siteintegration appears to be the avoidance of high IN concentrationsin its purification ( $~$ 125 µg/ml) and in the integrationassay (<50 nM). The results show that recombinant HIV-1 INmay not be significantly defective for full-site integration.The findings further suggest that a high concentration or possiblyaggregation of IN is detrimental to the assembly of correctnucleoprotein complexes for full-site integration.

INTRODUCTION

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Introduction

Integration of the retrovirus DNA genome into cellular chromosomesrequires the viral integrase (IN). In newly virus-infected cells,IN is localized in cytoplasmic nucleoprotein complexes termedpreintegration complexes (PIC) (2-4, 7). In the PIC, IN removestwo nucleotides from the 3' OH termini of linear blunt-endedviral DNA ( $~$ 10 kbp), producing recessed termini. Purified PICare capable of inserting their recessed DNA termini in a concertedfashion into exogenous target DNA, mimicking integration invivo (13, 14, 27, 29, 35, 42). The fidelity of the small hostduplications occurring upon concerted integration in vivo isalso observed with the PIC in vitro.

Early reconstitution experiments using purified IN, linear retrovirus-likeDNA donors, and target DNA were successful but inefficient forconcerted (full-site) integration (10, 15, 25). The efficiencyof the full-site integration reactions has been significantlyincreased by employing purified avian myeloblastosis virus (AMV)IN (32, 39-41), IN in nonionic detergent lysates of human immunodeficiencyvirus type 1 (HIV-1) virions (6, 17, 18), and recombinant INsof various retroviruses (1, 19, 21, 22, 33). The donor-targetproducts are visualized by agarose gel electrophoresis and characterizedby restriction enzyme analysis with radioactively labeled donors( $~$ 300 to 500 bp in length) (Fig. 1). Sequencing the donor-targetjunctions of individual recombinants verifies the fidelity ofthe small host duplications observed upon full-site integrationin vivo.

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FIG. 1. Schematic for nucleoprotein complex assembly and the half-site and full-site integration reactions. A radioactive 5'-end-labeled linear DNA donor (0.48 kbp) containing 3' OH recessed U5 and U3 LTR termini is assembled with IN on ice prior to the addition of supercoiled target DNA (2.8 kbp) (40). Half-site integration involves the insertion of one LTR end per target, and full-site integration involves the concerted insertion of two LTR ends per target. Restriction enzyme analysis resolves donor-target products.

The efficiency and fidelity of full-site integration observedwith the various reconstitution systems vary significantly.This variance could be due to either the differences betweenthe specific activities of virion and recombinant IN, the requirementof auxiliary viral or cellular proteins to mediate full-siteintegration, or both. AMV (39-41) and recombinant Rous sarcomavirus (RSV) PragueA (PrA) INs (33, 41) can efficiently mediatethe concerted insertion of 3' OH recessed long terminal repeat(LTR) termini from two linear donors into a circular target(bimolecular reaction) (Fig. 1) with high fidelity ( $~$ 95%) forhost site duplications. Efficient full-site integration is arbitrarilydefined as having $~$ 10% of the donor incorporated into a targetin 20 min at 37°C. The specific activities of AMV and recombinantRSV PrA INs for assembly and full-site integration appear equivalent(33). Recombinant simian immunodeficiency virus (SIV) IN canmediate efficient full-site integration possessing reasonablefidelity ( $~$ 84%) for the host site duplications (19). Recombinantmurine leukemia virus IN can mediate bimolecular full-site integrationwith high fidelity but with little efficiency (38). RecombinantRSV Schmitt-RuppinB IN performs full-site integration with highfidelity for the host site duplications by using blunt-endedtermini of a single linear donor ( $~$ 300 bp; unimolecular reaction)into a DNA target (1, 21, 22). But cellular proteins HMG 2 (high-mobilitygroup) and HMG-I(Y) are required to mediate efficient full-siteintegration. Recombinant HIV-1 IN also requires the viral nucleocapsid(NC) (6) or HMG-I(Y) (22) to perform efficient full-site integrationin vitro. Using similar-size LTR donor substrates describedin this report, recombinant HIV-1 IN by itself either failsto perform full-site integration (8, 28) or incorporates <1%of the donor into full-site products in 1- or 2-h reactionsat 37°C (6, 22). The fidelity of either recombinant (6,22) or virion (6, 18) HIV-1 IN for producing 5-bp host siteduplications upon full-site integration is $~$ 70% in vitro. Minorprotein-folding defects in recombinant HIV-1 IN or the aggregationof purified IN may prevent the correct assembly of complexeswith two viral DNA ends, which is necessary for full-site integration(9).

To investigate these two possible defects, we utilized differentpathways to determine whether recombinant wild-type (wt) HIV-1IN possesses the ability to mediate efficient full-site integrationwithout the addition of viral or cellular cofactors. To addressthe aggregation problem, we purified recombinant HIV-1 IN atlow protein concentrations (<125 µg/ml) and kept theconcentration of IN in the low nanomolar range (<50 nM) inthe reaction mixtures. The purification procedure involved lysisof bacteria without nonionic detergent, low-salt washes of "insoluble"IN-DNA pellet fractions followed by high-salt extraction ofIN, and further purification by column chromatography (33).In the second approach, we examined the effect of coexpressingprotein chaperones (GroEL and GroES) (16, 23, 30) with HIV-1IN in bacteria to investigate if possible protein-folding defectsin IN are corrected. With both approaches, highly purified recombinantHIV-1 IN was capable of efficiently performing both the half-siteand full-site integration reactions nearly equivalently withoutthe addition of purified cellular or viral proteins. The apparentkey factor for allowing recombinant HIV-1 IN to mediate full-siteintegration is the avoidance of high IN concentrations in itspurification and in the integration assay.

MATERIALS AND METHODS

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Materials and Methods

DNAs. The linear 480-bp LTR donor fragments were released by NdeIdigestion of plasmids and purified on agarose gels. The donorfragments contained 20 bp of wt U5 and wt U3 HIV-1 LTR sequencesat the termini (H-2) or only wt U5 sequences at the termini(H-5) (17-19). An additional donor containing 30 bp of wt U5and wt U3 sequences (P-2) was also used. The DNA donors containthe SupF amber gene for genetic selection of donor-target recombinantsusing MC1061/P3 cells (Invitrogen) (1, 22, 40). The LTR donorsalso contain a unique BglII site approximately 50 bp from theU3 terminus. The donors were 5' end labeled with [ ${gamma}$ -³²P]ATP andT4 polynucleotide kinase. The plasmid (pGroESL) expressing proteinchaperones GroEL and GroES (16) was kindly provided by A. A.Gatenby. The supercoiled target DNA was pGEM3.

Recombinant HIV-1 IN. Expression vector pET3a (Stratagene) containing the HIV-1 INcoding region (pNY clone) was provided by F. Bushman. The INcoding region was removed from pET3a by NdeI and BamHI digestionsand cloned into pET11a. The entire wt HIV-1 IN clone was sequencedto verify the construct. The pET11a-IN vector was transformedinto BL21(DE3) cells. Competent BL21(DE3) cells containing pET11a-INwere also cotransformed with pGroESL, and the cells were platedonto agarose plates containing carbenicillin (50 µg/ml)and chloramphenicol (34 µg/ml). Both plasmids replicatein the same cell because they possess different origins of replication.The presence of the two plasmids in individual colonies wasverified by restriction enzyme analysis and expression of bothsets of proteins. Individual colonies were grown in Luria-Bertanimedia with both antibiotics to an optical density at 600 nm(OD₆₀₀) of 0.8 and stored in glycerol at -70°C.

Purification of HIV-1 IN. Bacterial cells expressing HIV-1 IN, without and with coexpressionof pGroESL, were induced by the addition of IPTG (isopropyl-ß-D-thiogalactopyranoside;0.4 mM) at an OD₆₀₀ of 0.6 and harvested after 3 h by centrifugation.Purification of HIV-1 IN was very similar to procedures usedto purify recombinant RSV PrA IN (33). Briefly, HIV-1 IN wasextracted from cells (2 g wet weight) thawed on ice. After alight sonication in 4 ml of extraction buffer per g, the homogenizedmaterials were subjected to high-speed centrifugation and thepellets were suspended in 4 ml of a 0.1 M NaCl buffer. Aftera series of low-salt (0.1 M NaCl) washes and centrifugationsteps, IN was extracted from the pellet with 2 ml of a 1 M NaClbuffer per g wet weight of bacteria. The high-salt extract containedthe majority of IN at a reasonable purity (>60%) (see Fig.2, lane 8). Four-milliliter preparations of HIV-1 IN were loadedunto Pharmacia SP-Sepharose HiTrap (5-ml) columns for furtherpurification and removal of nucleic acids (33). The major nucleicacid peak preceding the elution of IN at 0.38 M NaCl was avoided;the protein peak (OD₂₈₀) representing the majority of IN wasloaded onto a second SP-Sepharose column. This procedure usuallyresulted in preparations with >90% purity. Heparin affinity(37) HP HiTrap (5-ml) columns were used to purify IN to nearhomogeneity. HIV-1 IN eluted from heparin-Sepharose columnsat 0.68 M NaCl (see Fig. 3). The standard chromatography bufferused for both columns was identical to that previously described(33) (50 mM HEPES-NaOH [pH 7.5], 1 mM dithiothreitol, 1 mM EDTA,10 mM MgSO₄) unless otherwise indicated. Linear gradients withvarious concentrations of NaCl in the above buffer were usedto elute IN from both columns. Fractions ( $~$ 0.5 ml) containingIN were divided into aliquots and frozen at -70°C for storage.Protein concentrations were determined by a filter paper techniqueusing Coomassie blue staining for submicrogram quantities (34)with bovine serum albumin as the reference standard.

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FIG. 2. Coomassie blue-stained SDS-PAGE gel showing HIV-1 IN coexpressed with protein chaperones at various steps of induction and purification. Lane 1, molecular weight standards from 15 to 190 kDa; lane 2, total-cell lysate uninduced; lane 3, total-cell lysate induced for 3 h; lane 4, supernatant after sonication and centrifugation of lysate; lane 5, supernatant of the second lysate after sonication of the IN-DNA pellet and centrifugation; lane 6, supernatant of the first wash of the pellet; lane 7, supernatant of the second wash of the pellet; lane 8, 1 M NaCl extraction; lane 9, purified GroEL. For lanes 4 to 7, 4 ml of buffer per g wet weight of bacteria was used; for lane 8, the 1 M NaCl extraction buffer volume was 2 ml per g wet weight of bacteria. A constant volume (10 µl) of extract was applied in lanes 4 to 8. The sizes of several molecular mass markers (in kilodaltons) are indicated on the left. Purified GroEL and IN are indicated on the right.

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FIG. 3. Purification of recombinant HIV-1 IN. (A) HIV-1 IN was purified by SP-Sepharose followed by heparin-Sepharose. Fractions 37 to 49 (heparin column) were analyzed for protein and strand transfer activities. The protein concentrations in fractions 40 to 44 are 48, 66, 53, 52, and 27 µg per ml, respectively (right). For fraction 41, the calculated concentration of IN is 1,030 nM. The percentages of donor incorporated into half-site and full-site strand transfer products are indicated (left; see panel B). (B) Aliquots (1 µl) of each indicated fraction shown in panel A (fractions 37 to 49 are represented in lanes 4 to 16, respectively) were preincubated with wt donor DNA (P-2) at 0°C for 12 min in 50-µl reaction volumes prior to strand transfer for 20 min at 37°C. The NaCl concentration varied from 0.63 to 0.82 M in fractions 40 to 44. Left, half-site, full-site, and donor-to-donor strand transfer products, along with the input donor. DNA products migrating slower than the half-site products contain two or more LTR donors inserted at different locations on the target DNA (40). Lanes 1 to 3, control reaction containing no IN, SIV IN at 100 nM, and molecular weight markers, respectively. The markers range in size from 0.5 to $~$ 10 kbp (Promega; 1-kbp DNA ladder set) (black dot, 4 kbp). (C) Aliquots (10 µl) of the indicated heparin-Sepharose fractions (lanes 1 to 9, fractions 38 to 46, respectively) were subjected to SDS-PAGE and stained with Coomassie blue.

Integration assay and analysis of full-site donor-target integration products. Standard integration assay conditions were previously described(18) except for several minor modifications. The MgCl₂, NaCl,and polyethylene glycol concentrations were changed to 15 mM,100 mM, and 10%, respectively. Zinc chloride was added to 25µM. The donor DNA concentration was decreased from 60to 15 ng per 20 µl. The reaction volumes were varied between20 and 100 µl to control the concentration of IN in thereaction mixture. The concentrations of all reactants and bufferswere held constant. In addition, the concentration of IN wasvaried while maintaining constant reaction volumes. Preincubationof IN with the donor was on ice for 12 min prior to the additionof the supercoiled target (100 ng of pGEM3 per 20 µl).The ratio of donor to target molecules was one. The strand transferreactions were carried out for 20 min at 37°C. Generally,50% of the reaction volumes was subjected to agarose gel electrophoresis.Scale-up reactions were performed to isolate sufficient quantitiesof donor-target recombinants for BglII digestion, for isolationof the 3.4-kbp full-site products (see Fig. 4; U5-target-U3),for genetic isolation of recombinants after transformation ofthe ligated 3.4-kbp product into MC1061/P3 cells, and for thesequencing of the donor-target junctions of individually isolatedrecombinants (see Table 1) (18, 40).

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FIG. 4. BglII restriction analysis of donor-target products produced by different preparations of HIV-1 IN. The strand transfer products produced by three preparations of HIV-1 IN were subjected to BglII digestion. Lanes 3 and 4, IN coexpressed with protein chaperones and purified through two SP-Sepharose columns; lanes 5 to 8, IN purified identically through heparin-Sepharose that was coexpressed with (lanes 5 and 6) or without (lanes 7 and 8) chaperones. All of the HIV-1 IN preparations were tested at 25 nM in the reaction mixtures. SIV IN concentrations were 25 (lanes 1 and 2) and 50 nM (lanes 10 and 11). The wt donor DNA (P-2; diagram on right) was used. The integration products were divided into two equal samples and were not (-) or were (+) digested by BglII. Left, half-site, full-site, and donor-to-donor products; right, full-site U5-target-U5, U5-target-U3, and U3-target-U3 products produced by BglII digestion. Equivalent quantities of each set were used for comparison purposes. Only the major U5-target half-site product (circle) is apparent on this gel because of the low activity of the U3 LTR end. Lane 9, DNA molecular mass markers as described for Fig. 3.

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TABLE 1. Sequence analysis of donor-target junction sites produced by HIV-1 IN^a

RESULTS

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Results

Expression of HIV-1 IN in bacteria. Recombinant HIV-1 IN was expressed in BL21(DE3) cells withoutand with coexpression of protein chaperones. Briefly, variousquantities of bacterial pellets (0.5 to 7 g wet weight) wereprocessed. The ratios of wet weight bacteria to lysis buffer(4 ml/g) and extraction buffer (2 ml/g) were held constant,which resulted in no apparent differences in either the relativeyield or purity of IN with the different-size bacterial preparations(Fig. 2). The expression levels and purity of IN coexpressedwith the chaperones and the subsequent lysis, wash, and extractionsteps (Fig. 2) were very similar to those for the IN expressedwithout the chaperones (data not shown). The 1 M NaCl extractionof IN from the IN-DNA pellet generally resulted with IN in the60 to 70% purity range (Fig. 2, lane 8). IN appears to be stablein the 1 M NaCl extracts at -70°C for at least 6 months.

Purification of HIV-1 IN and strand transfer analysis. A similarity between the purification procedures used in laboratoriesfor recombinant wt HIV-1 IN is the use of multiple grams wetweight of bacteria to purify milligram quantities of IN. Wewanted to avoid nonspecific interactions among recombinant HIV-1INs which result in aggregation of IN in solution at high proteinconcentrations (4, 24). We also observed that, upon the useof large amounts of initial 1 M NaCl bacterial extracts containingIN (>3 g [wet weight] of bacteria) (Fig. 2, lane 8) for SP-Sepharosecolumn (5 ml) chromatography, precipitation occurs in the fractionscontaining the majority of IN due to either nonspecific protein-proteinor protein-DNA interactions or both.

To avoid the precipitation of IN with our column purificationprocedures, we generally used the entire 1 M NaCl extract ofIN derived from $~$ 2 gm or less wet weight of bacteria for theinitial SP-Sepharose column step. The column fractions containingthe majority of IN (observed by measuring OD₂₈₀ and performingsodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE])were pooled and purified further by either an additional passageover another SP-Sepharose column (data not shown) (see Fig.4, lanes 3 and 4 for IN usage) or by heparin-Sepharose chromatography(see Fig. 3) prior to strand transfer analyses. The last chromatographysteps were necessary for further purification of IN, removalof contaminating DNA, and elimination of bacterial DNA endonucleases.

HIV-1 IN expressed in the absence of protein chaperones waspurified on a heparin-Sepharose column with the standard chromatographybuffer (Fig. 3). Strand transfer analysis demonstrated thatHIV-1 IN was capable of producing nearly equivalent amountsof half-site and full-site integration products using the P-2LTR donor and supercoiled DNA as the target (Fig. 3A and B).PhosphorImager analysis shows that $~$ 4 to 9% of the donor wasinserted into these products in the peak fractions (fractions41 to 43) (Fig. 3A). SDS-PAGE shows that IN is nearly homogeneous(Fig. 3C). With fractions 41 to 43, the concentrations of INin the reaction mixtures were calculated to be $~$ 20, 16, and 16nM, respectively. A significant amount of donor was also insertedby IN into other donor molecules ( $~$ 20% for donor-to-donor insertion)with these same fractions (Fig. 3B). In comparison, purifiedSIV IN (Fig. 3B, lane 2) at 100 nM incorporates approximately11 and 23% of the donor into half-site and full-site products,respectively, with supercoiled DNA as the target (19).

In summary, recombinant HIV-1 IN can be purified at low proteinconcentrations while remaining highly active and possessingthe ability to perform full-site integration without the additionof viral or cellular cofactors.

Independence of adding either Zn²⁺ or Mg²⁺ to buffers for purifying highly active HIV-1 IN capable of full-site integration. It was previously shown that Zn²⁺, Mg²⁺, and nonionic detergentsin the purification buffers differentially affected the interactionsbetween recombinant HIV-1 IN subunits at low concentrations(45 to 200 nM), subsequently affecting their half-site integrationproperties (11, 28). We tested whether different solution conditionsduring purification affected the stability and the ability ofrecombinant HIV-1 IN to perform strand transfer. Our standardchromatography buffer contains 50 mM HEPES, 1 mM dithiothreitol,1 mM EDTA, and 10 mM MgSO₄ with no nonionic detergents present(33). Recombinant HIV-1 IN was passed over two SP-Sepharosecolumns without MgSO₄ but with 10% glycerol in the final columnbuffer. SDS-PAGE analysis also shows that IN was purified tonear homogeneity (data not shown). The SP-Sepharose-purifiedenzyme (118 µg/ml) (Fig. 4, lanes 3 and 4) was stablefor over a year and, in reaction mixtures with two differentpreparations of HIV-1 IN purified through heparin-Sepharose,had strand transfer activities at 25 nM that were comparable(Fig. 4, lanes 5 to 8). These two preparations of IN were purifiedwith the standard chromatography buffer. IN was coexpressedwith chaperones (Fig. 4, lanes 5 and 6) or without them (lanes7 and 8). The concentrations of the stored enzymes ( $~$ 3 monthseach) were 66 and 101 µg/ml, respectively. The strandtransfer activities of the purified IN preparations were alsostable over several freeze-thaw cycles.

In summary, the stability of recombinant HIV-1 IN and the abilityof IN to efficiently perform half-site and full-site integrationat low concentrations appear independent of having added Zn²⁺,Mg²⁺, or glycerol in the purification buffers.

Fidelity of the full-site integration products for 5-bp host site duplications. DNA restriction enzyme analysis of the donor-target productsproduced by HIV-1 IN shows that the U5 terminus is used in preferenceto the U3 terminus for both the half-site and full-site integrationproducts (Fig. 4, lanes 3 to 8) (4, 6, 18, 31). With the P-2LTR donor as the substrate, the donor-target integration productsproduced by the three different purified HIV-1 IN preparationsgave the predicted BglII enzyme products (Fig. 4) (18). Digestionof integration products by BglII produced by all three preparationsof HIV-1 IN using the LTR donor (H-5) containing two U5 endsalso gave the predicted pattern (data not shown) (17, 18). SIVIN at 25 (Fig. 4, lanes 1 and 2) and 50 nM (Fig. 4, lanes 10and 11) is shown for comparison.

The fidelity of virion (6, 17, 18) and recombinant HIV-1 INwith cellular or viral protein cofactors (6, 22) for producingthe 5-bp host site duplications is $~$ 70%. We wanted to determineif the fidelity of the 5-bp host site duplications was increasedif HIV-1 IN was expressed in the presence of protein chaperones(Fig. 4, lane 3 and 4). Table 1 demonstrates the results oftwo independent experiments involving the sequencing of recombinantsproduced by HIV-1 IN coexpressed with chaperones. The linearLTR donor (H-5) containing two U5 ends was used for these sequencingexperiments (17, 18). The fidelity for producing the 5-bp hostsite duplications was not increased for HIV-1 IN in comparisonto results obtained with HIV-1 virions (6, 17) or recombinantHIV-1 IN with either cellular or viral cofactors (6, 22). Sequenceanalysis of the host site duplications produced by HIV-1 INnot expressed with chaperones was not performed.

In summary, the results show that HIV-1 IN under appropriatepurification and assay conditions has the capacity to efficientlyproduce the correct full-site integration products without theaddition of cellular or viral cofactors. The results also showthat coexpression with chaperones does not improve the fidelityof HIV-1 IN for producing 5-bp host site duplications.

Specific activities of the different HIV-1 IN preparations for full-site integration. We wanted to quantitatively determine if HIV-1 INs expressedwith and without protein chaperones have similar specific activitiesfor half-site and full-site integration. Various concentrationsof both IN preparations (5 to 50 nM) were assembled with theP-2 LTR donor at 0°C and assayed for strand transfer activities(Fig. 5). The quantities of the half-site and full-site productsproduced by both IN preparations appear to be similar with respectto protein concentrations. At 30 nM IN, the calculated IN dimer-to-donorend molar ratio is 7 to 1. With both preparations of HIV-1 INat $~$ 30 nM, levels of incorporation of donor into both productswere $~$ 12 and 7% for half-site and full-site products, respectively(Fig. 5, lanes 7 and 12). With SIV IN at 25 nM, levels of incorporationfor the half-site and full-site products are 3 and 9%, respectively(Fig. 5, lane 2). Generally, under optimum strand transfer conditionsfor HIV-1 IN, the total amount of input donor used for all strandtransfer products is $~$ 25 to 30% in 20 min, suggesting that alarge percentage of IN subunits are enzymatically active (Fig.5; data not shown). The lack of significant strand transferactivities (Fig. 5, lanes 3 and 9) below $~$ 8 nM IN in the reactionmixtures is a reproducible result suggesting that a criticalconcentration of IN subunits is needed for initiation of strandtransfer events. Further quantitative analyses of three independenttitration experiments using both IN preparations suggest thatIN expressed with chaperones is only slightly better than INnot expressed with chaperones for full-site rather than half-siteintegration (data not shown).

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FIG. 5. Quantitative comparison of strand transfer specific activities of HIV-1 IN expressed without and with protein chaperones. HIV-1 INs purified as described for Fig. 3 (heparin-Sepharose, -chaperones; SP-Sepharose, +chaperones) were simultaneously analyzed for strand transfer efficiencies. The wt donor DNA (P-2) was used. The reaction volume was 100 µl, and one-half of each sample was subjected to 1.5% agarose gel electrophoresis. Equivalent radioactive counts were loaded per lane. Lanes 1 and 2, no IN and SIV IN (25 nM), respectively; lanes 3 to 7, HIV-1 IN expressed without chaperones at 5.2, 10.4, 15.6, 20.8, and 31.2 nM IN, respectively; lane 8, molecular mass markers as described for Fig. 3; lanes 9 to 13, IN coexpressed with chaperones at 8.7, 17.4, 26.1, 34.8, and 52.2 nM, respectively. The wet gel was dried, exposed to X-ray film (6 h without an intensifying screen), and analyzed by a PhosphorImager. The half-site, full-site, and donor-to-donor products and the input donor are indicated on the left.

We investigated the effect of Zn²⁺ on recombinant HIV-1 IN full-siteintegration, which had previously been shown to affect the structuraland catalytic functions of IN (5, 26, 28, 43). Zn²⁺ promotesthe multimerization of IN subunits and slightly stimulates theMg²⁺-dependent 3' OH processing and half-site strand transferreactions. Figure 6 shows that Zn²⁺ also stimulates the half-siteand full-site integration reactions of HIV-1 IN (lanes 3 to6) in comparison to reactions performed without Zn²⁺ (lanes10 to 13). At 34 nM IN with Zn²⁺ (Fig. 6, lane 6), the percentagesof donor incorporated into half-site and full-site productswere 15 and 8%, respectively. Further quantitative analysisshows that Zn²⁺ stimulates both reactions from $~$ 20 to 40%, independentof whether IN was expressed with (compare lanes 5 and 6 to lanes12 and 13) or without (compare lanes 3 and 4 to lanes 10 and11) protein chaperones. SIV IN strand transfer activities wereonly slightly stimulated (Fig. 6, compare lanes 2 to 9). Theresults show that Zn²⁺ also enhances the ability of HIV-1 INto perform full-site integration as well as other strand transferactivities.

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FIG. 6. Effect of Zn²⁺ on recombinant HIV-1 IN full-site integration. HIV-1 INs coexpressed without protein chaperones (-ESL; purified by heparin-Sepharose) and with chaperones (+ESL; purified by SP-Sepharose) were tested for Zn²⁺ activation. The wt donor DNA (P-2) was used. Lanes 1 to 6, 25 µM Zn²⁺; lanes 8 to 13, no Zn²⁺. Control lanes 1 and 8, no IN; lanes 2 and 9, SIV IN at 25 nM; lanes 3 and 10 and 4 and 11, -ESL IN at 11 (lanes 3 and 10) and 22 nM (lanes 4 and 11); lanes 5 and 12 and 6 and 13, +ESL IN at 17 (lanes 5 and 12) and 34 nM (lanes 6 and 13). The half-site, full-site, and donor-to-donor products as well as the input donor are indicated on the left. The molecular mass markers (lane 7) are described in the legend for Fig. 3.

We also determined that the optimum NaCl concentration for assemblyand full-site integration by recombinant HIV-1 IN was $~$ 150 mM(data not shown). The titration experiments showed that recombinantHIV-1 IN at 32 nM displayed nearly equal responses to NaCl between110 and 170 mM for both half-site and full-site integrationactivities. Below $~$ 80 mM NaCl, only a minor population of full-siteintegration products are produced. In comparison, $~$ 40 mM NaClwas observed to be optimum for half-site and full-site integrationwith nonionic detergent HIV-1 lysates (6, 18) or recombinantHIV-1 IN (6, 22). In addition, results from expression and purificationof recombinant HIV-1 IN (strain HXB2) in bacteria showed thatHIV-1 IN derived from another wt HIV-1 IN gene possesses verysimilar specific activities for half-site and full-site integration(data not shown).

In summary, the results suggest that HIV-1 IN possesses similarspecific activities for performing full-site integration whenpurified at low protein concentrations with different buffersystems. The specific activities of HIV-1 IN preparations appearequivalent to those for SIV IN with respect to the quantityof full-site integration product produced. However, significantquantities of half-site integration products are produced bythe various HIV-1 IN preparations in comparison to what is foundfor SIV IN, suggesting that a majority of the HIV-1 IN subunitsare still not assembling correctly on the viral DNA ends toperform full-site integration (see the model in Fig. 7).

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FIG. 7. Model depicting the interactions of recombinant HIV-1 IN subunits in solution. White circles, IN monomers that interact to form dimers. This population of IN subunits possibly forms the majority of IN in recombinant HIV-1 IN preparations. The majority of these subunits can perform half-site integration but not full-site integration due to their inability to assemble two viral DNA ends correctly. Gray circles, IN monomers possessing the ability to form dimers and multimers which can correctly assemble on two viral DNA ends allowing full-site integration.

DISCUSSION

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Discussion

Recombinant wt HIV-1 IN purified from bacteria has the capacityto promote the half-site and full-site integration reactionsnearly equivalently. The efficient concerted insertion of twoLTR DNA ends into a circular target by recombinant HIV-1 INwithout the addition of viral or cellular protein cofactorssuggests that IN is not significantly defective for promotingfull-site integration. Further improvement in the assembly ofnucleoprotein complexes capable of full-site integration maybe related to the state of aggregation of HIV-1 IN, as suggestedby the avoidance of high IN concentrations during both its purificationand its use in the integration assay. Recombinant HIV-1 IN appearsnot to possess major protein-folding defects, as shown by ourprotein chaperone coexpression studies. Our reconstitution studiesdo not exclude the possibility that other proteins assist INwith integration in vivo.

With LTR donor substrates similar in size to those describedin this report, recombinant HIV-1 IN either failed to performfull-site integration (8, 28) or incorporated <1% of thedonor DNA into full-site integration products after 1- (6) or2-h (22) incubations at 37°C. We demonstrated that recombinantHIV-1 IN at similar protein concentrations ( $~$ 30 nM) is capableof incorporating 8 to 10% of the donor substrate into targetDNA, producing full-site integration products in 20 min. Thefidelity of the 5-bp host site duplication for full-site integrationcatalyzed by recombinant HIV-1 IN is comparable to that observedwith nonionic detergent lysates of HIV-1 virions (6, 17) orrecombinant HIV-1 IN enhanced by cellular or viral cofactors(6, 22). The reported amounts of half-site integration productsproduced by recombinant HIV-1 IN also vary significantly from $~$ 2 to 20% (6, 8, 22, 28). The total amounts of circular half-siteand donor-to-donor integration products produced by recombinantHIV-1 IN under optimum conditions are $~$ 25% (Fig. 5 and 6). Asdiscussed later, the majority of IN subunits are apparentlyhighly capable of performing half-site integration but onlya minority are capable of full-site integration (Fig. 7, model).Our report suggests that the concentrations of IN during purificationand in the reaction mixture play an important role in the assemblyof IN-DNA complexes favorable to full-site integration. It ispossible that nonspecific aggregation of IN results in incorrectnucleoprotein complexes for full-site integration (9).

What are the factors that influence the ability of IN to assembleon two viral DNA ends allowing the nucleoprotein complexes toperform full-site integration? Detailed biochemical analysesof recombinant HIV-1 IN (45 to 200 nM) derived from differentpurified preparations and in different solutions required forstrand transfer activities have given critical insights intofactors affecting the multimeric state of recombinant IN (11,28). Nonionic detergents appear to promote dissociation of INsubunits to monomers, while Zn²⁺ promotes multimerization todimers and tetramers (11, 28), protein states required for strandtransfer activities (4, 12, 24, 26, 36, 43). The minimum INstructure for 3' OH processing and half-site integration isa dimer (4), with the predicted minimum structure of one totwo tetramers for full-site integration (5, 9, 20, 39).

Several factors appear to impact the ability of recombinantwt HIV-1 IN in this report to perform efficient full-site integration.The concentration of HIV-1 IN upon purification was kept inthe 125 µg/ml or less range to prevent apparent aggregationof IN in solution. Our purification procedure (33) also lacksnonionic detergents, possibly allowing dimers and tetramersto be formed at low nanomolar concentrations (11, 28). Divalentmetal ion Mg²⁺ can be removed during the second SP-Sepharosechromatography step for HIV-1 IN, and it still remains fullyactive, even when stored in the presence of 1 mM EDTA (Fig.4, lane 3; Fig. 5, lanes 9 to 13). The lack of added Zn²⁺ duringpurification does not appear to be critical for IN stabilityalthough it enhances strand transfer activities (Fig. 6) (5,26, 28, 43). The multimeric state of purified IN in this reportis unknown. However, a minimum concentration of IN ( $~$ 8 nM) inthe reaction mixture (Fig. 5, lanes 3 and 9) is required fordetectable strand transfer activities, suggesting that multimerizationof IN on DNA ends is required for all strand transfer events.The actual concentrations of IN in our reaction mixtures areunknown because of the molecular crowding effects of polyethyleneglycol (10%), which significantly enhances full-site integrationactivity in vitro (3, 6, 40). In summary, our data suggest thatrecombinant HIV-1 IN can be purified at low concentrations indifferent buffers without a major effect on its ability to performfull-site integration.

Figure 7 predicts a general assembly process of IN subunitsin solution (11, 28). Conformational microheterogeneity of INsubunits may produce different populations of assembled subunits.The model may define the population of IN structures observedin our preparations as determined by the population of half-siteand full-site integration products produced by these preparations(Fig. 4 to 6). The majority of IN subunits (as dimers) (Fig.7, top) appear to be highly active for performing half-siteintegration because $~$ 25% of the input donor is incorporated in20-min reactions. It is currently not possible to define whetherall of the IN molecules are enzymatically active because ofthe high nonspecific DNA binding capabilities of IN (4). A minoritypopulation of IN (Fig. 7, bottom) in our preparations appearto have the capability of forming the higher-order multimerspredicted to be required for assembly of IN on two viral DNAends for full-site integration. The nonspecific interactionsof defective dimers (Fig. 7, top) with correctly formed dimers(Fig. 7, bottom), either in solution or in the assembly of nucleoproteincomplexes, may significantly influence the specific activitiesof different IN preparations for full-site integration. Thisminority population of HIV-1 IN subunits (Fig. 7, bottom) prefer $~$ 150 mM NaCl for correct assembly of two viral DNA ends, significantlyhigher than the $~$ 40 mM NaCl previously reported for HIV-1 virionlysates (6, 18) and recombinant IN (6, 22). It is not possibleto accurately compare the specific activities of recombinantHIV-1 IN to those of virion lysates because the lysates containnonionic detergents, which appear to be detrimental for correctIN subunit interactions (11, 28). However, under optimum conditions,recombinant HIV-1 IN appears to produce larger quantities ofall integration products (Fig. 5 and 6) than virion lysatesby severalfold (6, 17, 18) (data not shown).

In summary, further studies are necessary to improve the populationof IN subunits capable of assembling nucleoprotein complexespossessing the capacity to perform full-site integration. Subsequentsite-directed mutagenesis of HIV-1 IN may allow further insightsinto the multiple structure-functional relationships necessaryfor IN (2, 4, 7, 20) to mediate correct assembly of nucleoproteincomplexes capable of full-site integration.

ACKNOWLEDGMENTS

This work was supported by United States Public Health Servicesgrant AI31334 from NIAID and in part by grant 02750-30-RGT fromthe American Foundation for AIDS Research (amfAR).

We thank T. Mueser and C. C. Hyde for development of the procedureto purify RSV PrA IN (33) that was used to purify recombinantHIV-1 IN. We thank Jim Taylor for his studies expressing theHXB2 IN gene, Daria Hazuda for purified SIV IN, and Carl Friedenfor purified GroEL and GroES.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Molecular Virology, St. Louis University Health Sciences Center, 3681 Park Ave., St. Louis, MO 63110. Phone: (314) 577-8411. Fax: (314) 577-8406. E-mail: Grandgdp{at}SLU.EDU.

REFERENCES

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