Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M107365200 on October 3, 2001

J. Biol. Chem., Vol. 276, Issue 49, 45848-45855, December 7, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/49/45848    most recent
M107365200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Appa, R. S.
Right arrow Articles by Chow, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Appa, R. S.
Right arrow Articles by Chow, S. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Role of the Nonspecific DNA-binding Region and alpha  Helices within the Core Domain of Retroviral Integrase in Selecting Target DNA Sites for Integration*

Rupa Shree AppaDagger, Cha-Gyun Shin§, Philip Lee, and Samson A. Chow||

From the Department of Molecular and Medical Pharmacology, Molecular Biology Institute, and UCLA AIDS Institute, UCLA School of Medicine, Los Angeles, California 90095

Received for publication, August 2, 2001, and in revised form, September 19, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha  helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insertion of the viral genome into cellular DNA by the virally encoded protein, integrase, is a necessary step for the propagation of retroviruses. This essential event in the viral life cycle is termed integration and is a multistep process (for reviews see Refs. 1-3). The removal of the terminal two nucleotides of the 3'-viral DNA ends, termed 3'-end processing (4-6), is the first step of integration followed by the concerted cleavage and ligation of the processed viral 3'-ends to the target DNA. The latter step is referred to as either strand transfer or 3'-end joining (7-10). The completion of the integration process consists of the single-stranded gap repair at the site of concerted insertion followed by the joining of the 5'-viral ends to the target DNA. Retroviral integration results in the formation of a provirus flanked by a short direct repeat of cellular DNA.

Integration can occur into most sites of the chromosome (11-13). However, certain sites within a genome can be used at a frequency several hundred-fold greater than chance (11, 14, 15). This finding suggests that there are hot and cold spots for the insertion of retroviral DNA and that integration is not a random process (16). A great amount of effort and the use of sensitive PCR1 assays have only identified a weak consensus sequence for HIV-1 integration (17-20), revealing the nonspecific nature of retroviral integration. However, various other factors are known to have a more important impact on target site selection than DNA sequence (21-26). A compilation of many studies confirms that a distortion of the target DNA provides attractive sites to the integration machinery (for review, see Ref. 27). Widened major or minor grooves resulting from protein binding or DNA modifications are more frequently chosen for integration over the same sites in linear or unmodified B-form DNA. Although features of target DNA are influential on target site selection, integrase itself has a significant role in determining the DNA site for integration. In vitro, integrases from different retroviruses each display a distinct and unique choice of integration sites when given an identical target DNA (14, 28-30). Therefore, a target site determinant exists within integrase.

Mutational analysis and proteolysis studies on integrase have identified three discrete functional domains: an N terminus containing a zinc-binding domain, a central catalytic core domain, and a C terminus (31). The core domain contains the three catalytic residues that comprise the DD(35)E motif, a highly conserved arrangement of active site amino acids among the superfamily of polynucleotidyl transferases (32-35). Although some of their properties have been elucidated, the exact roles of the terminal domains are presently not well established. The N terminus is able to chelate zinc, which promotes the multimerization of integrase and increases its catalytic activity (36-40). The N terminus may also be necessary to specifically recognize and bind viral DNA (41-43). The C terminus binds nonspecific DNA, of either viral or target features (43-47), and may participate in the oligomerization of integrase (48, 49). Because it has been difficult to obtain an x-ray crystallographic structure of full-length integrase due to its poor solubility and tendency to aggregate, the interactions between integrase and viral or target DNA have not been fully delineated.

Despite the incomplete understanding of integrase, the available data on the function and structure of retroviral integrases allow us to use a chimeric integrase strategy to study its role in target site preference. Both HIV-1 and FIV integrases have similar biochemical properties and share 37% amino acid identity (see Fig. 1) but have different integration preferences when using an identical target DNA (30, 50, 51). Therefore, swapping corresponding domains between the two integrases will likely produce a functional protein and provide informative results. Using HIV-1/FIV chimeric integrases, the target site-specifying domain of integrase was mapped in the central core region, within amino acid residues 49-238 (30).

This previously identified target site selection determinant contains a portion of the nonspecific DNA-binding domain, comprising amino acids 213-266 (44, 45). Whether this particular DNA-binding region is involved in dictating integration site preference is not known. The core domain is also able to bind target DNA as observed in in vitro assays with the dumbbell disintegration substrate that mimics a single-ended integration event (52). In addition, specific peptides within the core have been identified to be in close proximity to target DNA in UV cross-linking studies (53). Therefore, new HIV-1/FIV chimeric integrases were prepared to determine if the nonspecific DNA-binding domain was responsible for target site selection. Another set of chimeric proteins was made to test for the involvement of certain alpha  helices of the core domain in target site selection. Analysis of the above-mentioned chimeric integrases further narrowed the target site-specifying domain to within amino acid residues 49-187 and indicated that the alpha 2 helix, residues 118-121, participates in target site selection.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of HIV-1/FIV Chimeric Integrases-- Chimeric integrases were constructed in a similar manner as previously described (30) by swapping regions between HIV-1 and FIV integrases (Fig. 1). The first set of chimeric proteins is identified by a four-letter code (Fig. 2, constructs b, c, e-g). The first and last letters represent the identity of the N- and C-terminal domains, respectively. The letters within parentheses represent the core region, with the second and third letter denoting residues 49-185 and 186-237 for HIV-1 integrase or residues 51-187 and 188-238 for FIV integrase, respectively. The identity of the domain is noted as H for HIV-1 integrase and F for FIV integrase. F/(FH)/H represents an exchange of the DraI-HindIII restriction fragment encoding residues 186-288 from an HIV-1 integrase clone, pT7-7 (His)H-IN (54), with that from an FIV integrase clone, pT7-7 (His)F-IN (50), cut with DraI and HindIII. H/(HF)/F represents the exchange of the DraI-BamHI fragment encoding residues 188-281 from the FIV integrase plasmid with that from the HIV-1 integrase clone. The N-terminal deletion mutant, Delta N/(FH)/H, was constructed in a similar manner as described above, except the DraI-HindIII fragment from the HIV-1 integrase clone was swapped into pT7-7 (His)Delta N/F-IN (30), which lacks the first 50 amino acids at the N terminus of FIV integrase.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 1.   Alignment of amino acid sequences of HIV-1 and FIV integrases. The shaded boxes are the N- and C-terminal domains. Vertical lines denote identical amino acids between the two proteins. Dashes represent gaps introduced by the alignment. The highly conserved HHCC and DD(35)E motifs are highlighted by asterisks. The amino acids within the six alpha  helices of the core domain are marked with brackets.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 2.   Primary structures of HIV-1 and FIV integrases and their chimeric derivatives. The integrases are divided into three major domains: N terminus, core, and C terminus. The position of the nonspecific DNA-binding region (residues 213-266) is denoted by a bracket. The thick dashed lines indicate the boundary of the target site-specifying domain identified previously (30). The thin dashed line denotes the exchange position of chimeric integrases b, c, e-g. Darkly shaded boxes depict peptides derived from HIV-1 integrase (H-IN), and lightly shaded boxes depict peptides derived from FIV integrase (F-IN). The numbers in parentheses correspond to the amino acid residues of the indicated wild-type integrases (constructs a and d) included in each chimeric protein. The nomenclature of the chimeric protein is as described under "Experimental Procedures."

Two other chimeric integrases, H/(FH)/H and F/(HF)/F, containing a swap of only residues 49-187 between HIV-1 and FIV integrases, were also made. H/(FH)/H was constructed by inserting the 896-bp BglII-StyI restriction fragment from pH/F/H, which encodes the N-terminal region of HIV-1 integrase (30), into F/(FH)/H that was previously cut with BglII and StyI. F/(HF)/F was constructed by ligating the 700-bp BglII-BalI restriction fragment encoding the N-terminal domain of FIV integrase from pF/H/F (30) into pH/(HF)/F that was previously cut with BglII and BalI.

An additional set of chimeric integrases (Fig. 2, constructs h-j), which contained an exchange of certain alpha  helices within the core domain, is termed by designating the source of the helix or helices swapped within the parentheses with the letters H and F representing HIV-1 and FIV integrases, respectively. All DNA fragments containing the desired region of exchange were obtained by PCR, and the primers for amplification are listed in Table I. H(Falpha 3,4) was made by amplifying the region inclusive of the alpha 3 and alpha 4 helices of FIV integrase (residues 125-166) from H/(FH)/H using H/Fc-Sca as the forward primer and H/Fc-Afl as the reverse primer (see Table I). The PCR product was digested with ScaI and AflII and cloned into pT7-7(His)H-IN previously cut with ScaI and AflII. H(Falpha 3*,4), H(Falpha 3*), and H(Falpha 4) were all constructed using an overlapping PCR that generates 5' and 3' fragments of integrase, each containing a portion of the sequence of the desired helix to be swapped. In one reaction, the region from a position in the 5'-end of integrase to the helix of interest was amplified. In the other reaction, the region from the 3'-end of integrase to the helix of interest was amplified. The two PCR products containing a common overlapping region were annealed together and extended in the presence of 1 mM dNTPs (United States Biochemicals) and 2.5 units of Pfu polymerase (PFU Turbo, Stratagene). The extension reaction was carried out in the thermocycler (MJ Research, Inc.) programmed for 30 cycles, each cycle consisting of 1-min denaturation at 95 °C, 2-min annealing at 62 °C, and 4-min extension at 72 °C. The extended product was subjected to another round of PCR using C56S and INHind as the primers (Table I). The PCR products were resolved on a 1% agarose gel containing Tris acetate-EDTA and purified using the Qiaex II gel extraction kit (Qiagen), digested with MscI and HindIII, and ligated into pT7-7 (His)H-IN to complete the cloning of the various chimeric constructs. H(Falpha 3*4) was generated using H(Falpha 3,4) as the template and primers C56S and FINa3NQas for the 5' fragment, and primers FINa3NQss and INHind for the 3' fragment. H(Falpha 3*) was made using H(Falpha 3*,4) as the template and primers C56S and FINa3Hb5 for the 5' fragment. The 3' fragment was generated using pT7-7 (His)H-IN as the template and HINb5ss and INHind as the primers. The 5' fragment of H(Falpha 4) was generated using pT7-7 (His)H-IN as the template and C56S and H-FIN4as as the primers, whereas the 3' fragment was made using H(Falpha 3,4) as the template and H-FIN4ss and INHind as the primers.

                              
View this table:
[in this window]
[in a new window]
 
Table I
DNA sequence of PCR primers used to generate HIV-1/FIV chimeric integrases

All chimeric constructs were verified by restriction analysis and DNA sequencing using the dideoxyribonucleotide chain termination method (Thermosequenase kit, Amersham Pharmacia Biotech).

Expression and Purification of Chimeric Integrases-- The chimeric integrase constructs were transformed into Escherichia coli BL21(DE3). The cells were grown between 32 and 35 °C in 3-6 liters of Luria broth (LB) containing 80 µg/ml ampicillin. Protein expression was induced with isopropyl-1-thiol-beta -D-galactopyranoside at a final concentration of 0.4 mM when the optical density at 600 nm reached 0.8. Cultures were grown for an additional 4 h and then harvested. The cell pellets were stored at -80 °C.

Frozen bacterial pellets were thawed and resuspended in 120-200 ml of lysis buffer (20 mM HEPES, pH 7.5, 10% glycerol, 5 mM 2-mercaptoethanol, 2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 M NaCl, 0.2 mM EDTA, 0.5% Igepal, and 0.2 µg/ml hen egg lysozyme). The cell suspensions were sonicated and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was dialyzed overnight against buffer A (20 mM HEPES, pH 7.5, 10% glycerol, 5 mM 2-mercaptoethanol, 1 M NaCl, and 0.1% Igepal). The dialysate was then bound to Ni2+-nitrilotriacetic acid-agarose resin (Qiagen) for 3 h and then washed four times with 10 ml of 50 mM imidazole plus buffer A. The resin was packed into an Econo-column (Bio-Rad), and protein was eluted using a linear gradient from 50 to 500 mM imidazole plus buffer A. Fractions were collected and analyzed by electrophoresis through a 12% SDS-polyacrylamide gel. Peak fractions containing integrase were pooled, and 10 mM CHAPS was added. The protein was concentrated by Centricon-10 columns (Amicon) and dialyzed against buffer C (20 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.5 M NaCl, 20% glycerol, 1 mM DTT, and 10 mM CHAPS). The histidine tag preceding the various integrases was removed by incubating the protein with 80-100 NIH units of human thrombin (Sigma Chemical Co.) per milligram of integrase and passing the digested protein through a cation exchange chromatography column packed with high performance SP-Sepharose (Amersham Pharmacia Biotech). The protein solution was diluted in buffer D (20 mM HEPES, pH 7.0, 0.1 mM EDTA, 10% glycerol, 10 mM DTT, and 10 mM CHAPS) to 0.1 mg/ml before being loaded onto the SP-Sepharose column. A gradient from 0 to 2 M NaCl in buffer D was used to elute the protein from the column. Peak fractions containing the non-His-tagged protein were pooled and concentrated by Centricon-10 columns (Amicon). The protein was then dialyzed against storage buffer (20 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.3 M NaCl, 20% glycerol, 10 mM DTT, and 10 mM CHAPS) overnight and stored at -80 °C. Protein concentrations were determined using bovine serum albumin as the standard in the Bradford assay (Bio-Rad).

In Vitro Assays for Integrase Activity-- All assays were performed as previously described (2). The HIV-1 Y-mer disintegration substrate was prepared by annealing T1 (5'-CAGCAACGCAAGCTTG-3') to T3 (5'-GTCGACCTGCAGCCCAAGCTTGCGTTGCTG-3'), H-V1/T2 (5'-ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC-3'), and H-U5V2 (5'-ACTGCTAGAGATTTTCCACAT-3'). Underlined letters denote the invariant CA/TG dinucleotide pair. The T1 strand was labeled at the 5'-end with [gamma -32P]ATP and T4 polynucleotide kinase (New England BioLabs). The substrate for 3'-end-joining activity, which mimics a pre-processed HIV-1 U5 end, was prepared by annealing H-U5V1-2 (5'-ATGTGGAAAATCTCTAGCA-3') to H-U5V2 (5'-ACTGCTAGAGATTTTCCACAT-3') or H-U5V1L-2 (5'-CCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3') to H-U5V2L (5'-ACTGCTAGAGATTTTCCACACTGACTAAAAGG-3'). The target DNA substrate was prepared by annealing T5 (5'-CGACGCGTGCTAGGCCTG-3') to T6 (5'-ACAGGCCTAGCACGCGTCG-3'). The annealed substrate was labeled at the 3'-end of T5 with [alpha -32P]TTP and exonuclease-free Klenow fragment of E. coli DNA polymerase I.

Typically, in a 20-µl volume, 0.1 pmol of the DNA substrate was incubated with integrase for 60 min at 37 °C in a reaction buffer containing a final concentration of 20 mM HEPES, pH 7.5, 10 mM MnCl2, 30 mM NaCl, 10 mM DTT, 0.01 mM EDTA, 1 mM CHAPS, and 0.05% Nonidet P40. The reaction was stopped by the addition of 18 mM EDTA, pH 8.0. The reaction products were mixed with an equal volume of loading buffer (98% deionized formamide, 10 mM EDTA, pH 8.0, 0.05% bromphenol blue, 0.05% xylene cyanol) and heated at 90 °C for 3 min before analysis by electrophoresis on 15% polyacrylamide gels with 7 M urea in Tris borate-EDTA buffer. Quantitation of the products was carried out with a Molecular Dynamics PhosphorImager.

PCR-based Integration Assay-- The PCR-based integration assay is used for analyzing the target DNA sites chosen for integration (26, 30). Individual integration events along a target DNA are amplified through PCR to show the distribution and frequency of integration. The donor substrate, a 30-mer that mimics the pre-processed HIV-1 U5 end, was prepared by annealing H-U5V1L-2 to H-U5V2L and preincubated with integrase for 5 min at room temperature. One microgram of target DNA, pBluescript KSII+, was added after preincubation, and the reaction proceeded for 50 min at 37 °C. The reaction was terminated with the addition of 15 mM EDTA and 10 µg of tRNA. The DNA was extracted with phenol-chloroform, ethanol-precipitated, and resuspended in 40 µl of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 7.5. A 5-µl aliquot was then added to a reaction buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.001% gelatin, 1.5 mM MgCl2, 200 µM of dNTPs, 5 pmol of primers, and 1 unit of Taq polymerase (Taq 2000, Stratagene) in a final volume of 20 µl. The forward primer for the PCR reaction, H-U5VL-2, is complementary to the U5 donor substrate and was prepared by mixing 0.05 µM 5'-end-labeled H-U5VL-2 and 0.20 µM unlabeled H-U5VL-2. The reverse primer in the reaction, BS+ (5'-CATTAATGCAGCTGGCACGA-3'), anneals to a position on the plus strand (nucleotides 988-969) of the pBluescript KS II+ and was used at a concentration of 0.25 µM. Integration events were amplified by 30 cycles of PCR: 45 s at 94 °C, 1 min at 55 °C, and 2 min at 72 °C. The radiolabeled PCR products were resolved on a 5% denaturing polyacrylamide gel containing 7 M urea in a Tris borate-EDTA buffer and analyzed with a PhosphorImager (Molecular Dynamics).

Western Immunoblotting-- The wild-type or chimeric protein was suspended in lysis buffer (62.5 mM Tris-HCl, pH 6.8, 0.2% SDS, 5% 2-mercaptoethanol, 10% glycerol) and loaded onto a 12% SDS-polyacrylamide gel. After electrophoresis, the protein was transferred to nitrocellulose membranes (pore size, 0.45 µm; Micron Separations, Inc.) for probing with anti-integrase antibody. HIV-1 immune antiserum was purchased from the Scripps Research Institute. A monoclonal antibody against FIV integrase was obtained from Dr. John Elder at the Scripps Research Institute. Western blot analysis was carried out with an alkaline phosphatase detection kit as specified by the manufacturer (Bio-Rad).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification and Catalytic Activity of Chimeric Integrases-- All chimeric integrases were purified using nickel-chelating affinity chromatography followed by a second purification step using cation exchange chromatography. Western blot analyses using a monoclonal antibody to FIV integrase or HIV-1 immune antiserum showed that the apparent masses of all proteins were consistent with those predicted by the amino acid sequence, at ~32 kDa (Fig. 3A). The chimera, Delta N/(FH)/H, migrated faster at around 26 kDa due to the absence of the first 50 amino acid residues at the N terminus.


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 3.   Western blot analysis and catalytic activity of HIV-1/FIV chimeric integrases. A, immunoblotting. Purified wild-type HIV-1 integrase and HIV-1/FIV chimeric integrases (20-40 pmol) were electrophoresed on a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and then probed with HIV-1 immune antiserum as described under "Experimental Procedures." All chimeric proteins except Delta N/(FH)/H (lane 3) had a mobility about the size of the full-length wild-type HIV-1 integrase (lane 1) at 32 kDa. Delta N/(FH)/H lacks the first 50 amino acids of integrase and had a mobility around 26 kDa. B, catalytic activity. The reaction was carried out with 5 nM of the Y-oligomer disintegration substrate and the indicated concentration of wild-type or chimeric integrase. The open arrowhead indicates the position of the 5'-end-labeled T1 strand (16-mer) of the substrate, and the filled arrowhead indicates the position of the disintegration product (30-mer). The products between the 16-mer and 30-mer are either the result of reintegration of the released viral end from the substrate (43, 55) or nonspecific alcoholysis of the disintegration product (57). A diagrammatic depiction of the disintegration reaction is shown on the right. Thick lines represent viral DNA sequences, and thin lines represent target DNA sequences. Asterisks denote the positions of the label. Disintegration is a coupled cleavage-ligation that results in rejoining of the target DNA and the release of the viral DNA.

The catalytic activity of the various chimeric integrases was determined using the disintegration assay that measures the ability of the protein to revert a substrate resembling the integration intermediate to its viral and target DNA components (55). Identical to the 3'-end-joining reaction, disintegration is a concerted cleavage-ligation reaction (55, 56). Disintegration has been useful in studying the catalytic and kinetic properties of mutant and truncated integrases and, therefore, was chosen to determine the catalytic activity of chimeric integrases (Fig. 3B). All proteins except H(Falpha 3,4) and H(Falpha 3*,4) were active as evidenced by their ability to convert the labeled 16-mer in the Y-mer substrate into a labeled 30-mer product. Chimeric proteins Delta N/(FH)/H, F/(FH)/H, and H/(FH)/H displayed between 40 and 80% activity of that of wild-type HIV-1 or FIV integrase. H/(HF)/F and F/(HF)/F displayed a lower disintegration activity, between 5 and 10% of that seen with both wild-type integrases. The bands between the substrate and product might be the result of nonspecific reintegration of the viral DNA sequence into the labeled DNA (43, 55) or nonspecific nuclease activity by integrase (57).

H(Falpha 3,4) had a very weak disintegration activity, and 3'-end-joining activity was barely detectable using the sensitive PCR-based assay (data not shown). This chimera showed a poor solubility and a strong tendency to aggregate. We hypothesized that unfavorable interactions between the adjacent beta  sheets of HIV-1 integrase and the exchanged alpha  helices from FIV integrase might cause misfolding and improper conformation of the active site. This problem was further investigated by constructing a molecular model using Insight II (Biosym). Three amino acid residues, Met-126, Glu-127, and Met-133, within the unexposed face of the FIV alpha 3 helix were identified to possibly affect the folding of integrase due to steric hindrance. In addition, H(Falpha 3*,4) contains the first two amino acids of the FIV alpha 3 helix, Asn-125 and Gln-126, which were previously excluded in H(Falpha 3,4). However, reversions to the corresponding HIV-1 residues Val-126, Lys-127, and Ala-133 did not restore stability or integration activity of the new chimeric protein H(Falpha 3*,4) (data not shown). Because of the lack of activity, these chimeric integrases were not further analyzed.

The Target Site-specifying Motif Resides within Amino Acid Residues 49-187 of the Core Domain-- To identify the region responsible for target site selection, integration patterns of chimeric integrases, as defined by the distribution and frequency of integration events (30), were determined using both the oligonucleotide-based and the PCR-based 3'-end joining assays and compared with those of wild-type HIV-1 and FIV integrases. New chimeric integrases were designed to assess whether the nonspecific DNA-binding region, amino acid residues 213-266 (44, 45), dictates the selection of host integration sites, because a portion of this domain is included in the previously identified target site-specifying domain of residues 49-238 (30). The integration pattern from the oligonucleotide-based 3'-end joining assay was only informative for chimeric proteins F/(HF)/F, Delta N/(FH)/H, and F/(FH)/H (Fig. 4A, lanes 3, 5, and 6, respectively). The activity of H/(HF)/F and H/(FH)/H (Fig. 4A, lanes 2 and 7, respectively) was very low, and an integration pattern for these proteins could not be determined. In this assay, an unlabeled, pre-processed substrate that mimics the HIV-1 U5 long terminal repeat was used as the donor DNA, and a 19-mer of arbitrary sequence labeled at one of the 3' ends served as the target DNA. The slower migrating products above the labeled target substrate were indicative of integration events into the target DNA. HIV-1 integrase formed integration products ranging from 20 to 26 nucleotides in length with the 26-mer being the most abundant product (Fig. 4A, lane 1). There were also the two characteristic doublet products, one at the 29-mer and 30-mer, and the other at the 32-mer and 33-mer. With FIV integrase (Fig. 4A, lane 4), the most abundant product was the 22-mer, and the other less abundant products were 20, 21, 23, and 26 nucleotides in length. Chimeric proteins Delta N/(FH)/H (Fig. 4A, lane 5) and F/(FH)/H (Fig. 4A, lane 6) displayed a pattern similar to that of FIV integrase, with the 22-mer being the most prominent product followed by the 21-mer and 23-mer products. The integration pattern of F/(HF)/F (Fig. 4A, lane 3) was similar to that of HIV-1 integrase, with the 26-mer being the most abundant product.


View larger version (69K):
[in this window]
[in a new window]
  		Fig. 4.   Patterns of integration site preference of HIV-1/FIV chimeric integrases containing an exchange of the nonspecific DNA-binding region. A, oligonucleotide-based assay. A pre-processed 21-bp HIV-1 U5 substrate (H-U5V1-2/H-U5V2, 15 nM) was preincubated in the absence (-, lane 8) or presence of 200 nM of wild-type or chimeric integrase for 10 min at room temperature in the standard reaction buffer. This was followed by adding 5 nM 3'-end-labeled target DNA (T5/T6) and incubating at 37 °C for 60 min. The open arrowhead indicates the position of the labeled T5 strand (19-mer) of the substrate. The integration products are marked by IP, and the lengths in nucleotides are indicated on the right. The asterisk corresponds to a contaminant present in the unreacted, labeled substrate and is not an integration product. B, PCR-based assay. A pre-processed 30-bp HIV-1 U5 DNA (H-U5V1L-2/H-U5V2L, 15 nM) was preincubated with wild-type or chimeric integrase for 5 min at room temperature. The concentrations of the wild-type or chimeric integrase used were 150 (HIV, lanes 2, 4, and 6; FIV, lanes 3, 8, 10, and 12), 300 (Delta N/(FH)/H, lane 9; F/(FH)/H, lane 11; H/(FH)/H, lane 13), and 500 nM (H/(HF)/F, lane 5; F/(HF)/F, lane 7). This was followed by the addition of 1 µg of pBluescript KS II+ as the target DNA and incubation for 50 min at 37 °C. The reaction products were amplified by PCR as described under "Experimental Procedures." The identities of the wild-type and chimeric integrases are as labeled above each lane. Lane 1 represents a complete reaction without the addition of enzymes. The upper and lower horizontal lines on the left represent the positions of the DNA size markers at 234 and 194 nucleotides, respectively. Filled circles denote the integration hot spots characteristic of HIV-1 integrase, and open circles denote characteristic FIV integration hot spots.

Integration patterns of wild-type HIV-1 and FIV integrases and all chimeric proteins were also obtained using the PCR-based integration assay. H/(HF)/F and F/(HF)/F produced a similar integration pattern to that of HIV-1 integrase (Fig. 4B, lanes 4-7). The characteristic HIV-1 integration events displayed by H/(HF)/F and F/(HF)/F are denoted as filled circles at 170, 210, and between 230 and 280 nucleotides. On the other hand, integration patterns from Delta N/(FH)/H, F/(FH)/H, and H/(FH)/H were similar to that of wild-type FIV integrase (Fig. 4B, lanes 8-13). These chimeras displayed the most characteristic FIV integration events that are marked as open circles at 180, around 200, and between 230 and 260 nucleotides. Thus, results from both oligonucleotide- and PCR-based assays clearly indicated that residues 49-187 of integrase contain the target site-specifying motif and that the nonspecific DNA-binding region located within residues 213-266 is not involved in target site selection. In addition, these data confirmed the previous finding that the N and C termini are not involved in the selection of target sites during integration (30).

The Involvement of Specific alpha  Helices within the Core Domain of Integrase in Target Site Selection-- To identify the minimal motif responsible for target site selection, predicted target DNA-binding regions within the core were investigated for their participation in the choice for integration sites. Based on the x-ray crystal structure of the truncated HIV-1 integrases (58-60) and the molecular model of an N-terminal-truncated HIV-1 integrase bound to viral and target DNA (61), we propose that the target site-specifying motif may be located within the alpha 2, alpha 3, or alpha 4 helix. To test this hypothesis, the helix of interest in HIV-1 integrase was replaced with the corresponding helix of FIV integrase resulting in chimeras H(Falpha 2), H(Falpha 3*), and H(Falpha 4) (Fig. 2, constructs h-j).

Chimeric proteins H(Falpha 3*) and H(Falpha 4) were active in disintegration assays (Fig. 5A). H(Falpha 4) displayed ~5% of the level of activity of HIV-1 and FIV integrases, whereas H(Falpha 3*) displayed a similar level of activity to both wild-type integrases. Because H(Falpha 3*) retained a high level of activity, the target site preference of this chimeric protein could be assessed by the oligonucleotide-based integration assay (Fig. 5B). This assay was identical to that described previously for Fig. 4A, except that the unlabeled donor U5 DNA was 30-nt in length instead of 19-nt. Therefore, the integration patterns of wild-type HIV-1 and FIV integrases in Figs. 4A (lanes 1 and 4, respectively) and 5B (lanes 2 and 6, respectively) were identical except that the products in Fig. 5B were shifted by 11 nucleotides. Due to the length difference of the donor DNA, the integration pattern of HIV-1 integrase in Fig. 5B (lane 2) is characterized by the most abundant product of 37 nucleotides in length and the two doublets at 40/41 and 43/44 nucleotides. FIV integrase (Fig. 5B, lane 6) had its most abundant product at the 33-mer followed by the other characteristic integration products of a lesser frequency of 31, 32, 34, and 37 nucleotides in length. The integration pattern of H(Falpha 3*) (Fig. 5B, lane 4) was identical to that observed with HIV-1 integrase, whereas the activity of H(Falpha 4) was too weak to be detected using the oligonucleotide-based assay (Fig. 5B, lane 5). In the PCR-based assay, both H(Falpha 3*) (Fig. 5C, lane 1) and H(Falpha 4) (Fig. 5C, lane 8) displayed a target site preference typical of that of wild-type HIV-1 integrase (Fig. 5C, lanes 2 and 7) as marked by filled circles at 170, 210, and between 230 and 280 nucleotides. Neither of the chimeric proteins displayed the characteristic FIV integration events marked as open circles (Fig. 5C, lanes 4 and 9). These results indicated that the target site-specifying motif is not located within the alpha 3 and alpha 4 helices of the core domain.


View larger version (46K):
[in this window]
[in a new window]
  		Fig. 5.   Catalytic activity and integration pattern of HIV-1/FIV chimeric integrases containing an exchange of alpha  helices in the core domain. A, catalytic activity. The reaction was carried out as described in Fig. 3. The concentration of the wild-type or chimeric integrase was 25 nM. The open arrowhead indicates the position of the 5'-end-labeled T1 strand (16-mer) of the substrate, and the filled arrowhead indicates the position of the disintegration product (30-mer). B, oligonucleotide-based assay. The assay was essentially identical to that described earlier in Fig. 4A, except a 30-mer pre-processed HIV-1 U5 substrate was used as the donor DNA. Symbols have the same significance as in Fig. 4A. C, PCR-based assay. The reaction condition was identical to that described earlier in Fig. 4B, except that the enzyme concentration in all reactions is 25 nM. Symbols have the same significance as in Fig. 4B. Lane 5 contains the DNA size markers with the lengths in nucleotides on the left, and lane 6 represents a reaction carried out in the absence of enzymes. Integration hot spots shared between H(Falpha 2) (lane 3) and HIV-1 integrase (lanes 2 and 7) and between H(Falpha 2) and FIV integrase (lanes 4 and 9) are denoted by filled and open triangles, respectively.

The position of the alpha 2 helix in the core domain also appeared to be a good candidate for involvement in target site selection, although only one out of the four amino acids is different between HIV-1 and FIV integrases. Amino acid 119 in HIV-1 integrase is a Ser, and the corresponding residue in FIV integrase is a Pro that is in close proximity to the active site residue Asp 116. H(Falpha 2) was made by replacing the Ser residue at position 119 in HIV-1 integrase to a Pro residue to resemble the alpha 2 helix of FIV integrase. Like H(Falpha 3*), H(Falpha 2) was active in catalyzing disintegration and 3'-end joining (Fig. 5). The integration pattern produced by H(Falpha 2) using the oligonucleotide-based assay (Fig. 5B, lane 3) was intermediate to those of HIV-1 and FIV integrases (Fig. 5B, lanes 2 and 6, respectively). For instance, both the 33-mer (the most prominent for FIV integrase) and the 37-mer (the most prominent for HIV-1 integrase) products were of equivalent abundance. Also, the relative intensity of integration products between 32 and 34 nucleotides resembled that displayed by FIV integrase, whereas the pattern between the positions of 38 and 44 nucleotides resembled that seen with HIV-1 integrase. In the PCR assay, H(Falpha 2) displayed several typical integration products for FIV integrase marked as open triangles at 180, around 200, and between 230 and 260 nucleotides, as well as some characteristic HIV-1 integration events marked as filled triangles at 170, 210, and between 230 and 280 nucleotides (Fig. 5C, lane 3). Although the alpha 2 helix alone was not able to dictate the sites chosen for integration on target DNA, the intermediate integration pattern obtained suggests that a target site-specifying motif comprises multiple regions or a larger region inclusive of the alpha 2 helix is responsible for target site selection.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integration of retroviral DNA into the host chromosome is inherently a mutagenic event. Integration can have significant consequences to the well-being of the host cell, as exemplified by the ability of retroviruses to produce tumors in infected animals after natural or experimental infections (1). Because integration is essential for productive viral infection, the sites chosen for integration may also be relevant to the virus to ensure its perpetuation. Integrases from different retroviruses have different target site preferences in vitro when using an identical target DNA substrate DNA (14, 28-30). Therefore, a motif within integrase dictating this preference must exist, and integrase may play a significant role in selecting target DNA sites for integration in vivo. We hypothesized that the target site-specifying motif is either part of a nonspecific DNA-binding region or comprises residues that are in close proximity to target DNA. HIV-1/FIV chimeric integrases were constructed for this study, and integration data from oligonucleotide- and PCR-based assays showed that target site selection resides within residues 49-187 of the integrase core and not within the nonspecific DNA-binding region, residues 213-266 (44, 45). Additional chimeras were also made to test whether regions that were predicted to be in close proximity to target DNA within residues 49-187 were involved in target site selection. Integration data revealed that alpha  helices 3 and 4 of the core domain (residues 123-166) were not responsible for choosing integration sites on target DNA. However, the alpha 2 helix appeared to be involved in target site selection, because the chimeric integrase H(Falpha 2) preferred integration sites favored by both HIV-1 and FIV integrases. Although alpha 2 helix alone is not sufficient for conferring target site specificity, we believe that the alpha 2 helix is part of a larger or multiple motifs responsible for target site selection.

The location of the target and viral DNA-binding regions of integrase has not been well established, and very little is known about the interaction between integrase and target DNA. A nonspecific DNA-binding region in the C terminus consisting of amino acid residues 213-266 was identified using deletion mutants of integrase in filter binding assays and Southwestern blotting (45-47, 62, 63). The nonspecific binding to DNA by this region is independent of divalent metal ion as observed in UV cross-linking studies (44). Our results showed that, despite the ability to bind target DNA, the nonspecific DNA-binding domain within residues 213-266 is not involved in target site selection.

Another DNA-binding region is identified in the core domain and consists of amino acid residues 50-186. The DNA binding of this region is divalent cation-dependent and substrate-specific. In the presence of Mn2+, this region is able to bind a disintegration substrate that mimics the single-ended integration event (44, 52). This DNA-binding region recognizes key features of viral DNA substrates and may be responsible for viral DNA specificity (19, 41, 52). In addition to viral DNA, UV cross-linking studies with HIV-1 integrase and a dumbbell disintegration substrate have identified residues 49-69 and 139-152 to be in close proximity to target DNA. The alpha 2 (residues 118-121), alpha 3 (residues 123-133), and alpha 4 (residues 148-166) helices were chosen as candidates for involvement in target site selection based on their position in the core domain and close proximity to target DNA by molecular modeling (58, 61). In this model, specific residues Ser-153 and Lys-160 of the alpha 4 helix were proposed to be involved in hydrogen bonding with phosphate oxygens of nucleotides in target DNA. Also, type II restriction endonucleases and HIV-1 integrase share some structural similarities, and the alpha 3 helix of HIV-1 integrase has been suggested to correspond to the DNA-binding long helices of endonucleases (64). In addition, neither the alpha 3 nor the alpha 4 helix is highly conserved between HIV-1 and FIV integrases, further suggesting that these helices are ideal for dictating a characteristic integration pattern. In the case of the alpha 2 helix, not only is it positioned in close proximity to target DNA and the active site residue Asp-116, it has one divergent residue that is structurally different between the HIV-1 and FIV integrases. Amino acid 119 is a Ser in HIV-1 integrase, and the corresponding residue in FIV integrase is a Pro. Substituting the Ser at position 119 with Thr or Gly is one of the most frequently observed integrase variants in viral isolates from HIV-1-infected patients (65).

The integration patterns produced by the chimeric integrases containing specific exchanges of alpha  helices confirmed the importance of the alpha 2 helix in target site selection and ruled out the involvement of the alpha 3 and alpha 4 helices. These results indicated that, although the residues Ser-153 and Lys-160 of the alpha 4 helix may be positioned to bind target DNA, they are not involved in target site selection.

The intermediate integration pattern obtained by H(Falpha 2) in the integration assay opens the door for further experiments to identify the minimal target site-specifying motif. The change from the inherent Ser at position 119 in HIV-1 integrase to Pro alters the side-chain moiety significantly and thereby may affect the interactions with the phosphodiester backbone of the target and the subsequent choice of integration sites. Perhaps the rest of the target site-specifying motif lies in proximity to the active site and comprises either a larger region encompassing the alpha 2 helix or multiple regions of integrase, such as the alpha 1 helix, beta 1 or beta 4 sheets of the core domain. Additional chimeric integrases can be constructed by exchanging regions outside of residues 123-166 together with the alpha 2 helix. Because of its location in the core domain and its involvement in maintaining the dimer interface, it seems less likely that residues 167-187 would be involved in target site selection. Therefore, future studies on identifying the minimal target site-specifying motif should probably focus on regions between residues 49-122.

The mechanisms of nonspecific and specific DNA recognition by catalytic molecules, such as transposases, ribozymes, and the restriction endonucleases vary, and our understanding of this process is limited. Nonspecific DNA binding in many cases does not require a metal ion, in contrast to specific recognition of a target site for proper catalysis (66, 67). Many class type II restriction endonucleases, such as BamHI and EcoRI, bind DNA nonspecifically and scan the substrate by either linear or facilitated diffusion to locate their cognate sites (68), whereas the type IIe endonuclease EcoRII mediates specific DNA recognition allosterically (69, 70). Perhaps in the case of integrase, the nonspecific DNA-binding site in the C terminus is responsible for scanning the target DNA by linear or facilitated diffusion and for stabilizing the recognition of the integration site by the target site-specifying motif in the core domain. It is also a possibility that a conformational change induced by the binding of divalent metal ions or viral DNA is necessary to unmask the target site-specifying motif (71).

Because DNA distortion and local DNA structures rather than primary DNA sequence are important factors in target site selection, perhaps specific local structures are more preferred by one integrase than the other. The target DNA may be complementary or made to fit the shape of the DNA-binding region, providing an energetically favorable interaction that results in DNA recognition. Shape recognition resulting in DNA binding is seen for many DNA-binding proteins and enzymes in crystal structures and molecular modeling (72). Therefore, the target site-specifying motifs of HIV-1 and FIV integrases need not be so divergent for choosing characteristic sites since a simple kink or bulky side-chain moiety may be enough to alter DNA recognition. In addition, small differences in residue composition can alter the spatial arrangement of waters, which may affect DNA interactions through hydrogen bonding.

Attempts to modify restriction endonucleases to alter cleavage at a sequence similar to its cognate site have generally been unsuccessful. For instance, even though BglII and BamHI share a similar catalytic core topology, replacement of BglII's DNA recognition motif with that of BamHI does not alter the catalysis to the cognate BamHI site (73, 74). The result indicates that DNA recognition is not solely dependent on the direct contacts made between the amino acids and the nucleotides but that the overall conformation of the protein also plays a critical role as seen in crystallographic studies (74). This may also be a reason why we were not able to see a complete switch in integration patterns with the H(Falpha 2) chimeric integrase. Changing the other amino acids involved in stabilizing the active conformation or other DNA contacts may be necessary to see a complete switch for the choice of integration sites.

Protein-DNA recognition is key for many cellular and viral processes, and identification of the motif involved in retroviral target site selection will provide a better understanding of the various mechanisms used by enzymes for this process. Further knowledge in this field may reveal new ways to manipulate the DNA specificity of enzymes for their use as therapeutics or molecular tools. Furthermore, pinpointing the target site-specifying motif within integrase will also help further evolve the model of the molecular interactions between integrase and target DNA, because structural data have been difficult to obtain. Currently, the chimeric protein approach has allowed us to test the role of several possible target DNA-binding regions for their involvement in target site selection and has narrowed the target site-specifying determinant within specific regions of the core domain.

    ACKNOWLEDGEMENTS

We thank Michael Hankinson for assistance on molecular modeling, Reid Johnson for helpful discussion, Sonia Lee for technical assistance, and Diane Martin for graphic support.

    Note Added in Proof

The importance of residue 119 in the alpha 2 helix of HIV-1 integrase in target site selection has also been described by Harper et al. (75).

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant R01 CA68859.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of an Esther Hayes Student Research Award from the UCLA AIDS Institute (National Institutes of Health Grant AI28697).

§ Visiting Professor, Department of Biotechnology, Chung-Ang University, S. Korea.

|| To whom correspondence should be addressed: Dept. of Molecular and Medical Pharmacology, Molecular Biology Institute and UCLA AIDS Institute, UCLA School of Medicine, 23-133 CHS, 10833 Le Conte Ave., Los Angeles, CA 90095. Tel.: 310-825-9600; Fax: 310-825-6267; E-mail: schow@mednet.ucla.edu.

Published, JBC Papers in Press, October 3, 2001, DOI 10.1074/jbc.M107365200

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; HIV-1, human immunodeficiency virus type 1; FIV, feline immunodeficiency virus; bp, base pair(s); dNTP, deoxyribonucleotide triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; nt, nucleotide(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Brown, P. O. (1997) in Integration. Retroviruses (Coffin, J. M. , Hughes, S. H. , and Varmus, H. E., eds) , pp. 161-203, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
2. Chow, S. A. (1997) Methods 12, 306-317[CrossRef][Medline] [Order article via Infotrieve]
3. Katz, R. A., and Skalka, A. M. (1994) Annu. Rev. Biochem. 63, 133-173[CrossRef][Medline] [Order article via Infotrieve]
4. Brown, P. O., Bowerman, B., Varmus, H. E., and Bishop, J. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2525-2529[Abstract/Free Full Text]
5. Fujiwara, T., and Mizuuchi, K. (1988) Cell 54, 497-504[CrossRef][Medline] [Order article via Infotrieve]
6. Katzman, M., Katz, R. A., Skalka, A. M., and Leis, J. (1989) J. Virol. 63, 5319-5327[Abstract/Free Full Text]
7. Bushman, F. D., Fujiwara, T., and Craigie, R. (1990) Science 249, 1555-1558[Abstract/Free Full Text]
8. Craigie, R., Fujiwara, T., and Bushman, F. (1990) Cell 62, 829-837[CrossRef][Medline] [Order article via Infotrieve]
9. Katz, R. A., Merkel, G., Kulkosky, J., Leis, J., and Skalka, A. M. (1990) Cell 63, 87-95[CrossRef][Medline] [Order article via Infotrieve]
10. Bushman, F. D., and Craigie, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1339-1343[Abstract/Free Full Text]
11. Withers-Ward, E. S., Kitamura, Y., Barnes, J. P., and Coffin, J. M. (1994) Genes Dev. 8, 1473-1487[Abstract/Free Full Text]
12. Shih, C.-C., Stoye, J. P., and Coffin, J. M. (1988) Cell 53, 531-537[CrossRef][Medline] [Order article via Infotrieve]
13. Hughes, S. H., Shank, P. R., Spector, D. H., Kung, H. J., Bishop, J. M., Varmus, H. E., Vogt, P. K., and Breitman, M. L. (1978) Cell 15, 1397-1410[CrossRef][Medline] [Order article via Infotrieve]
14. Pryciak, P. M., Muller, H. P., and Varmus, H. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9237-9241[Abstract/Free Full Text]
15. Pryciak, P. M., Sil, A., and Varmus, H. E. (1992) EMBO J. 11, 291-303[Medline] [Order article via Infotrieve]
16. Craigie, R. (1992) Trends Genet. 8, 187-190[CrossRef][Medline] [Order article via Infotrieve]
17. Bor, Y.-C., Miller, M. D., Bushman, F. D., and Orgel, L. E. (1996) Virology 222, 283-288[CrossRef][Medline] [Order article via Infotrieve]
18. Carteau, S., Hoffmann, C., and Bushman, F. (1998) J. Virol. 72, 4005-4014[Abstract/Free Full Text]
19. Esposito, D., and Craigie, R. (1998) EMBO J. 17, 5832-5843[CrossRef][Medline] [Order article via Infotrieve]
20. Zhou, H., Rainey, G. J., Wong, S. K., and Coffin, J. M. (2001) J. Virol. 75, 1359-1370[Abstract/Free Full Text]
21. Bor, Y.-C., Bushman, F. D., and Orgel, L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10334-10338[Abstract/Free Full Text]
22. Katz, R. A., Gravuer, K., and Skalka, A. M. (1998) J. Biol. Chem. 273, 24190-24195[Abstract/Free Full Text]
23. Kitamura, Y., Ha Lee, Y. M., and Coffin, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5532-5536[Abstract/Free Full Text]
24. Pruss, D., Bushman, F. D., and Wolffe, A. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5913-5917[Abstract/Free Full Text]
25. Pruss, D., Reeves, R., Bushman, F. D., and Wolffe, A. P. (1994) J. Biol. Chem. 269, 25031-25041[Abstract/Free Full Text]
26. Pryciak, P. M., and Varmus, H. E. (1992) Cell 69, 769-780[CrossRef][Medline] [Order article via Infotrieve]
27. Holmes-Son, M. L., Appa, R. S., and Chow, S. A. (2001) Adv. Genet. 43, 33-69[Medline] [Order article via Infotrieve]
28. Bushman, F. D., and Wang, B. (1994) J. Virol. 68, 2215-2223[Abstract/Free Full Text]
29. Katzman, M., and Sudol, M. (1995) J. Virol. 69, 5687-5696[Abstract]
30. Shibagaki, Y., and Chow, S. A. (1997) J. Biol. Chem. 272, 8361-8369[Abstract/Free Full Text]
31. Engelman, A., Bushman, F. D., and Craigie, R. (1993) EMBO J. 12, 3269-3275[Medline] [Order article via Infotrieve]
32. Fayet, O., Ramond, P., Polard, P., Prère, M. F., and Chandler, M. (1990) Mol. Microbiol. 4, 1771-1777[CrossRef][Medline] [Order article via Infotrieve]
33. Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. P. G., and Skalka, A. M. (1992) Mol. Cell. Biol. 12, 2331-2338[Abstract/Free Full Text]
34. Engelman, A., and Craigie, R. (1992) J. Virol. 66, 6361-6369[Abstract/Free Full Text]
35. Polard, P., and Chandler, M. (1995) Mol. Microbiol. 15, 13-23[CrossRef][Medline] [Order article via Infotrieve]
36. Burke, C. J., Sanyal, G., Bruner, M. W., Ryan, J. A., LaFemina, R. L., Robbins, H. L., Zeft, A. S., Middaugh, C. R., and Cordingley, M. G. (1992) J. Biol. Chem. 267, 9639-9644[Abstract/Free Full Text]
37. Bushman, F. D., Engelman, A., Palmer, I., Wingfield, P., and Craigie, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3428-3432[Abstract/Free Full Text]
38. Lee, S. P., and Han, M. K. (1996) Biochemistry 35, 3837-3844[CrossRef][Medline] [Order article via Infotrieve]
39. Lee, S. P., Xiao, J., Knutson, J. R., Lewis, M. S., and Han, M. K. (1997) Biochemistry 36, 173-180[CrossRef][Medline] [Order article via Infotrieve]
40. Zheng, R., Jenkins, T. M., and Craigie, R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13659-13664[Abstract/Free Full Text]
41. Katzman, M., and Sudol, M. (1998) J. Virol. 72, 1744-1753[Abstract/Free Full Text]
42. van den Ent, F. M. I., Vos, A., and Plasterk, R. H. A. (1999) J. Virol. 73, 3176-3183[Abstract/Free Full Text]
43. Vincent, K. A., Ellison, V., Chow, S. A., and Brown, P. O. (1993) J. Virol. 67, 425-437[Abstract/Free Full Text]
44. Engelman, A., Hickman, A. B., and Craigie, R. (1994) J. Virol. 68, 5911-5917[Abstract/Free Full Text]
45. Puras Lutzke, R. A., Vink, C., and Plasterk, R. H. A. (1994) Nucleic Acids Res. 22, 4125-4131[Abstract/Free Full Text]
46. Vink, C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1993) Nucleic Acids Res. 21, 1419-1425[Abstract/Free Full Text]
47. Woerner, A. M., Klutch, M., Levin, J. G., and Markus-Sekura, C. J. (1992) AIDS Res. Hum. Retroviruses 8, 2433-2437
48. Andrake, M. D., and Skalka, A. M. (1995) J. Biol. Chem. 270, 29299-29306[Abstract/Free Full Text]
49. Puras Lutzke, R. A., and Plasterk, R. H. A. (1998) J. Virol. 72, 4841-4848[Abstract/Free Full Text]
50. Shibagaki, Y., Holmes, M. L., Appa, R. S., and Chow, S. A. (1997) Virology 230, 1-10[CrossRef][Medline] [Order article via Infotrieve]
51. Vink, C., van der Linden, K. H., and Plasterk, R. H. A. (1994) J. Virol. 68, 1468-1474[Abstract/Free Full Text]
52. Gerton, J. L., and Brown, P. O. (1997) J. Biol. Chem. 272, 25809-25815[Abstract/Free Full Text]
53. Heuer, T. S., and Brown, P. O. (1997) Biochemistry 36, 10655-10665[CrossRef][Medline] [Order article via Infotrieve]
54. Goulaouic, H., and Chow, S. A. (1996) J. Virol. 70, 37-46[Abstract]
55. Chow, S. A., Vincent, K. A., Ellison, V., and Brown, P. O. (1992) Science 255, 723-726[Abstract/Free Full Text]
56. Gerton, J. L., Herschlag, D., and Brown, P. O. (1999) J. Biol. Chem. 274, 33480-33487[Abstract/Free Full Text]
57. Katzman, M., and Sudol, M. (1996) J. Virol. 70, 2598-2604[Abstract]
58. Chen, J. C., Krucinski, J., Miercke, L. J., Finer-Moore, J. S., Tang, A. H., Leavitt, A. D., and Stroud, R. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8233-8238[Abstract/Free Full Text]
59. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R., and Davies, D. R. (1995) Science 266, 1981-1986
60. Goldgur, Y., Dyda, F., Hickman, A., Jenkins, T. M., Craigie, R., and Davies, D. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9150-9154[Abstract/Free Full Text]
61. Heuer, T. S., and Brown, P. O. (1998) Biochemistry 37, 6667-6678[CrossRef][Medline] [Order article via Infotrieve]
62. Mumm, S. R., and Grandgenett, D. P. (1991) J. Virol. 65, 1160-1167[Abstract/Free Full Text]
63. Woerner, A. M., and Marcus-Sekura, C. J. (1993) Nucleic Acids Res. 21, 3507-3511[Abstract/Free Full Text]
64. Venclovas, C., and Siksnys, V. (1995) Nat. Struct. Biol. 2, 838-841[CrossRef][Medline] [Order article via Infotrieve]
65. Skinner, L. M., Lamers, S. L., Sanders, J. C., Eyster, M. E., Goodenow, M. M., and Katzman, M. (1998) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 19, 99-110[Medline] [Order article via Infotrieve]
66. Su, L. J., Qin, P. Z., Michels, W. J., and Pyle, A. M. (2001) J. Mol. Biol. 306, 655-668[CrossRef][Medline] [Order article via Infotrieve]
67. Brännvall, M., and Kirsebom, L. A. (1999) J. Mol. Biol. 292, 53-63[CrossRef][Medline] [Order article via Infotrieve]
68. Pingoud, A., and Jeltsch, A. (1997) Eur. J. Biochem. 246, 1-22[Medline] [Order article via Infotrieve]
69. Krüger, D. H., Kupper, D., Meisel, A., Tierlich, M., Reuter, M., and Schroeder, C. (1995) Gene 157, 165[CrossRef][Medline] [Order article via Infotrieve]
70. Reuter, M., Kupper, D., Meisel, A., Schroeder, C., and Krüger, D. H. (1998) J. Biol. Chem. 273, 8294-8300[Abstract/Free Full Text]
71. Gao, K., Butler, S. L., and Bushman, F. (2001) EMBO J. 20, 3565-3576[CrossRef][Medline] [Order article via Infotrieve]
72. Rhodes, D., Schwabe, J. W., Chapman, L., and Fairall, L. (1996) Phil. Trans. Royal Soc. London. Ser. B: Biol. Sci. 351, 501-509
73. Dorner, L. F., Bitinaite, J., Whitaker, R. D., and Schildkraut, I. (1999) J. Mol. Biol. 285, 1515-1523[CrossRef][Medline] [Order article via Infotrieve]
74. Lukacs, C. M., Kucera, R., Schildkraut, I., and Aggarwal, A. K. (2000) Nat. Struct. Biol. 7, 134-140[CrossRef][Medline] [Order article via Infotrieve]
75. Harper, A. L., Skinner, L. M., Sudol, M., and Katzman, M. (2001) J. Virol. 75, 7756-7762[Abstract/Free Full Text]

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
C. L. Woodward, S. Prakobwanakit, S. Mosessian, and S. A. Chow
Integrase Interacts with Nucleoporin NUP153 To Mediate the Nuclear Import of Human Immunodeficiency Virus Type 1
J. Virol., July 1, 2009; 83(13): 6522 - 6533.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. A. Wilkinson, K. Januszyk, M. L. Phillips, S. S. Tekeste, M. Zhang, J. T. Miller, S. F. J. Le Grice, R. T. Clubb, and S. A. Chow
Identifying and Characterizing a Functional HIV-1 Reverse Transcriptase-binding Site on Integrase
J. Biol. Chem., March 20, 2009; 284(12): 7931 - 7939.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Kim, N. Kim, B. Dong, D. Boren, S. A. Lee, J. Das Gupta, C. Gaughan, E. A. Klein, C. Lee, R. H. Silverman, et al.
Integration Site Preference of Xenotropic Murine Leukemia Virus-Related Virus, a New Human Retrovirus Associated with Prostate Cancer
J. Virol., October 15, 2008; 82(20): 9964 - 9977.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
K. K. Pandey, S. Bera, J. Zahm, A. Vora, K. Stillmock, D. Hazuda, and D. P. Grandgenett
Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate
J. Virol., November 15, 2007; 81(22): 12189 - 12199.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
T. L. Diamond and F. D. Bushman
Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture
J. Virol., December 15, 2005; 79(24): 15376 - 15387.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. Lu, H. Z. Ghory, and A. Engelman
Genetic Analyses of Conserved Residues in the Carboxyl-Terminal Domain of Human Immunodeficiency Virus Type 1 Integrase
J. Virol., August 15, 2005; 79(16): 10356 - 10368.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. P. Grandgenett
Symmetrical recognition of cellular DNA target sequences during retroviral integration
PNAS, April 26, 2005; 102(17): 5903 - 5904.
[Full Text] [PDF]

Home page
 J. Virol.Home page
R. Lu, A. Limon, H. Z. Ghory, and A. Engelman
Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase
J. Virol., February 15, 2005; 79(4): 2493 - 2505.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
K. Gao, S. Wong, and F. Bushman
Metal Binding by the D,DX35E Motif of Human Immunodeficiency Virus Type 1 Integrase: Selective Rescue of Cys Substitutions by Mn2+ In Vitro
J. Virol., July 1, 2004; 78(13): 6715 - 6722.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
W. Tan, K. Zhu, D. J. Segal, C. F. Barbas III, and S. A. Chow
Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
J. Virol., February 1, 2004; 78(3): 1301 - 1313.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
R. Chiu and D. P. Grandgenett
Molecular and Genetic Determinants of Rous Sarcoma Virus Integrase for Concerted DNA Integration
J. Virol., June 1, 2003; 77(11): 6482 - 6492.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. L. Harper, M. Sudol, and M. Katzman
An Amino Acid in the Central Catalytic Domain of Three Retroviral Integrases That Affects Target Site Selection in Nonviral DNA
J. Virol., March 15, 2003; 77(6): 3838 - 3845.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/49/45848    most recent
M107365200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Appa, R. S.
Right arrow Articles by Chow, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Appa, R. S.
Right arrow Articles by Chow, S. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement