©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Human Spuma Retrovirus Integrase by Site-directed Mutagenesis, by Complementation Analysis, and by Swapping the Zinc Finger Domain of HIV-1 (*)

(Received for publication, April 21, 1994; and in revised form, November 29, 1994)

Armin Pahl Rolf M. Flügel (§)

From the Abteilung Retrovirale Genexpression, Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69009 Heidelberg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human spuma retrovirus or foamy virus integrase (HFV IN) is an enzymatically active protein consisting of domains similar to other retroviral integrases: an amino-terminal HH-CC finger, a centrally located region with the conserved D, D-35-E protein motif required for catalytic activity and oligomerization, and at least one DNA binding domain implicated in the 3` DNA processing activity and integrase. Recombinant, purified HFV IN protein carrying 10 histidine residues displays a site-specific endonuclease, an integrase, and a disintegrase activity with oligonucleotide substrates that mimic the viral long terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV IN residues of the catalytic domain had increased endonuclease and disintegrase activities. Deletion mutants at both ends of the HFV IN protein were generated, purified, and characterized. Unexpectedly, it was found that the HFV integrase and disintegrase activities require an intact NH(2)-terminal sequence and that COOH-terminal deletions led to an increase in disintegrase activity. The HH-CC finger of HFV IN was exchanged with that of the human immunodeficiency virus-1 (HIV-1) IN protein. The resulting chimeric IN had a 3` processing activity that utilized the HFV LTR instead of the HIV LTR, indicating that the central domain is crucial for substrate recognition. Functional complementation of the amino-terminal deletion mutant of HFV IN was achieved by a carboxyl-terminal deletion mutant of the chimeric IN, resulting in high levels of integrase activity.


INTRODUCTION

Retroviral integration is a key step during viral replication that at least in vitro requires only one protein, the integrase (IN), (^1)and involves the covalent insertion of a DNA copy of the viral DNA genome into a host cell chromosome (for reviews, see (1, 2, 3, 4, 5, 6) ). The IN proteins are encoded by the pol genes that in most retroviruses are synthesized as gag-pol fusion proteins from which the integrases are generated by proteolytic processing. Early work indicated that the Rous sarcoma virus IN protein can form dimers(7) . Recent data from several groups show that IN forms dimers and/or oligomers(8, 9, 10, 11, 12, 13) . Three enzymatic activities have been shown to reside in retroviral IN proteins. (i) A site-specific endonuclease removes the two terminal nucleotides at the end of each long terminal repeat (LTR) prior to integration. (ii) A DNA strand transfer activity covalently joins the 3` recessed termini of each strand to the 5` ends of target host DNA. (iii) Recently, a third activity, the disintegrase, was found to be detectable in vitro and is responsible for part of the reverse reaction of integration, also called disintegration. During the disintegration reaction, the LTR-DNA target junction is cleaved, thereby releasing the LTR and covalently joining the nick between the DNA strands. The disintegrase activity can be monitored by using appropriate branched or Y-shaped model substrates(14) .

Retroviral integrases consist of several domains(8, 9, 15, 16, 17, 18) . The amino-terminal HH-CC finger region has been reported to bind Zn ions stabilizing the conformation of that domain(19, 20) . A number of apparently conflicting reports on the functional role of the HH-CC finger domain have been published. Recently, it was reported that this domain is capable of binding the LTR deoxyoligonucleotides with high affinity in the absence of divalent cations(13) . It was found that wild type tetrameric HIV-1 IN molecules can be cross-linked to the U5 LTR, whereas an IN form that had been mutated in one of the Cys residues of the finger domain showed reduced oligonucleotide binding, indicating that the HH-CC finger may be a DNA binding domain for the 3` processing activity under the conditions used (13) . In contrast, efficient DNA strand transfer activity was restored to a mutant Rous sarcoma virus integrase lacking the HH-CC domain by fusion to various short peptides(18) .

The centrally located, catalytic domain with the characteristic D, D-35-E motif overlaps and coincides with the dimerization or tetramerization domain(8, 9, 12, 13) . The carboxyl-terminal regions of HIV-1 IN were reported to contain a low affinity or aspecific DNA binding domain(16, 17) .

In view of the distinct human foamy virus (HFV) IN sequence, we are interested in defining the minimal sequences required for the individual enzymatic activities of the human spuma or foamy virus integrase. The HFV IN protein has been reported to have a size of 39 kDa in wild type infected cells(21) . Recombinant HFV IN protein expressed from Escherichia coli that carries a tag of 10 His residues at the amino terminus was purified by Ni chelate affinity chromatography and shown to possess all three enzymatic activities of the intact HFV integrase(21) . The 41-kDa recombinant HFV IN protein has a relatively high degree of homology compared to other retroviral IN(22) . While most of the amino acid residues conserved in other retroviral IN proteins are also invariant in the HFV integrase, there are some features and motifs that set it apart(23) . In particular, the HFV HH-CC domain itself and adjacent sequences are longer and different from those of other complex human retroviruses. In an effort to analyze and characterize the HFV IN protein, we used deletion mutants, complementation analysis of differently shortened mutant IN proteins, site-directed mutagenesis, and domain swapping to define the functions of the HFV IN domains. Our results show that for full enzymatic activity of the HFV integrase, sequences flanking the HH-CC finger domain were required.


EXPERIMENTAL PROCEDURES

Construction and Growth of HFV pET16b IN

The preparation and purification of recombinant HFV integrase protein was performed as reported previously(21) . To obtain wild type modified or truncated versions of the HFV IN protein, plasmid pET16b (Novagen) was digested with NdeI and BamHI. The DNA fragment containing the HFV IN was obtained from pHSRV clone C55 (22) by PCR using the Pfu DNA polymerase (Stratagene). This enzyme has been reported to be of higher fidelity than the Taq DNA polymerase(24) . The sequences of the antisense and the sense primer were 5`-CAGTATAATTGGATCCTTCTG-3` and 5`-CAGGGTCATCATATGAAAGGA-3`, respectively, for HFV genome positions 5429-6598. PCR amplifications were done at 92 °C for 1 min, at 52 °C for 45 s, and at 72 °C for 90 s; the cycle was repeated 35 times. The resulting PCR fragments were digested with NdeI and BamHI and ligated into the corresponding cloning sites of pET16b(25) . The correct orientation of the insert was confirmed by dideoxy sequencing of both ends of the vector-HFV insert borders. Plasmid pET16bIN was used to express the HFV IN insert in E. coli BL21 (DE3) cells. Numbering of the recombinant HFV IN protein sequence starts at the proline residue 1 (shown in bold letters below) that was introduced by the cloning procedure used. The NH(2)-terminal region of the HFV IN sequence deduced from nucleotide sequencing is as follows, with the HFV IN sequences in italics and the vector-derived residues in uppercase: MGHHHHHHHHHHSSGHIEGRHMKGYPQYTYFLEDGKVKVSRPEGVKIIPPQSDRQKIVLQAHNLAHGREAT . . . The 2 His residues of the HH-CC finger domain conserved in all known retroviruses are underlined. The remaining HFV IN sequence has been reported previously(21) . The designation of the IN deletion mutants follows this numbering, i.e. in deletion mutant Delta1-34, all residues upstream of the Val marked by double underlining were deleted.

The truncated forms of the HFV IN protein were synthesized in an analogous way as described above for the recombinant intact HFV IN protein. A NdeI site was created at the 5` end and a BamHI site at the 3` end. The sequences of the primer pair used for Delta1-34 were 5`-ACAAAAACATATGCTTCAAG-3` as sense primer and 5`-CAGGGTCATCATATGAA-AGGA-3` as antisense primer (HFV genomic positions 5429-6598); for Delta1-74, sense primer 5`-AGCGCCCGCCATATGCAACAGTGTTTAATC-3`, antisense primer 5`-CAGGGTCAT-CATATGAAAGGA-3` (genomic positions 5658-6598); Delta323-366, sense primer 5`-CAGTATAATTGGATCCTTCTG-3`, antisense primer 5`-CTCTCCTGGATCCATTGGCC-3` (genomic positions 5429-6410); Delta297-366, sense primer 5`-CAGTATAATTGGATCCTT-CTG-3`, antisense primer 5`-AGTCCTTGGATCCAACACCT-3` (genomic positions 5429-6334). To construct the double mutant Delta1-34/Delta323-366, DNA of pET16bDelta1-34 and pET16bDelta323-366 vectors were digested with EcoRI. The 6020-bp DNA backbone of pET16bDelta1-34 containing the NH(2)-terminal deletion was ligated to the 540-bp COOH-terminally truncated fragment of pET16bDelta323-366.

Site-directed Mutagenesis

The site-specific mutations of the HFV- and HIV-1 int genes were synthesized by the PCR method described previously(26) . Synthetic oligonucleotides were designed that formed the desired single amino acid changes or restriction sites. PCR was carried out with the C55 DNA as template using Pfu DNA polymerase. The mutated HFV and HIV int genes were cloned into the pET16b vector and confirmed by DNA sequencing.

Construction of the Chimeric and Mutated HFVDeltaHH-CC/HIV-1 HH-CC int Gene

The HFV IN gene was amplified by PCR with clone C55 DNA as template. A new NdeI site was introduced at the 5` end, a BamHI site at the 3` end, and a HindIII site 32 nucleotides downstream of the C(X)(2)C motif of the HH-CC finger domain by two subsequent PCR steps. The same strategy was used for the amplification and mutation of the HIV IN gene except that DNA of the HIV-1 HXB2 clone was used as template and the HindIII site was located 24 nucleotides downstream of the C(X)(2)C motif of the metal finger. The HindIII site were generated without changing the reading frame after swapping the corresponding HFV and HIV DNA fragments. The HFV and HIV IN genes were separately ligated into the NdeI-BamHI cloning site of pET16b (the HindIII site at position 29 was destroyed). Thereafter, the pET16bHFVHindIII and the pET16bHIVHindIII vectors were digested with BamHI and HindIII. The 908-bp BamHI/HindIII DNA fragment of HFV IN was ligated into the pET16bHIVHindIII vector backbone. Correct orientation and cloning was confirmed by DNA sequencing and restriction enzyme analysis. To construct the chimeric HIV/HFV Delta323-334 and Delta297-334 mutants, a BamHI site by PCR was introduced with the pET16-containing int recombinant as template and the same HIV sense primer as for the construction of the chimeric IN recombinant. The same antisense primers as for the corresponding HFV IN deletion mutants were used (see above). The resulting PCR-amplified reaction product was cloned from the pET16b vector.

Protein Expression and Purification

To purify HFV IN protein in greater amounts, 1 liter of BL21 (DE3) pET16bIN (or other inserts) cells were grown at 37 °C in LB medium (supplemented with 50 mg/ml carbenicillin) to an A of 0.6-0.8. To induce IN protein expression, isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 1 mM; bacteria were grown for another 4 h and harvested by low speed centrifugation. The pellet was resuspended in 40 ml of 20 mM Tris, pH 7.6, 1 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM imidazole (IMAC5). Cells were lysed with a Dounce homogenizer, sonicated for 3 min, and centrifuged at 39,000 times g and 4 °C for 20 min to remove cell debris. The supernatant was loaded on a column containing Ni-charged His Bind Resin (Novagen). The flow rate was 10 ml/h. The resin was washed with IMAC200 (the same as IMAC5 except 200 mM imidazole), and the specifically bound protein was eluted with IMAC500. The eluate was dialyzed against 10% glycerol, 20 mM MES, pH 6.2, 80 mM KCl, 6 mM MnCl(2), 10 mM dithiothreitol, stored at -70 °C, and further purified by cation exchange chromatography on a Mono S column (Pharmacia Biotech Inc.). To elute the IN protein, a linear salt gradient from 0.2 to 1.0 M NaCl in HEPES at pH 7.6 (10 mM beta-mercaptoethanol) was employed. The HFV IN protein was recovered from a peak that was eluted at 600 mM NaCl. The eluate was dialyzed against 10% glycerol, 20 mM MES, pH 6.2, 80 mM KCl, 6 mM MnCl(2), 10 mM dithiothreitol and stored at -70 °C. The protein concentrations were determined at A in a spectrophotometer (LKB Ultrospec II) using different bovine serum albumin concentrations as standards according to Bradford(27) . The purity of the different IN preparations was monitored by SDS-polyacrylamide gel electrophoresis as described previously(21) . The individual IN mutant protein preparations were found to migrate as single and homogeneous bands.

Synthesis, Purification, and Quantitation of Oligodeoxynucleotides

Oligodeoxynucleotides were synthesized on a Millipore Cyclon Plus apparatus, gel-purified, and eluted from the gel slices as described previously(21) . Oligodeoxynucleotide concentrations were spectrophotometrically determined at A. To determine the amounts of the reaction products of the three IN enzymatic activities, the same amounts of substrates were used in the individual reactions under standard assay conditions. The total concentration of substrates plus reaction products were set to 100%. The intensities of the individual bands were determined within a single lane only. As the amounts of aliquots taken and and loaded onto a gel particular gel pocket varied slightly, band intensities in different lanes cannot be compared.

Endonucleolytic Cleavage Assay

The assay conditions were optimized for Mn, KCl, and pH value. The standard endonuclease assay was carried out as follows. A gel-purified synthetic 20-nucleotide oligodeoxynucleotide corresponding to the 3` end of the U5 region of the plus strand of HFV LTR (Fig. 1, F2) was labeled at the 5` terminus using T4 polynucleotide kinase and [-P]ATP (222 TBq/mmol). The labeled strand was annealed with a 4-fold excess of a gel-purified synthetic 20-mer oligodeoxynucleotide corresponding to the 3` end of the minus strand U5 region of the HFV LTR (Fig. 1, F1). Subsequently, 1.3 pmol of F1/F2 (panelB) was used in a reaction mixture containing 20 mM MES, 85 mM KCl, 10 mM dithiothreitol, 10% glycerol, 6 mM MgCl(2) or 6 mM MnCl(2), and 370 ng of HFV IN in a total volume of 15 µl, pH 6.2. The mixture was incubated for 90 min at 37 °C, stopped by addition of 15 µl of dye-containing formamide, and heated to 80 °C for 5 min. Samples were loaded (6 µl each) on a 12% denaturing polyacrylamide gel and electrophoresed in 89 mM Tris, 89 mM boric acid, 2 mM EDTA. Reaction products were visualized by autoradiography for 1 or 2 h. The same standard assay conditions were used for analysis of the different substrates except that instead of HFV U5 LTR, the substrates listed in Fig. 1were employed. The intensity of the bands on the autoradiogram were quantitated by an optical densitometric scanner (Elscript 400, Hirschmann). The calculation of the enzymatic activity was done as described previously (15) . For complementation assays, pairs of mutated IN proteins were mixed at equimolar concentrations before adding the substrates. The final concentration was 370 ng of purified, HFV mutant IN proteins.


Figure 1: Structure of oligonucleotides substrates of the HFV and HIV-1 integrase. Substrates of disintegrase of HFV (A) and HIV (D), integrase of HFV (B) and HIV (E), and endonuclease of HFV (C) and HIV-1 (F). The substrates in panels B and C were derived from the 3` end of the HFV U5 LTR (E) and the HIV U5 LTR (F). The branched substrates A and D represent random target sequences shown as horizontal strands; the 3` end of the HFV U5 LTR region and the 3` end of the HIV U5 LTR region (I1, 21 nucleotides and I4, 33 nucleotides in length) are shown diagonally. The asterisks mark the P label. G, autoradiogram of the reaction products of the enzymatic activities of the HFV IN. The substrates shown in A-C were incubated in the absence (lanes 1, 3, and 5) and in the presence of the IN protein for 90 min under standard conditions. Lane2, endonuclease with substrate C; lane4, disintegrase with substrate B; lane6, integrase activity with substrate A; the brackets mark the integration products. For further details, see ``Experimental Procedures.''



Integration Assays (Standard Assay Conditions)

To assay for DNA joining activity, an 18-bp oligonucleotide corresponding to the 3` end of the HFV U5 plus strand but lacking 2 nucleotides at the 3` terminus (Fig. 1, F3) was labeled at the 5` end and annealed to a 4-fold excess of F1. The reaction conditions used were identical to those of the endonucleolytic cleavage assay except that 13.6 pmol of annealed F1/F3 (Fig. 1C) was employed as substrate. Time of exposure was 15 h.

Disintegration Assays (Standard Assay Conditions)

The disintegration assay was done under conditions identical to those for the endonuclease assay, except that 13.6 pmol of a Y-shaped oligonucleotide (Fig. 1) that was shortened by 2 bp at the 5` end of T1 were used as substrate. To form the Y-shaped oligomer, 13.6 pmol of the T1 target oligomer was labeled with P at its 5` end; subsequently, 54.4 pmol of F1, V4, and F2 (Fig. 1) were added, heated to 80 °C, and slowly cooled to 30 °C. F4 was a 33-mer and derived from F3 (Fig. 1) plus a target sequence. The branched oligomer was precipitated and used for disintegration assays.


RESULTS

To determine the role of the different domains for the three enzymatic activities of the HFV integrase, we constructed deletions at either terminus of the IN protein to analyze which of the three enzymatic activities, if any, was lost or perturbed.

The mutated versions of the HFV IN were expressed in E. coli and purified by Ni chelate affinity chromatography and cation exchange chromatography (21) to near homogeneity. Subsequently, the purified HFV IN proteins were assayed for endonuclease, disintegrase, and DNA strand transfer activity by using the appropriate oligonucleotide substrates shown in Fig. 1. The oligonucleotides are model substrates derived from the termini of the LTRs of either the HFV or HIV-1 DNA genome for assaying the 3` processing and integrase activity. The branched, Y-shaped substrates consisting of four different oligonucleotides were used for assaying the disintegrase activity of HFV or HIV-1 IN (Fig. 1, A and D). Quantitation of substrates and reaction products was done by determining the intensities of individual bands of the same lane only separately after electrophoresis. Total concentration of substrates and reaction products were set to 100% (see ``Experimental Procedures''). Fig. 1G shows autoradiograms of the reaction products of the three enzymatic activities of the HFV IN protein with the corresponding substrates C, A, and B under standard conditions. The result shows that the three oligomers served as substrates for the individual enzymatic activities in the presence (lanes 2, 4, and 6) of the HFV IN protein but not in its absence (lanes1, 3, and 5).

Effects of the Mutations in Conserved Regions of HFV IN Protein

The rationale for this experimental series was to determine if the enzymatic activities of the HFV IN protein were affected when non-conserved amino acid residues were mutated to those residues invariant in most retroviral integrases and that had previously been shown to play an essential role for the enzymatic activities(11, 23) . To this end, two HFV IN regions of homology were selected. This is illustrated by the Ile residue at position 106 of HFV IN that was changed into a Thr residue, since it is conserved in most retroviral IN (Fig. 2A). The mutant I106T showed a slight increase in the disintegrase activity of HFV IN, but the endonuclease activity was decreased to 34% whereas the integrase activity was low but still detectable (Fig. 2, panels A and B).


Figure 2: Structure of point mutations in highly conserved regions of the HFV IN protein and enzymatic analysis. Panel A schematically presents the HH-CC finger domain, the positions of point mutations in two different centrally located DYIG and SDQG motifs of the catalytic domain, and the resulting enzymatic activities given in percentage of activity of the intact HFV IN protein set to 100% (for details of quantitation, see ``Experimental Procedures''). Panel B shows the autoradiograms of the endonuclease, panel C the disintegrase, and panel D the strand transfer reaction: wild type HFV IN (lane 1), I106T106 (lane 2), S159T159 (lane 3), and S159/N161T159/Q161 (lane 4).



The second conserved region analyzed was the TDNG motif, which is highly conserved among retroviral integrases. Point mutations of this motif led to either a complete loss or a drastic reduction of the 3` processing and integrase activities as reported for HIV-1(23, 28) . The HFV integrase differs at two positions from this motif (Fig. 2A); therefore, mutagenesis from the HFV sequence to the retroviral consensus sequence was carried out in two subsequent steps. First, the Ser residue at position 159 was changed into Thr and the enzymatic activities were determined. Subsequently and additionally, Gln-162 was changed to an Asn residue. The three enzymatic activities were separately determined for the two mutant proteins. The first amino acid exchange had nearly no effect on the endonuclease and integrase activities, but the disintegrase increased to 161%. The second additional amino acid mutation caused no major change in the integration and endonuclease activity compared with those of the single exchange mutant. Both the nucleolytic cleavage and the disintegration activity increased to 118 and 143%, respectively, of that of the intact HFV IN (Fig. 2, panels A and D).

The results obtained demonstrate that point mutations at defined positions can lead to a strong enhancement of disintegration but to reduce DNA cleavage activity. One point mutation (I106T) only slightly changed the disintegrase and affected the endonuclease and integrase activities, reducing the integrase activity to almost background values, whereas the endonuclease was still detectable at 34% of the intact HFV IN (Fig. 2, A and B).

Enzymatic Activities of Truncated HFV IN Proteins

Two HFV IN proteins differently truncated at the NH(2) terminus were studied first. The Delta1-34 protein lacked the first 34 amino acid residues leading up to the HH-CC finger domain (for numbering, see ``Experimental Procedures''). This deletion of the HFV IN protein reduced the endonuclease activity to 13%, whereas the integrase activity was detectable but only at a low level compared to that of the intact HFV IN (Fig. 3, panels B and D). The disintegrase activity reached 33% (Fig. 3, panels A and C). The Delta1-74 deletion mutant constructed to determine the influence of the HH-CC finger domain contained an even larger deletion, encompassing almost the entire metal finger region. It was found that the Delta1-74 deletion mutant of HFV IN had barely detectable levels of the three enzymatic activities. The results so far indicate that even residues upstream of the HH-CC finger domain affected the three enzymatic activities more strongly than corresponding HIV IN deletion mutants. This is particularly valid for the HFV disintegrase (Fig. 3, panels A and C) compared to that of HIV-1 IN(9, 23) .





Figure 3: Schematic representation of locations of terminal deletions of the HFV IN protein and analysis of the corresponding reaction products. Topline in part A shows full-length HFV integrase domains. The resulting activities of the three enzymatic activities of the deleted HFV IN protein are compiled in the right part of panel A; intact HIV IN activities were set to 100%. The purified mutant proteins were assayed as described under ``Experimental Procedures.'' Panels B-D, autoradiograms of endonuclease, disintegrase, and integrase: intact HFV IN (lane 1), Delta1-34 HFV IN (lane 2), Delta1-74 HFV IN (lane 3), Delta323-366 HFV IN (lane 4), and Delta297-366 HFV IN (lane 5).



To determine the influence of the residues located at the opposite end of HFV IN, two COOH-terminally truncated HFV IN proteins were constructed, expressed, and purified. Analysis of the corresponding enzymatic activities showed that the HFV IN mutant (Delta323-366) that lacks 43 carboxyl-terminal residues up had an 3` processing activity of 32% compared to that of full-length HFV IN (Fig. 3B). The integrase was at a low level but clearly detectable. Strikingly, the disintegration activity of Delta323-366 increased to 230% (Fig. 3, A and B).

In another terminally truncated HFV IN mutant (Delta297-366) studied, 69 amino acids were deleted from the COOH-end that should include a presumed DNA binding domain located at approximately the same region as in HIV IN(16, 17) . The solubility of the mutant Delta297-366 protein was lower compared with the intact and the other truncated forms of HFV IN protein. The endonuclease activity was 11% compared with that of the intact IN (Fig. 3B); the integrase activity was barely detectable, while the disintegrase had approximately half of the activity of the Delta323-366 deletion mutant but was still active at about the same level as intact HFV IN (Fig. 3C). An IN mutant that had both ends shortened (Fig. 3, bottomline of panel A) did have detectable disintegrase activity (8%) but had less than 3% of 3` processing and strand transfer activities of the intact HFV IN (data not shown).

The results show that a full-length HFV IN protein is required for any DNA strand transfer activity. The endonucleolytic activity was decreased by truncating either the NH(2) or the COOH terminus. In contrast to HIV-1, the disintegration activity of the HFV IN requires an NH(2)-terminal region upstream of the HH-CC finger domain for full activity. It is remarkable that small deletions at the COOH terminus increased the disintegrase whereas, the 3` processing activities were negatively influenced by deletions at either end of HFV IN.

Construction and Enzymatic Activity of a Chimeric HFV/HIV-1 IN Protein

To study the role of the HH-CC finger domain of HFV IN protein, the metal finger region of HFV was exchanged with the corresponding finger domain of HIV-1 IN. It has been shown previously that the HH-CC finger domain of HIV-1 is essential for the integration and endonuclease activity but not for the disintegrase activity(9, 15, 19) . To synthesize the chimeric IN protein, a HindIII site was introduced into the tether region between the finger domain and the catalytic domain of the HIV-1 and HFV integrase. The HindIII site was positioned in a way that no change of the number and the codons occurred after ligating the HIV-1 metal finger domain to the catalytic domain of HFV integrase. The DNA of the chimeric IN protein was ligated into a pET16b vector and transformed into BL21(DE3) cells. The bacterially expressed protein was purified by Ni-chelate chromatography with an additional step of cation exchange chromatography to virtual homogeneity as described for the intact HFV IN (21) and checked for purity by gel electrophoresis.

Endonucleolytic Activity of the Chimeric IN Protein

The endonucleolytic assay was performed with the HIV-1 and HFV LTR substrates (Fig. 1) at two different pH values (pH 6.2 and 7.6) and with either Mg or Mn as divalent cations. An endonucleolytic activity of the chimeric IN protein was detectable with the HFV preprocessed substrate C only, in the presence of Mn, and at both pH values but not with the HIV-1 substrate E. The activities of the chimeric IN at pH 6.2 and 7.6 were 59 and 43% of that of the intact HFV IN protein activity, respectively (Fig. 4B). Thus, the HH-CC finger of HIV-1 IN was able to replace partially the endonucleolytic cleavage activity of the metal finger domain of HFV IN. The substrate and divalent cation specificity of the HFV IN protein was not altered by the substitution. The pH range was extended to the alkaline range, where the intact HFV IN endonuclease is not active at pH 7.6 (Fig. 9).


Figure 4: Endonucleolytic cleavage activity of the chimeric integrase HFV Delta HH-CC/HIV HH-CC IN protein. Assays were done at different pH values; intact HFV IN activity was set to 100%. Panel A, Mg, pH 6.2, HIV LTR substrate E (lane 1); Mg, pH 6.2, HFV LTR substrate B (lane 2); Mg, pH 7.6 substrate E (lane 3); Mg, pH 7.6, substrate B (lane 4); Mn, pH 6.2, substrate E (lane 5); Mn, pH 6.2, substrate B (lane 6); Mn, pH 7.6, substrate E (lane 7); and Mn, pH 7.6, substrate B (lane 8). For the structures of the different substrates, see Fig. 1. In panel B, the endonucleolytic cleavage activities are compiled.




Figure 9: Determination of the optimal concentration of KCl, Mn, and of the pH maximum of the HFV IN endonuclease activity. The assays were done as described under ``Experimental Procedures'' under standard assay conditions.



Integration Activity of the Chimeric IN Protein

The integration activity was determined with the HFV U5LTR substrate B and HIV 20-mer substrate F (Fig. 1) and Mg or Mn as divalent cations. The integrase of the chimeric IN protein did not show much of a difference for either of the divalent cations used, 26 and 19% activity of that of intact HFV IN (Fig. 5). The HFV substrate B showed an increase in cation-dependent transfer activity of 42-53% in comparison to the HIV-1 U5LTR substrate F (Fig. 5). The HIV-1 HH-CC finger domain was capable of replacing the HFV finger domain in both the endonucleolytic and integrase activity.


Figure 5: Integrase activity of the chimeric HFV Delta HH-CC/HIV HH-CC IN protein. Standard assay conditions were as described under ``Experimental Procedures'' with different substrates and divalent cations. Panel A, Mg and HIV U5LTR substrate F (lane 1), Mn and HFV 20-mer substrate C (lane 2), Mn and substrate F (lane 3), Mg and substrate C (lane 4). The brackets mark the integration products longer than the 18-nucleotide substrate. Panel B, both preprocessed substrates C and F were utilized in the DNA strand transfer reaction in the presence of either Mn or Mg with the HFV DNA as the preferred substrate. Intact HFV IN activity was set to 100%.



Disintegration Assays of the Chimeric IN Protein

The disintegration assays were carried out with the HFV branched substrate A and the HIV-1 Y-shaped substrate D (Fig. 1) in the presence of the chimeric IN protein or the intact HFV IN protein. The chimeric IN protein showed low levels of enzymatic activity of 6 and 13%, with either the HFV- or HIV Y-shaped substrate (Fig. 6). There was only a slight difference in the level of disintegrase activity of the intact HFV IN protein with the HIV-1 branched substrate (84% of intact HFV activity) or HFV Y-shaped substrate (Fig. 6). It is of great interest that the HIV-1 HH-CC finger domain was not capable of fully substituting for the metal finger region of HFV IN with respect to disintegrase. These results correlate and are consistent with the data on the NH(2)-truncated HFV mutants, which also show a drastically reduced disintegration activity. This is in contrast to the HIV-1 IN protein that still had disintegrase activity without the HH-CC finger domain (9, 15, 19) but decreased disintegrase when the carboxyl-terminal end of the enzyme was shortened.


Figure 6: Disintegrase activity of the chimeric HFV/HIV-1 IN protein. Assays were performed as described under ``Experimental Procedures'' in the presence of Mn. Panel A, chimeric HIV-1 HH-CC finger IN activity with the branched HIV substrate D (lane 1), HFV substrate A (lane 3), intact HFV IN activity with HIV substrate D (lane 2), and HFV substrate A (lane 4); for the structure of the Y-shaped substrates, see Fig. 1. Panel B, disintegrase activity.



Functional Complementation of HFV Deletion Mutants

To determine the potential of different deletion mutants to complement each other, pairs of HFV IN mutant proteins were mixed and assayed for the three enzymatic activities. Endonucleolytic cleavage activity was detectable in the mutant combinations (Fig. 7A), indicating that a partial complementation occurred. However, the mixture of the Delta1-34 and Delta323-366 mutants displayed low levels even of the disintegrase activity, although mutant Delta323-366 by itself showed abundant levels of disintegrase activity (Fig. 7, A and C). Further analysis of IN mutant mixtures revealed that most of the mutant pairs were capable of functional complementing the endonuclease activity but not the integrase activity (Fig. 7). The data can be explained by assuming an interaction between the different HFV IN mutant proteins restoring partially active enzymatic activities including the disintegrase.


Figure 7: Complementation experiments of enzymatic activities of HFV IN mutant proteins. The purified mutant proteins were mixed at equimolar amounts and assayed as described under ``Experimental Procedures.'' The resulting enzymatic activities of the deleted HFV IN protein mixtures are compiled at right. Integrase, endonuclease, and disintegrase activities are given as the percent of wild type activity: +++, 50-100%; ++, 10-50%; +, 3-10%; -, <3%.



Functional Complementation of HFV IN Deletion Mutants by Chimeric HIV/HFV Deletion Mutants

In contrast to HIV IN, functional complementation of HFV integrase activity was not detectable after mixing pairs of the corresponding HFV IN deletion mutants (see above and Fig. 7). In order to determine whether functional complementation of HFV integrase can be performed by means of the chimeric HIV/HFV IN deletion mutants, two differently truncated versions of the chimeric IN proteins were generated by deleting parts of the COOH terminus. The first chimeric mutant Delta291-334 retained high levels of integrase but no disintegrase activity, whereas the second chimeric mutant Delta265-334 did not show any enzymatic activity (Fig. 8).



Figure 8: Enzymatic activities of intact and mutant chimeric HIV/HIV IN proteins and functional complementation of the HFV IN deletion mutants by the chimeric HIV/HFV IN mutant proteins. Assays were done under standard conditions. The resulting enzymatic activities of the deleted HFV IN protein mixtures are compiled in the right part of panel A. Panel B, chim HIV/HFV 1-334 (lane 1), chim mutant HIV/HFV Delta291-334 (lane 2), chim HIV/HFV Delta291-334 plus HFV Delta1-34 (lane 3), chim HIV/HFV Delta291-334 plus HFV Delta1-74 (lane4), chim HIV/HFV Delta265-334 (lane5), chim HIV/HFV Delta265-334 plus HFV Delta1-34 (lane6), and HIV/HFV Delta265-334 plus HFV Delta1-74 (lane7). Integrase and disintegrase activities are given as the percent of wild type activity: +++, 50-100%; ++, 10-50%; +, 3-10%; -, <3%.



Functional complementation of the HIV/HFV IN mutant Delta265-334 was achieved with both NH(2)-terminal HFV IN deletion mutants Delta1-34 and Delta1-74 (Fig. 8). This result is remarkable, since neither the short nor the longer HFV IN protein was active by itself. Thus, functional complementation of the integrase activity of an inactive chimeric HIV/HFV IN deletion mutant was efficient and accomplished by mixing it with one of the NH(2)-terminally truncated HFV IN proteins.

The HH-CC finger does influence the pH range of the endonucleolytic cleavage activity, since the chimeric enzyme was active at pH 7.6 in the presence of Mn. The strand transfer activity of the chimeric IN protein was active with both the HIV and HFV preprocessed LTR substrates and both of the two divalent cations. This result is consistent with the known fact that integrases generally lack DNA target specificity. This is best illustrated by comparing the disintegrase activities of the different mutant proteins, which seem to be unmasked by COOH-terminal deletions.

In Fig. 9(A-C), the results of determining the optima for KCl concentration, divalent cation, and pH for the 3` processing activity are summarized. It was found that the optimal KCl concentration was broad ranging from 20 to 160 mM (panel A). The optimal range for Mn was between 2 and 8 mM (panel B). The pH optimum of the HFV endonuclease was found at 6.2; at pH 7.6 activity, was not detectable (panel C).


DISCUSSION

The results of this report, which include the analysis of point and deletion mutations, indicate that the in vitro DNA nucleolytic cleavage and strand transfer activities of the HFV IN protein cannot be separated into independent domains. This is consistent with data on the properties of HIV integrase from several groups(8, 9, 15) . Concerning the HFV IN point mutations with increased disintegrase activity, it is interesting that particular HIV IN mutants also showed elevated disintegrase activities surpassing those of wild type HIV-1 IN(23) .

Close examination and comparison of the three enzymatic activities of the HFV IN deletion mutants with those of HIV-1 IN revealed clear differences. Analysis of truncated HIV-1 IN proteins showed that even a short deletion of 5 residues at both the NH(2) and COOH termini (8, 9, 19) abolished both endonuclease and integrase activities of HIV-1 IN, while disintegrase activity was still detectable after deleting of up to 50 amino acid residues at both ends, although at a level lower than that of the wild type IN. In contrast, the HFV IN deletion mutants displayed a different pattern of activities. It is noteworthy that the Delta1-34 HFV IN mutant lost DNA strand transfer activity but retained detectable activity, not only of 33% disintegrase but also of 13% of the 3` processing activity. This HFV IN mutant is directly comparable to the HIV-1 DeltaN5 mutant(9) , since the deletions in both mutants are located 4 and 3 residues upstream of the first His residue of the corresponding viral HH-CC domains. The lower activity of the HFV Delta1-34 mutant indicates that for full activity the HFV disintegrase requires not only the HH-CC finger but also upstream sequences of this domain encompassing the major part of the residues deleted. Removal of additional 40 residues as in mutant HFVDelta1-74 led to a complete loss of detectable levels of the three enzymatic activities, including the disintegrase activity. This is in contrast to the corresponding DeltaN38 mutant HIV IN that still retained residual disintegrase activity(17) . The results suggest that the HH-CC finger and flanking sequences of the HFV IN protein contribute substantially to full disintegrase activity.

On top of this difference with NH(2)-terminal deletion mutant IN proteins, HFV IN COOH-terminal deletion mutants showed properties completely different from the corresponding HIV mutants. It was found that COOH-terminally deleted HFV IN mutants showed a dramatic increase in disintegrase activity that even surpassed the levels of the intact HFV IN protein. It has been reported that 50-91 carboxyl-terminal residues of HIV-1 IN could be deleted with some disintegrase activity retained(17) . A 2-3-fold increase over the activity of the intact HFV IN protein as in the case of HFV IN mutants was not found for HIV IN deletion mutants(19) .

We assume that several factors contribute to the difference between the HFV and HIV IN activities. These factors include sequence differences of the individual domains of the retroviral IN analyzed and, in addition, functional redundancies that are likely caused by the oligomeric nature of the IN proteins.

As to the amino terminus, there are 32 additional residues in the HFV IN that include 26 residues upstream of the metal finger domain and 6 extra residues within the finger region. Compared to that of HIV-1, the HFV IN sequence encodes 7 additional residues directly downstream of the C(X)(2)C motif of the metal finger in the region preceding the catalytic domain. The higher disintegrase activity of the COOH-terminally truncated HFV IN mutant Delta323-366 might be explained by assuming that the mutated IN protein has a better capacity to recognize and bind to the branched DNA substrates because of higher accessibility of the substrates to the DNA binding domains(16, 17) . The HFV mutant Delta297-366 that probably lacks the aspecific DNA binding domain of the COOH-terminal domain has, nevertheless, full disintegrase activity. Based upon this result, it is concluded that another HFV IN region may function as DNA binding domain at least for this mutant.

The chimeric HIV/HFV IN protein that contains the HH-CC finger domain of HIV-1 had relatively high levels of integrase activity with the preprocessed substrates but comparatively low disintegrase activity with the HIV and particularly with HFV LTR substrates (Fig. 8B), again indicating that for full activity the HFV disintegrase requires not only the HH-CC finger but also sequences flanking this domain. Since the chimeric HIV/HFV IN endonuclease was active with the HFV LTR substrate only, it appears that the HH-CC finger does not play an essential role in the recognition of the DNA 3` processing substrate. The chimeric protein had about 60% of the endonuclease activity of that of the intact HFV IN. Thus, the chimeric IN protein is functionally equivalent to at least the integrase activity of the wild type HFV IN protein. This result is agreement with data on functionally active Rous sarcoma virus mutants that contain foreign peptide sequences instead of the HH-CC finger(18) . On the basis of these results, we conclude that the specificity of the DNA cleavage reaction is not determined by the HH-CC finger domain. Instead, the central region of the HFV IN is responsible and essential for the site-specificity of the endonucleolytic cleavage reaction. While the precise boundaries of the central catalytic domain of HFV IN remain to be defined, it seems that in analogy to other well characterized IN proteins it extends from the C(X)(2)C motif approximately to residue 297. The extended pH range of the chimeric HIV/HFV IN also indicates that the HH-CC finger domain is in close contact to the catalytic center of the active oligomeric forms of the IN protein. However, the precise role of the NH(2)-terminal IN sequences that include the H(X)(n)H motif (n = 3-6, including IN sequences of retrotransposons) is still not fully understood.

The disintegrase activity of the chimeric IN was 2-fold higher with the HIV-1 LTR substrate than with the HFV LTR. Thus, the part of the chimeric IN that contributes to HFV disintegrase activity appeared to reside more to the amino-terminal part when compared to the HIV-1 IN protein consistent with the results of the NH(2)-terminal HFV IN deletion mutants.

To analyze why HFV IN mutant proteins deleted at either end were not capable of complementing each other in integrase activity as reported for the corresponding HIV IN mutants(8) , a chimeric HIV/HFV IN COOH-terminal deletion mutant that by itself had no integrase activity was separately complemented by two inactive HFV IN mutants that had different truncations at the NH(2) terminus. The result of the positive functional complementation indicates that DNA binding sites can be supplied in trans. In addition, the HH-CC finger domain seems to be active when present in trans. This did not hold for the disintegrase activity, which was not restored, probably because of the very low level of this activity of the intact chimeric IN protein itself.

Close inspection of the data of the complementation assays revealed that again the amino-terminal deletions behave differently when compared to those of the carboxyl-terminal truncated forms of the HFV IN protein. The low efficiency of complementation of different HFV deletion mutants is due to the requirement of the NH(2)-terminal sequence of the HH-CC finger domain and those upstream of it. This was proven by the functional complementation of the HFV mutant Delta1-74 by the chimeric IN mutant Delta297-366 that restored integrase activity to high levels. In addition, the endonucleolytic activity of HFV IN point mutant I106T was reconstituted to nearly wild type activity by complementation with either NH(2)- or COOH-terminal HFV IN mutants (data not shown).

Our results are in agreement with those reported for the MLV IN protein (28, 29) . MLV IN proteins with mutations in the HH-CC region were inactive in integrase function but not in disintegration activity (30, 31, 32) . In fact, the HFV IN protein sequence has a higher degree of homology to that of MLV compared to that of HIV-1 IN as reported previously(33) .


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel.: 6221-424611; Fax: 6221-424852.

(^1)
The abbreviations used are: IN, integrase; LTR, long terminal repeat; HFV, human foamy virus; MLV, mouse leukemia virus; HIV-1, human immunodeficiency virus-1; PCR, polymerase chain reaction; bp, base pair(s); MES, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

We wish to thank Dr. Jan Mous (HoffmannLaRoche, Basel) for providing an HIV-1 IN DNA clone and Robert D. Wells for critically reading the manuscript.


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