JBC

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


     

Originally published In Press as doi:10.1074/jbc.M400349200 on January 20, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14509-14519, April 9, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/15/14509    most recent
M400349200v1
Right arrow Alert me when this article is cited
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Segal, D. J.
Right arrow Articles by Barbas, C. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Segal, D. J.
Right arrow Articles by Barbas, C. F., III
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?

Attenuation of HIV-1 Replication in Primary Human Cells with a Designed Zinc Finger Transcription Factor*

David J. Segal{ddagger}§, João Gonçalves¶, Scott Eberhardy{ddagger}, Christina H. Swan||, Bruce E. Torbett||, Xuelin Li{ddagger}, and Carlos F. Barbas, III{ddagger}**

From the {ddagger}The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry and the ||Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the URIA-Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, 1649-019 Lisboa, Portugal

Received for publication, January 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite stunning advances in the treatment of HIV-11 disease achieved with the introduction of highly active antiretroviral therapy (HAART), virus resistance, toxicity, side-effects of the drug mixture, and patient compliance are problematic for a significant percentage of the patient population (Ref. 1 and references therein). Although the antiviral effects of HAART therapy are often immediate and profound, virus is not eliminated, and one or more reservoirs of latently infected cells persist even in patients that show no detectable viral load for sustained periods during treatment (2, 3). Typically, virus replication rebounds rapidly after drug withdrawal, making HAART therapy a life-long treatment. These results underscore the importance of developing adjunct strategies of retarding viral entry and/or inhibiting of viral replication. One potential strategy involves the genetic modification of cells to endow them with viral resistance via gene delivery. If the primary target cells of HIV-1 can be genetically engineered to express antiviral genes continuously and without toxicity, such cells might escape virus destruction, immune impairment, and preferentially expand in vivo. In particular, intracellular "immunization" of CD34 stem cells and/or CD4 T cells with antiviral genes might lead to long term reconstitution of the immune system in individuals with protease inhibitor resistant viruses or adverse drug reactions.

Inhibition of viral transcription has long been recognized as an important goal in HIV-1 therapy. This was the driving force behind the development of antisense oligonucleotides for HIV-1 therapy (4). Other strategies that are well advanced are based on the concept of intracellular immunization (5). These include siRNA (6, 7), ribozymes (8, 9), dominant-negative approaches (10), and intrabodies (11). The anti-RNA approaches are most successful when they target essential transcripts or gene products that are produced at a low level. Often their function in intracellular immunization strategies is very dependent on achieving high levels of intracellular expression. This is in contrast to control at the transcriptional level, which presents the advantage that only a single DNA site needs to be occupied as compared with targeting the many RNA messages that might be produced from a single gene. As with any approach that attempts specific inhibition in HIV-1, the mutation rate and genetic diversity found in vivo presents formidable obstacles. Despite this, there are regions within the HIV-1 genome that are conserved over all clades and provide suitable targets for the exploration of this approach.

Our approach is based on the recognition of the structural features unique to the Cys2-His2 class of nucleic acid-binding, zinc finger proteins. To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences and have described the generation of zinc finger domains recognizing sequences of the 5'-(G/A)NN-3' subset of a 64-member zinc finger alphabet (12–14). These domains can be used as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. Methods for the rapid construction of polydactyl proteins have been developed that, together with this predefined set of zinc finger domains, provides ready access to over a billion novel proteins that bind the 5'-(G/ANN)6-3' family of 18-bp DNA sites (12, 15, 16).

Appending effector domains, such as the activation domain VP16 (17) and the repression domain KRAB (18), creates potent artificial transcription factors. In previous work, we and others (12, 19–22) have demonstrated that both gene repression and activation can be achieved by targeting designed transcription factors to a single site within endogenous genes. Such regulation has been demonstrated in human, mouse, rat, monkey, Arabidopsis, and tobacco cells as wells as in transgenic Arabidopsis and tobacco plants (23, 24). Furthermore, temporal control of expression can be achieved via chemical control of gene expression imparted by appending ligand-binding domains derived from human steroid-hormone receptors (25). As an alternative to designing and testing individual transcription factors, combinatorial libraries of designed transcription factors have been recently used to select effective transcriptional regulators in the absence of detailed chromatin accessibility information and to identify novel gene regulatory pathways (26).

In this study, we used our recently developed methodologies to prepare optimized transcriptional regulators designed to present transcriptional blockades to the HIV-1 lifecycle. Our work extends a recent study by Reynolds and co-workers (27) in which they observed repression of HIV-1 replication by an engineered zinc-finger transcription factor. These findings strongly support a transcription factor-based approach to intracellular immunization of CD34 stem cells and/or CD4 T cells (28).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Custom DNA-binding Proteins—DNA-binding proteins containing six zinc finger domains were assembled onto an Sp1C zinc finger scaffold (29) using methods and domains described previously (12–16). Briefly, overlapping PCR primers were designed to encode zinc finger domains that had been previously modified to bind unique 3-bp sites. Three-finger proteins were assembled by overlap PCR then assembled into six-finger proteins by AgeI/XmaI ligation. For in vitro characterization, the constructs were cloned into the prokaryotic expression vector pMAL-c2 (New England Biolabs). Fusions with the maltose-binding protein were expressed and purified using the Protein Fusion and Purification System (New England Biolabs). Multitarget ELISA specificity assays and electrophoretic mobility shift affinity assays were performed as described previously (14, 15).

Transient Transfection Assays—Effector plasmids containing the KRAB and SID effector domains have been described previously (15). Effector plasmids consisted of the mammalian expression vector pcDNA3 (Invitrogen) containing the gene for a C-terminal designed six-finger protein cloned in-frame with an N-terminal effector domain. All constructs also encoded a nuclear localization signal from SV40 large T antigen (PKKKRKV) between the effector and zinc finger domains, as well as a C-terminal epitope tag from influenza hemagglutinin (HA tag, YPYDVPDYAS). GenBankTM accession numbers for KRAB-HLTR1, KRAB-HLTR3, and KRAB-HLTR6 transcription factors are AY518586 [GenBank] , AY518587 [GenBank] , and AY518588 [GenBank] , respectively. Reporter plasmids were based on pGL3-control (Promega) and contained a luciferase gene under control of the SV40 promoter (pGL3-control), the erbB-2 promoter (erbB-2 (–758 to –1) (15)), or the HIV-1 LTR promoter. The HIV-1 HXB2 LTR was amplified by PCR from the plasmid pIIIenv3–1 (National Institutes of Health AIDS Research & Reference Reagent Program, NARRRP (30)) using the primers 5'-ggatccggaggggacggggccggagccgcagtgggggtagaaatggaagggctaattcactcc-3' and 5'-ctcctcctcctcctcggatccatggtggcgcccacgccgcccacgccactgctagagattttccacactgactaaaaggg-3'. The LTR fragment was cloned between the BglII/NcoI sites of pGL3-control. The plasmid pSV2tat72 (NARRRP (31)) was included in all experiments as a source of TAT protein.

For all transfections, HeLa cells (American Type Culture Collection, ATCC) were used at a confluency of 40–60%. Typically, cells were transfected with 100 ng each of effector and reporter plasmid and 50 ng of pSV2tat72 per well in a 24-well dish by using LipofectAMINE transfection reagent (Invitrogen). Cell extracts were prepared ~48 h after transfection. Luciferase and {beta}-galactosidase activity were measured with corresponding assay reagent kits (Promega) in a MicroLumat LB96P luminometer (EG&G Berthold, Gaithersburg, MD). Luciferase activity was normalized to total extract protein.

For determination of protein expression, ~2 x 106 HeLa cells were plated onto 10-cm plates and transfected with 2 µg of zinc finger protein expression plasmid. Cell extract was prepared by the addition of cell lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and Complete Protease Inhibitor (Roche Applied Science)). Protein concentration was measured by the Bradford assay, and 50 µg of cell extract was separated by SDS-PAGE and blotted onto Hybond-P membrane (Amersham Biosciences). The blot was probed with an anti-HA antibody (Roche Applied Science), then stripped and reprobed with an anti-{beta}-actin antibody (Sigma) as a loading control. Relative zinc finger protein expression was determined using ImageQuaNT (Molecular Dynamics).

Inhibition of transiently transfected, plasmid-based HIV expression was examined using 293T cells (ATCC) at a confluency of 50–60%. Cells were co-transfected with 500 ng each of HIV-1 plasmid (pNL4–3 (32) or pYK-JRCSF (33), NARRRP), pSIN-KRAB-HLTR3 (a lentiviral delivery vector, described below) and 100 ng of the luciferase reporter plasmid pGL3-Control (Promega) in a 6-well dish by using FuGENE transfection reagent (Roche Applied Science). Medium was changed at 24 h post-transfection, and p24 antigen was measured in the supernatant at 48 h post-transfection. Cell extracts were prepared, and luciferase activity was measured with corresponding assay reagent kits (Promega) in a Labsystems luminometer. Luciferase activity was normalized to total extract protein.

Generation of Stable PM1 Cell Clones—pMAL-HLTR3 was digested with SfiI, and the HLTR3 zinc finger gene was cloned between the two SfiI sites of pMX-KRAB-E2C-IRES-GFP (19), replacing the E2C zinc finger gene. This retroviral vector expresses a single bicistronic message for the translation of the zinc finger protein and, from an internal ribosome entry site (IRES), the green fluorescent protein (GFP). The vector additionally appends an HA-tag to the C-terminal of the zinc finger gene. pMX-KRAB-HLTR3-IRES-GFP and pMD-G, a plasmid expressing the envelope glycoprotein G of the vesicular stomatitis virus (kindly provided by Inder Verma, the Salk Institute (34)), were co-transfected into the Gag-Pol-293 packaging cell line (Clontech) using LipofectAMINE Plus (Invitrogen). After 48 h of incubation, culture supernatants were used for infection of PM1 cells (NARRRP (35)) in the presence of 8 mg/ml Polybrene. PM1 cells are a clonal derivative of HUT78. These cells are permissive for growth of macrophage and T-cell tropic viruses. Cells were cloned by limiting dilution. Expression levels of KRAB-HLTR3 was determined by Western analysis on 100 µg of total soluble cell protein using the ECL Western blotting detection system (Amersham Biosciences) with a mouse anti-HA primary antibody (Roche Applied Science) and a goat anti-mouse-IgG horseradish peroxidase secondary antibody conjugate (Sigma).

HIV-1 Challenge of Stable T-cell Clones—Stocks for the murine heat-stable antigen-expressing (HSA-expressing), CCR5-tropic HIV-1 reporter virus, NFN-SX-HSAS (36) were produced by infecting PM1 T-cell lines with NFN-SX-HSAS virus generated by calcium phosphate transfection of 293T cells (ATCC) with a proviral plasmid. Viral supernatants were stored at –80 °C. The p24 value of the virus stock was 2649 ng/ml. 5 x 105 PM1 T-cells were exposed to NFN-SX-HSAS (140 µl) for 3 h at 37 °C, washed three times, and then cultured in 3 ml RPMI (10% fetal bovine serum, 1% penicillin/streptomycin/glutamine, and 1% HEPES).

HIV-1 infection was determined by flow cytometry analysis on cells isolated from culture on days 2, 4, 9, and 13 post-infection. Cells were stained with rat anti-mouse CD24 (heat-stable antigen) antibody labeled with R-phycoerythrin (BD Pharmingen). Rat IgG2k labeled with R-phycoerythrin was used as an isotype control. Cells were fixed with 2% paraformaldehyde prior to flow cytometry analysis. Flow cytometry was performed on a FACScan flow cytometer (BD Biosciences Immunocytometry Systems) and analysis replied on the Cell Quest Software program.

Supernatant obtained on days 2, 4, and 9 post-infection were also assessed for the presence of p24. Culture medium containing infected cells was pelleted at 2000 rpm for 2 min. 50 µl of cell-free supernatant was added to 450 µl of p24 sample buffer for the p24 assay. Quantification of HIV-1 p24 protein production was performed using the antigen capture ELISA test (Coulter Corp.) according to the manufacturer's instructions.

Construction of pSIN-KRAB-HLTR3—To clone pSIN-KRAB-HLTR3, the DNA sequence for KRAB-HLTR3 was amplified by PCR from the pcDNA KRAB-HLTR3 plasmid, using the primers 5'-atcgcttagggatccgctagcatggatgctaagtca-3' and 5'-atcgcttaggtcgacaagctttcaagaagcgtagtc-3', which contain restriction sites for BamHI (5') and SalI (3'). The PCR product was purified and digested with BamHI and SalI and ligated into BamHI/SalI-digested pRRLPGK-GFP SIN-18 (36), so that the KRAB-HLTR3 sequence replaced the GFP gene. The final plasmid was sequenced to ensure that no mutations were introduced during PCR.

Lentiviral Vector Production and Titration—The HIV-derived packaging construct pCMVdeltaR8.91 encodes the HIV-1 gag and pol precursors as well as the regulatory proteins tat and rev (kindly provided by Inder Verma, the Salk Institute) (33). Pseudotyped lentiviruses were produced by transient calcium phosphate co-transfection of 293T cells with pCMVdeltaR8.91, pMD-G, and the zinc finger-expressing lentiviral transfer vector pSIN-ZF. Viral supernatants were harvested 48–60 h after transfection, concentrated by ultracentrifugation, and resuspended in serum-free RPMI 1640 culture medium. Viral titers were determined from the p24 antigen. Preparation of peripheral blood mononuclear cells (PBMCs) were purified from the fresh blood of healthy donors by Ficoll-Paque (Amersham Biosciences) density gradient centrifugation. Non-adherent peripheral blood leukocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C, 5% CO2. They were stimulated for 3 days with recombinant human interleukin-2 (20 IU/ml; Roche Applied Science) and phytohemagglutinin (5 µg/ml) and then cultured in the presence of human interleukin-2 (20 IU/ml).

Lentiviral Transduction—Transductions of PM1 cells and PBMCs were performed in 48-well plates with addition of 5 µg/ml Polybrene. The cells were exposed to lentiviral concentrates at titers of 100 ng/ml p24 antigen concentration at a density of 5 x 105 cell/ml. After 6 h of transduction, the cells were washed with phosphate-buffered saline and further incubated in fresh tissue culture medium. The transduction protocol was repeated two more times in the following 48 h.

HIV-1 Infection of PM1 Cells and PBMC—M-tropic (YK-CSF), T-tropic (NL4–3), and dual tropic (89.6) HIV-1 viruses (NARRRP (37)) were produced in 293T cells and used to infect the transduced PM1 cells and PBMCs at a multiplicity of infection (m.o.i.) of 0.1 (11). The m.o.i. values were determined in a separate experiment by fluorescence-activated cell sorting analysis using pRRLPGK-GFP SIN-18. It was estimated that 1 ng of p24 corresponds to 2000–3000 transducing units as m.o.i. is a statistical function of the number of virus able to infect a population of cells (38). As determined by trypan blue staining technique, the viability of cells was higher than 95%. Where indicated, fresh PBMCs were added at day 7 to rescue viable HIV particles.

After 6 h of incubation at 37 °C, the infected lymphocytes were washed once and resuspended in fresh medium. Cell culture supernatants were collected from the infected cells at various time points, and the concentration of the p24 protein was determined by ELISA (Innotest). Cell viability was determined using the Cell Proliferation Reagent WST-1 (Roche Applied Science) according the manufacturer's instructions. The activity of mitochondrial dehydrogenases, an indicator of cell viability, was measured in 10,000 cells by the conversion of the tetrazolium salt WST-1 to the red dye formazan. Formazan accumulation is detected by increased absorbance at 440 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Target Site Selection in HIV-1—The 5'-LTR of HIV-1 contains the promoter for all viral transcripts (39–41). The primary transcript can be either packaged into new virus particles or spliced to create the mRNA for the viral gene products. The LTR consists of three regions, U3, R, and U5. The cis-acting regulatory elements of the promoter are contained in the U3 region, which can be further subdivided into the negative regulatory region (nucleotides 1–350 as numbered in the genome of strain HXB2), the enhancer (nucleotides 351–376), and the core promoter (nucleotides 377–454). An array of transcription factors has been shown to interact with the U3 region (reviewed in Ref. 42), and variations in their binding sites have been implicated in modulating promoter strength and replication kinetics (43, 44). Transcription initiates at the first nucleotide of the R region, and polyadenylation occurs immediately after the R in the 3'-LTR. The R region also codes for the transacting response element (TAR), a stem-loop structure found in all viral mRNAs to which the transactivator protein Tat will bind. U5 spans the region between R and the binding site for tRNALys, which is used during reverse transcription.

In a systematic characterization, we previously defined the zinc finger domains that specifically bind to 3-bp sites of the 5'-(G/A)NN-3' type (12–14). After searching 5'-LTR sequences around the promoter region for existence of 18 contiguous nucleotides that are targetable with our defined domains using a search program of our own design,2 we found excellent coverage of most of the promoter region (Fig. 1). Potential target sites were checked for the presence of identical or similar sites in the human genome by a BLAST search of the Homo sapiens DNA sequences in the GenBankTM data base (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1.
Region of HIV-1 targeted by designed transcription factors. A, diagrammatic representation of the 5'-LTR of HIV-1. All vertical lines indicate 18-bp sites that can be targeted with the currently available lexicon of modified zinc finger domains. The three sites studied in this report are indicated (HLTR1, 3, and 6), as are several features of the HIV-1 promoter (binding sites for NF-{kappa}B (N), Sp1 (S), TATA binding protein (T), transcription initiation site (arrow), and TAR loop (TAR)). Combined DNase I hypersensitive and micrococcal nuclease sensitive sites are shown as gray boxes (45). B, sequence of the 5'-LTR in the targeted region for the prototypic HIV-1 strain, HXB2. Target sites for designed transcription factors are overlaid on promoter features.

 
The parameters defining a productive target site for gene regulation have not been fully resolved at this time. However, we reasoned that a good target site should be located in chromatin where it is accessible to DNA-binding proteins and where the sequence is highly conserved. Some transcription factors have been shown to have little access to their DNA binding sites in chromatin. There have been reports correlating the ability of designed 3-finger proteins to regulating gene expression and the location of their target sites in accessible chromatin structures (20, 21). Accessible target sites may be located in the linker region between positioned nucleosomes and are sensitive to micrococcal nuclease digestion or appear as DNase I-hypersensitive sites. For HIV-1, the nuclease-sensitive regions in the promoter have been well characterized (Fig. 1A) (45). However, HIV presents an additional challenge in that the viral genome mutates with each replication cycle. A comparison of 5'-LTR sequences from different viral isolates and various subfamilies or clades of HIV-1 reveals that mutations arise with varying frequency, even in known transcription factor binding sites (Table I, HIV Sequence Data base (available at hiv-web.lanl.gov), and Ref. 46). However, there are some regions of the 5'-LTR that show remarkably high sequence conservation across all clades, probably due to their role in the viral life cycle. For example, the HIV-1 LTR target site 1 (HLTR1) is extremely well conserved (for examples within clade B see Table I), perhaps due to its proximity to the highly conserved TAR region (Fig. 1B). Therefore, by following these guidelines we were able to select target sites that were both accessible and conserved.


View this table:
[in this window]
[in a new window]
  		TABLE I
Sequence conservation of designed transcription factor target sites among members of clade B of HIV-1

 
Combining the micrococcal and DNase I data, there are essentially four accessible regions in the 400 bp that surround the start of transcription. Using the genome of viral isolate HXB2 as a reference, the accessible regions approximately include nucleotides 200–350, 375–450, 500–575, and 600–775 (Fig. 1A). Three target sites were selected (HLTR1, 3, and 6). Their binding site sequence and position are shown in Fig. 1B. Site HLTR3 overlaps with two of the three binding sites for the transcriptional activator Sp1. Sites HLTR1 and 3 are in regions expected to be accessible. Site HLTR6 had good sequence conservation, but it was less clear that this site would be accessible in chromatin. Examples of the sequence conservation for the various sites are shown in Table I. Although data are shown only for 49 members of the B clade (to save space), the extent of sequence conservation is representative of that observed across all clades.

Identifying the Most Potent Repressor—Six-finger proteins that specifically recognize each proposed 18-bp target site were assembled as maltose-binding protein fusion proteins and purified. Their DNA binding specificity and affinity were assessed in multitarget ELISA assays, and electrophoretic mobility shift assays as previously described (12, 15). The KD values of the six-finger proteins HLTR1, 3, and 6 for their DNA targets were determined to be 10, 1, and 6 nM, respectively (Fig. 2A). Transcriptional repressors were prepared by attaching several human-derived repressor domains to the designed zinc finger proteins. The Krüppel-associated box (KRAB) domain (18) and the Mad mSIN3 interaction domain (SID) (47) both showed good repressive activity in previous studies of imposed erbB-2 regulation (15, 19).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 2.
A, electrophoretic mobility shift assay to determine binding constants for the designed zinc fingers. B, transient reporter assays comparing the repression potentials of various designed transcription factor using a KRAB (left) or SID (right) repression domain. HeLa cells were transfected with an HIV LTR/luciferase reporter plasmid, a TAT-expression plasmid, and a plasmid expressing the protein indicated. "No ZF" = only LTR/luciferase reporter and pSV2tat72 plasmids. "No Tat" = only LTR/luciferase reporter plasmid. Error bars represent the mean value of duplicate experiments. C, protein expression of KRAB-HLTR zinc fingers. Cell extracts from HeLa cells transfected with KRAB-HLTR1, KRAB-HLTR3, or KRAB-HLTR6 or empty vector were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blots were probed with an antibody that recognize the HA tag on the zinc finger proteins or a {beta}-actin antibody. Relative protein expression was calculated by normalizing zinc finger expression to {beta}-actin expression in each sample.

 
To assess the potential for transcriptional repression of each of the transcription factors, HeLa cells were transiently co-transfected with 1) an effector plasmid expressing the designed transcription factor, 2) a reporter plasmid expressing luciferase under control of the HIV-1 LTR promoter, and 3) a TAT-expressing plasmid. Luciferase levels were measured 48 h post-transfection. A 13-fold reduction in reporter expression was observed when KRAB-HLTR3 was present, but less than 2-fold for KRAB-HLTR1 and KRAB-HLTR2 (Fig. 2B, left). Repression was more dramatic for HLTR1 when a SID repression domain was used (Fig. 2B, right), whereas HLTR6 showed very little repression with this effector. However, SID-HLTR3 again showed the most impressive repression potential (~6-fold). In addition, a control six-finger artificial transcription factor that recognizes the sequence GACGGGGCTGCTGCAGAC was tested for its ability to affect the HIV-1 LTR. The recognition sequence for this protein is not found in the HIV-1 LTR, and so this protein should not affect transcription from the LTR. As expected, no repression of the reporter construct was observed with either the KRAB or SID domains fused to the control zinc finger. Finally, to determine if the differences in repression seen with the HLTR proteins was not due in part to differences in protein expression, Western blots were performed. These studies revealed a modest difference in the level of protein expression of HLTR3 (~1.5-fold greater) as compared with the other proteins (Fig. 2C).

KRAB-HLTR3 Specificity and Activity in Chromatin—Nonspecific transcriptional repression might result in cellular toxicity, precluding the use of this anti-viral protein for long term expression in a potential intracellular immunization strategy. To examine the specificity of regulation, KRAB-HLTR3 was assayed on three different promoters (Fig. 3A). A 10-fold repression of the HIV-1 LTR was observed. However, no repression was observed with an SV40 or erbB-2 promoter, demonstrating that repression by this protein effectively targeted the HIV-1 LTR promoter.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3.
Transient reporter assays comparing the activity of KRAB-HLTR3 on various promoters. A diagram of the reporter construct appears above each graph. A, HeLa cells were transfected with a luciferase reporter plasmid containing the indicated promoter, a TAT-expression plasmid, and a plasmid expressing the protein indicated. "No ZnFn" = only LTR/luciferase reporter and pSV2tat72 plasmids. B, P4R5 MAGI cells, containing an HIV-1/lacZ reporter integrated into the genome, were co-transfected with a TAT-expression plasmid and a plasmid expressing the protein indicated. 293T cells were transfected with a plasmid form of the genome for NL4–3 (C) or YK-CSF (D), a plasmid expressing the protein indicated, and a constitutive luciferase expression plasmid. E, cell viability as indicated by luciferase activity.

 
The chromatinized state of plasmid DNA in mammalian cells can be quite different than that of the chromosome, where nucleosomes and DNA topology might limit accessibility of binding proteins. Other groups have reported differences in targeting proteins to sites in plasmid or chromosomal DNA (20, 48). We therefore co-transfected the KRAB-HLTR3-expressing plasmid with the Tat-expressing plasmid into the HeLa derivative, P4R5 MAGI (49), which contains a chromosomally integrated HIV-1 LTR driving expression of a lacZ reporter gene. We again observed a 10-fold reduction in reporter gene expression when KRAB-HLTR3 was present (Fig. 3B). A 3-fold repression was also observed when HLTR3 was expressed in these cells without the KRAB domain. This effect was likely due to the fact that the HLTR3 binding site overlaps two Sp1 sites, allowing HLTR3 to interfere with the binding of these transcriptional activators (see "Discussion").

To evaluate the ability of zinc finger factor to inhibit plasmid-based viral production, a transient assay was performed in 293T cells by co-transfection of a KRAB-HLTR3-expressing plasmid with an HIV-1 NL4–3-expressing plasmid or an HIV-1 YK-CSF-expressing plasmid. At 48 h post-transfection, expression of KRAB-HLTR3 produced strong inhibition of virus production (~99%) (Fig. 3, C and D). This effect was not due to different transfection efficiencies or cytotoxic effects specific to individual zinc finger transcription factors as indicated by transfection studies using a luciferase reporter lacking the recognition sequences of the transcription factors (Fig. 3E).

Stable Expression of KRAB-HLTR3 Inhibits HIV-1 Replication—To assess the ability of the transcription factor to inhibit viral replication, stable cells lines were produced and challenged with HIV. The PM1 T-cell line was used, because it expresses CD4, CCR5, and CXCR4, allowing this line to be infected by both R5 and X4 HIV strains. Stable expression of the transcription factor in PM1 cells was achieved using a pMX-KRAB-HLTR3-IRES-GPF retroviral vector. This virus uses a Moloney murine leukemia virus promoter, which does not contain a site for KRAB-HLTR3 and is, therefore, not expected to be regulated by the expressed transcription factor. KRAB-HLTR3 expression in stable PM1 cell clones was confirmed by Western analysis. Clonal lines 8 and 19 were selected for further study based on their high and low expression of KRAB-HLTR3, respectively (Fig. 4).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 4.
Expression of KRAB-HLTR3 in stable PM1 cell clones. PM1 T-cells transduced with pMX-KRAB-HLTR3-IRES-GFP retrovirus were cloned. Expression of KRAB-HLTR3 in parental PM1 or clones was shown by Western analysis using an antibody directed against the HA tag on the C terminus of protein.

 
The clones and untransduced PM1 cells were challenged with the CCR5-tropic, HSA-reporter virus, NFN-SX-HSAS. This virus is based on the HIV strain NL4–3, which does contain a site for KRAB-HLTR3 in its promoter and is, therefore, expected to be regulated by the transcription factor. As shown in Fig. 5 (A and B) the majority of clone-8 T-cells expressing the KRAB-HLTR3 protein demonstrated marked inhibition of HIV replication over time, whereas both the parental PM1 and clone-19 T-cell lines readily supported NFN-SX-HSAS replication, as shown by HSA expression. At 13 days post-infection, parental and clone-19 PM1 cells were highly infected and showed signs of virus-induced pathology, i.e. reduced HSA expression, increased numbers of dead cells in culture, and reduced proliferation. Infection of the parental and clone-19 PM1 T-cell lines was further confirmed by p24 analysis of cultures supernatants over time (Fig. 5C). Although ~20% of the KRAB-HLTR3-overexpressing clone-8 PM1 T-cells showed HSA protein expression, the number of cells infected and the mean fluorescent HSA intensity, an indication of the level of viral infection per cell, at day 13 of culture was 1.5 log lower than that of the parental and clone-19 PM1 T-cell lines (Fig. 5B). Moreover, by day 9 post-infection, the parental and clone-19 PM1 T-cell lines had an average of 1363 ng/ml of p24 in culture supernatant, whereas supernatants from the clone-8 cells contained 2.6 ng/ml of p24, a 524-fold drop in p24 concentration (Fig. 5C). These results demonstrate that KRAB-HLTR3 is able to inhibit HIV replication in T-cell lines. The results also suggest that long term constitutive expression of KRAB-HLTR3 is not toxic to T-cells.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 5.
KRAB-HLTR3 expression inhibits HIV-1 replication in PM1 T-cell clones. A, flow cytometry analysis showing that PM1 T-cells stably expressing KRAB-HLTR3 (clone 8, thick line), but not parental PM1 T-cells (thin line) or a non-zinc-finger expressing PM1 clone (clone 19, dotted line), are protected from challenge with the NFN-SX-HSAS reporter HIV virus at m.o.i. 1. Viral replication was measured using a fluorescently-tagged antibody to the HSA marker. For reference, cultured, but not HIV-1 exposed, parental PM1 T-cells (dashed line) are shown. B, graphical representation of flow cytometry analysis of HSA expression. Lines are as in (A). C, p24 levels over time. Culture supernatants were collected on days 2, 4, and 9 post-infection and measured by ELISA. Lines are as in A.

 
Primary Human Cells Transduced with KRAB-HLTR3 Are Protected from HIV-1 Replication—To assess the ability of the transcription factor to inhibit viral replication, primary human peripheral blood mononuclear cells (PBMCs) and a T-cell line (PM1) were transduced with a lentiviral vector expressing KRAB-HLTR3 (pSIN-KRAB-HLTR3) or empty vector (pSIN) and subsequently challenged with various strains of HIV-1. Note that the transcription factor transgene in this delivery vector is under control of a cytomegalovirus promoter, which does not contain a site for KRAB-HLTR3 and is, therefore, not expected to be regulated by the expressed transcription factor. Levels of HIV-1-expressed p24 were measured at various time points post-infection as an indicator of viral replication. The HIV-1 strains used were NL4–3 (X4 tropic), YK-CSF (R5 tropic), and 89.6 (R5X4 tropic). The sequence of the HLTR3 target sites in these strains is indicated in Table I. ELISA and electrophoretic mobility shift assay studies indicated that the HLTR3 protein bound double-stranded DNA oligonucleotides displaying the sequences derived from each of these viruses with an affinity similar to that of the native sequence. In both the PM1 T-cell line (Fig. 6A) and primary PBMC (Fig. 6B), a 10- to 100-fold reduction in p24 levels was observed for all KRAB-HLTR3-transduced cells compared with non-transduced cells. Moreover, the repressive effect was maintained even at 18 days post-HIV-1 infection.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 6.
Inhibition of HIV-1 replication by KRAB-HLTR3 in the T-cell line PM1 (A) or primary human PBMCs (B). Cells were transduced with the lentiviral vector pSIN-KRAB-HLTR3 (open symbols) or empty pSIN vector (solid symbols) (100 ng/ml of lentiviral p24 antigen per 5 x 105 cell/ml), before infection with HIV-1 strains NL4–3 (circles), YK-CSF (triangles), or 89.6 (squares) at an m.o.i. of 0.1. HIV-1-produced p24 levels were monitored at days 3, 7, 10, 14, and 20. Fresh PBMCs were added at day 7 to rescue viable HIV particles. Error bars represent the mean value of triplicate experiments.

 
To determine if these effects were due to the KRAB-HLTR3 protein, the experiments were repeated using PBMC transduced with empty vector, KRAB-HLTR3, or KRAB-Aart, a repression construct containing an control zinc finger Aart. Protein Aart is a six-finger protein designed to bind an artificial AT-rich sequence and is not expected to have any binding site in the human genome (12). Intracellular staining, using an antibody directed against the C-terminal HA tag on the zinc finger transcription factor, revealed expression of the transgene in ~35% of PBMCs (Fig. 7). However, co-staining with an anti-CD4 antibody demonstrated that this 35% contained 70% of the CD4 cell population. That is to say, 70% of potentially HIV-permissive cells expressed a zinc finger transcription factor.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 7.
Lentiviral transduction of PBMC. Two-dimensional flow cytometry data of PBMC that were untransduced (A), transduced with empty pSIN lentiviral vector (B), pSIN-KRAB-Aart (C), a vector expressing KRAB fused to an irrelevant zinc finger gene (Aart), or pSIN-KRAB-HLTR3 (D). Fluorescence intensity on the x-axis represents intracellular staining for the zinc finger fusion protein using an antibody directed against the C-terminal HA tag. Intensity on the y-axis represents surface CD4 expression. The percentage of total cells for each quadrant is indicated.

 
PBMCs exposed to the KRAB-HLTR3-expressing lentivirus displayed dramatic resistance to replication of HIV-1 strains NL4–3 and YK-CSF (Fig. 8A). Cells exposed to KRAB-Aart behaved similar to empty vector controls. To rule out the possibility that viral replication was reduced due or decreased due to KRAB-HLTR3-induced cytotoxicity, cell viability was measured at various time points post-HIV-1 infection. All cell samples were found to be similarly viable at all time point (Fig. 8B).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 8.
Second HIV-1 challenge assay in PBMCs. PBMCs were transduced with lentiviral vector pSIN-KRAB-HLTR3 (open symbols), a vector expressing KRAB fused to an irrelevant zinc finger gene (Aart, dashed line), or empty pSIN vector (solid symbols), before infection with HIV-1 strains (A) NL4–3 (circles) or YK-CSF (triangles) at an m.o.i. of 0.1. HIV-1-produced p24 levels were monitored at days 3, 7, 12, and 18. B, viability of cells from the above experiments as determined using the WST-1 assay for mitochondrial dehydrogenase activity.

 
Repeating the transduction procedure three times instead of once provided only a modest increase in repression of KRAB-HLTR3, and had no effect on the other samples (data not shown). However, these results do demonstrate that attempts to boost the expression level of the anti-viral protein are tolerated but are not necessary to attenuate viral replication.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
What Makes KRAB-HLTR3 Such a Potent Transcriptional Repressor of HIV-1?—A number of factors have likely contributed to the effectiveness of HLTR3 as a transcriptional repressor as compared with HLTR1 and HLTR6. HLTR1, 3, and 6 should all bind in chromatin-accessible regions, and the proteins have similar affinity for their target sites (10, 1, and 8 nM, respectively) (Fig. 2A). Although these affinity differences are modest, in our original studies of endogenous gene regulation using designed six-finger transcription factors (single-site regulators), we did observe potent endogenous gene regulation with transcription factors that bound their target with ~1 nM dissociation constants and little to no regulation with proteins that bound their target DNA with KD values of 10 nM or higher (19). Thus, DNA affinity is likely to be a significant factor contributing to the success of HLTR3. Additionally, study of the expression of the transcription factors revealed that HLTR3 is also expressed 1.5-fold more efficiently than the other KRAB-bearing transcription factors (Fig. 2C). With respect to target site position as it relates to the start of transcription, the HLTR1 target site is actually more proximal to the transcription start site than the HLTR3 target site. A priori, it might be reasonable to expect that HLTR1 and 6 might be less effective repressors, because they lie downstream of the transcription initiation site. However, several of the most potent designed transcriptional regulators bind in a similar position relative to their target promoter (50). Similarly, HLTR1 and 6 both bind the minus strand of the promoter, whereas HLTR3 binds the plus strand. However, numerous potent regulators have been reported on either strand (12, 19).

The binding site of HLTR3 also overlaps two Sp1 binding sites. The Sp1 sites, together with the TATA box, comprise the core promoter and have been shown to be essential for the function of HIV-1 proviral DNA-mediated gene expression (44). The Sp1 elements also seem to play a role in the Tat inducibility of expression (51). It has been previously shown that one mechanism by which repression can be achieved is to interfere with the binding of critical elements of the transcriptional machinery (52). Therefore, in addition to localizing a repression effector domain, a protein targeted to the HLTR3 site may have the additional advantage of being able to inhibit transcription by competing with the binding of endogenous Sp1 activator proteins. In support of this notion, HLTR3 (lacking a repression domain) demonstrated a significant 3-fold inhibition of the HIV-1 LTR in transient reporter assays (Fig. 3B). A significant role of the repression domain is revealed, however, by the differential repressive capability of HLTR3 bearing SID and KRAB repression domains indicating that the effector domain plays a critical role in transcriptional repression beyond that imparted by direct competition with Sp1 activators.

The results of the reporter assays identified HLTR3 as the most potent HIV-1 inhibitor. Although the target site sequence for this protein is not as well conserved as that for HLTR1 and 6, there appear to be many viral isolates for which the HLTR1 and 6 target sites occur in gapped regions and are thus completely absent (Table I). Based on the most current version (2002) of the HIV Sequence Data base, the HLTR3 site occurs in virtually all sequenced isolates in all clades (data not shown). Therefore, among the three candidate repressors, HLTR3 is not only the most potent but also provides the greatest potential to target the most viral strains. It should be noted that artificial engineered HIV-1 variants completely devoid of Sp1 sites have been shown to be replication competent in cell culture suggesting a functional redundancy of activation elements within the HIV-1 LTR (53).

Co-transfection of 293T cells with plasmids expressing KRAB-HLTR3 and HIV resulted in a 100-fold repression of HIV replication (Fig. 3, C and D). This dramatic repressive effect is likely due to the inhibition of basal HIV transcription prior to significant expression of TAT transactivator protein. This explanation is consistent with the results of the conceptually similar reporter assays (Fig. 3, A and B), in which the transcription of a TAT-activated HIV promoter was reduced only 10-fold by KRAB-HLTR3. These results suggest that prior expression of KRAB-HLTR3 may be required prior to HIV infection to produce the dramatic repression of HIV-1 replication observed in the PM1 and PBMC experiments (Figs. 4, 5, 6, 7, 8).

In the experiment shown in Fig. 5, stable PM1 clones were exposed to HIV at an approximate m.o.i. of 1. A modest amount of HIV replication was observed in these cells (albeit 50- to 500-fold less than controls). In the experiments shown in Figs. 6 and 8, PBMCs were exposed to HIV at an approximate m.o.i. of 0.1. At this multiplicity of infection, HIV replication was almost completely repressed. The lower m.o.i. might be considered more physiologically relevant in some cases, particularly in individuals undergoing highly active antiretroviral therapy (HAART). The latter results are also significant, because they suggest that, under some conditions, viral replication can be repressed even if KRAB-HLTR3 is not expressed in every cell.

PBMCs expressing KRAB-HLTR3, KRAB-Aart, or empty transduction vector were found to have similar viability after HIV-1 challenge (Fig. 8B). These results demonstrate that the observed inhibition of viral replication is not due to decreased cell viability. The apparent lack of cytotoxicity is further supported by the fact that a PM1 T-cell clone could be propagated indefinitely despite expressing high levels of functional KRAB-HLTR3 protein (Figs. 4 and 5, clone 8). Together, these results provide evidence that expression of KRAB-HLTR3 is not cytotoxic, even at levels sufficient to repress HIV-1 replication.

The Potential of KRAB-HLTR3 as an Antiviral Therapeutic—In an independent study, Reynolds and co-workers (27) showed that a different designed transcription factor, HIVBA'-KOX, targeted near the Sp1 sites in the HIV-1 LTR was able to inhibit, following transient transfection, the replication of HIV-1 strain HXB2 in the human glioma cell line NP2 by ~3.5-fold. Their optimal factor similarly contained six zinc fingers (modified for site-specific recognition by a somewhat different strategy) fused to a KRAB repression domain in accord with our early studies on endogenous gene regulation with artificial zinc finger transcription factors (15, 19). Although issues of specificity, toxicity, and breadth of activity in primary human cells were not directly addressed, their results corroborate those of our study. A significant difference between the protein HIVBA' described by Reynolds et al. (27) and HLTR3 described here, is that in the absence of a repression domain HLTR3 significantly represses transcription (~3-fold), whereas HIVBA' activates transcription (~2-fold) suggesting that HIVBA' does not directly compete with Sp1 factors for binding to the LTR. This differential activity in the absence of repression domains suggests that the precise placement of an inhibitory factor is key in studies that attempt to interfere with endogenous factor binding. Cooperative DNA-binding and synergistic activation of non-competing transcription factors has been noted before and likely explains the undesirable activation seen with HIVBA' (54). Nonetheless, taken together, the two studies present compelling evidence that zinc finger-based transcriptional repressors targeted to the Sp1 sites of HIV-1 are able to achieve dramatic and reproducible repression of HIV-1 replication.

As an alternative to directed transcriptional repression at the DNA level with designed zinc finger transcription factors, RNA interference strategies targeting HIV-1 viral RNA have been reported (55, 56). Although these studies have demonstrated successful inhibition of HIV-1 replication through targeting viral RNA, they have also demonstrated that single point mutations within the viral genome allow for the virus to escape RNA interference strategies (57). Given the propensity of HIV-1 to mutate, it has been suggested that effective RNA interference strategies will require the delivery of multiple interfering RNAs. Significantly, in the zinc finger transcription factor strategy explored here, we have demonstrated tolerance of 1 and 2 mutations within the viral sequence targeted by HLTR3. Nonetheless, the development of resistance to zinc finger transcription factors should also be anticipated and an effective therapeutic strategy based on this approach will likely require the delivery of multiple transcription factors. We believe, however, that a highly effective and long lasting transcriptional blockade of HIV-1 can be obtained with a smaller mixture of transcription factors than siRNA molecules. These approaches are, of course, not mutually exclusive, and a therapeutic mixture consisting of both transcription factors and siRNA molecules should be considered.

In conclusion, the results of this study have identified KRAB-HLTR3 as a designed transcription factor capable of potent, specific, and extended inhibition of HIV-1 replication in primary human lymphocytes. Expression of the transcription factor was able to dramatically suppress replication of various HIV-1 strains for nearly 3 weeks, during which time no cellular toxicity was observed. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM065059 and R01 AI49165. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to GenBankTM/EBI Data Bank with the accession number(s) AY518586 [GenBank] , AY518587 [GenBank] , and AY518588 [GenBank] . Back

§ Present address: Dept. of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721. Back

** To whom correspondence should be addressed: The Scripps Research Institute, BCC-550, North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9098; Fax: 858-784-2583; E-mail: carlos{at}scripps.edu.

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; HAART, highly active antiretroviral therapy; siRNA, small interference RNA; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; NARRRP, National Institutes of Health AIDS Research & Reference Reagent Program; LTR, long terminal repeat; IRES, internal ribosome entry site; GFP, green fluorescent protein; HSA, heat-stable antigen; PBMC, peripheral blood mononuclear cell; m.o.i., multiplicity of infection; TAR, transacting response element; HLTR1, HIV-1 LTR target site 1. Back

2 D. J. Segal, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Roberta Fuller for technical assistance. The following reagent were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: P4.R5 MAGI from Dr. Nathaniel Landau, PM1 from Dr. Marvin Reitz, pIIIenv3–1 from Dr. Joseph Sodroski, pSV2tat72 from Dr. Alan Frankel, pNL4–3 from Dr. Malcolm Martin, p89.6 from Dr. Ronald G. Collman, and pYK-JRCSF from Dr. Irvin S. Y. Chen and Dr. Yoshio Koyanagi.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gea-Banacloche, J. C., and Clifford Lane, H. (1999) Aids 13, Suppl. A, S25–S38
  2. Pierson, T., Hoffman, T. L., Blankson, J., Finzi, D., Chadwick, K., Margolick, J. B., Buck, C., Siliciano, J. D., Doms, R. W., and Siliciano, R. F. (2000) J. Virol. 74, 7824–7833[Abstract/Free Full Text]
  3. Schrager, L. K., and D'Souza, M. P. (1998) J. Am. Med. Assoc. 280, 67–71[Abstract/Free Full Text]
  4. Stein, C. A., and Cheng, Y. C. (1993) Science 261, 1004–1012[Abstract/Free Full Text]
  5. Baltimore, D. (1988) Nature 335, 395–396[Medline] [Order article via Infotrieve]
  6. Jacque, J. M., Triques, K., and Stevenson, M. (2002) Nature 418, 435–438[CrossRef][Medline] [Order article via Infotrieve]
  7. Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) Nat. Biotechnol. 20, 500–505[Medline] [Order article via Infotrieve]
  8. Wong-Staal, F., Poeschla, E. M., and Looney, D. J. (1998) Hum. Gene Ther. 9, 2407–2425[Medline] [Order article via Infotrieve]
  9. Amado, R. G., Mitsuyasu, R. T., Symonds, G., Rosenblatt, J. D., Zack, J., Sun, L. Q., Miller, M., Ely, J., and Gerlach, W. (1999) Hum. Gene Ther. 10, 2255–2270[CrossRef][Medline] [Order article via Infotrieve]
  10. Davis, B. R., Saitta, F. P., Bauer, G., Bunnell, B. A., Morgan, R. A., and Schwartz, D. H. (1998) Hum. Gene Ther. 9, 1197–1207[Medline] [Order article via Infotrieve]
  11. Goncalves, J., Silva, F., Freitas-Vieira, A., Santa-Marta, M., Malho, R., Yang, X., Gabuzda, D., and Barbas, C., 3rd. (2002) J. Biol. Chem. 277, 32036–32045[Abstract/Free Full Text]
  12. Dreier, B., Beerli, R. R., Segal, D. J., Flippin, J. D., and Barbas, C. F., III (2001) J. Biol. Chem. 276, 29466–29478[Abstract/Free Full Text]
  13. Dreier, B., Segal, D. J., and Barbas, C. F., III (2000) J. Mol. Biol. 303, 489–502[CrossRef][Medline] [Order article via Infotrieve]
  14. Segal, D. J., Dreier, B., Beerli, R. R., and Barbas, C. F., III (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2758–2763[Abstract/Free Full Text]
  15. Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F., III (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14628–14633[Abstract/Free Full Text]
  16. Segal, D. J. (2002) Methods 26, 76–83[CrossRef][Medline] [Order article via Infotrieve]
  17. Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988) Nature 335, 563–564[CrossRef][Medline] [Order article via Infotrieve]
  18. Margolin, J. F., Friedman, J. R., Meyer, W. K., Vissing, H., Thiesen, H. J., and Rauscher, F. J., 3rd. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4509–4513[Abstract/Free Full Text]
  19. Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1495–1500[Abstract/Free Full Text]
  20. Zhang, L., Spratt, S. K., Liu, Q., Johnstone, B., Qi, H., Raschke, E. E., Jamieson, A. C., Rebar, E. J., Wolffe, A. P., and Case, C. C. (2000) J. Biol. Chem. 275, 33850–33860[Abstract/Free Full Text]
  21. Liu, P. Q., Rebar, E. J., Zhang, L., Liu, Q., Jamieson, A. C., Liang, Y., Qi, H., Li, P. X., Chen, B., Mendel, M. C., Zhong, X., Lee, Y. L., Eisenberg, S. P., Spratt, S. K., Case, C. C., and Wolffe, A. P. (2001) J. Biol. Chem. 276, 11323–11334[Abstract/Free Full Text]
  22. Ren, D., Collingwood, T. N., Rebar, E. J., Wolffe, A. P., and Camp, H. S. (2002) Genes Dev. 16, 27–32[Abstract/Free Full Text]
  23. Stege, J. T., Guan, X., Ho, T., Beachy, R. N., and Barbas, C. F., 3rd. (2002) Plant J. 32, 1077–1086[CrossRef][Medline] [Order article via Infotrieve]
  24. Guan, X., Stege, J., Kim, M., Dahmani, Z., Fan, N., Heifetz, P., Barbas, C. F., 3rd., and Briggs, S. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13296–13301[Abstract/Free Full Text]
  25. Beerli, R. R., Schopfer, U., Dreier, B., and Barbas, C. F., III (2000) J. Biol. Chem. 275, 32617–32627[Abstract/Free Full Text]
  26. Blancafort, P., Magnenat, L., and Barbas, C. F., III (2003) Nat. Biotechnol. 21, 269–274[CrossRef][Medline] [Order article via Infotrieve]
  27. Reynolds, L., Ullman, C., Moore, M., Isalan, M., West, M. J., Clapham, P., Klug, A., and Choo, Y. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 1615–1620[Abstract/Free Full Text]
  28. Wu, H., Yang, W.-P., and Barbas, C. F., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 344–348[Abstract/Free Full Text]
  29. Desjarlais, J. R., and Berg, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2256–2260[Abstract/Free Full Text]
  30. Sodroski, J., Goh, W. C., Rosen, C., Campbell, K., and Haseftine, W. A., (1986) Nature 322, 470–474[CrossRef][Medline] [Order article via Infotrieve]
  31. Frankel, A. D., and Pabo, C. O. (1988) Cell 55, 1189–1193[CrossRef][Medline] [Order article via Infotrieve]
  32. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284–291[Abstract/Free Full Text]
  33. Koyanagi, Y., Miles, S., Mitsuyasu, R. T., Merrill, J. E., Vinters, H. V., and Chen, I. S. Y. (1987) Science 236, 819–822[Abstract/Free Full Text]
  34. Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997) Nat. Biotechnol. 15, 871–875[CrossRef][Medline] [Order article via Infotrieve]
  35. Lusso, P., Cocchi, F., Balotta, C., Markham, P. D., Louie, A., Farci, P., Pal, R., Gallo, R. C., and Reitz, M. S., Jr. (1995) J. Virol. 69, 3712–3720[Abstract]
  36. Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B. E., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 805–810[Abstract/Free Full Text]
  37. Collman, R., Balliet, J. W., Gregory, S. A., Friedman, H., Kolson, D. L., Nathanson, N., and Srinivasan, A. (1992) J. Virol. 66, 7517–7521[Abstract/Free Full Text]
  38. Zufferey, R., Dull, T., Mandel, R., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D. (1998) J. Virol. 72, 9873–9880[Abstract/Free Full Text]
  39. Taube, R., Fujinaga, K., Wimmer, J., Barboric, M., and Peterlin, B. M. (1999) Virology 264, 245–253[CrossRef][Medline] [Order article via Infotrieve]
  40. Jeang, K. T., Xiao, H., and Rich, E. A. (1999) J. Biol. Chem. 274, 28837–28840[Free Full Text]
  41. He, G., Ylisastigui, L., and Margolis, D. M. (2002) DNA Cell Biol. 21, 697–705[CrossRef][Medline] [Order article via Infotrieve]
  42. Pereira, L. A., Bentley, K., Peeters, A., Churchill, M. J., and Deacon, N. J. (2000) Nucleic Acids Res. 28, 663–668[Abstract/Free Full Text]
  43. Jones, K. A., Kadonaga, J. T., Luciw, P. A., and Tjian, R. (1986) Science 232, 755–759[Abstract/Free Full Text]
  44. Berkhout, B., and Jeang, K. T. (1992) J. Virol. 66, 139–149[Abstract/Free Full Text]
  45. Verdin, E., Paras, P., Jr., and Van Lint, C. (1993) EMBO J. 12, 3249–3259[Medline] [Order article via Infotrieve]
  46. Kuiken, C., Foley, B., Hahn, B., Korber, B., Marx, P., McCutchan, F., Mellors, J., and Wolinksy, S. (eds) (2001) HIV Sequence Compendium, Los Alamos National Laboratory, Los Alamos, NM
  47. Ayer, D. E., Laherty, C. D., Lawrence, Q. A., Armstrong, A. P., and Eisenman, R. N. (1996) Mol. Cell. Biol. 16, 5772–5781[Abstract]
  48. Rossi, F., Charlton, C. A., and Blau, H. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8405–8410[Abstract/Free Full Text]
  49. Charneau, P., Mirambeau, G., Roux, P., Paulous, S., Buc, H., and Clavel, F. (1994) J. Mol. Biol. 241, 651–662[CrossRef][Medline] [Order article via Infotrieve]
  50. Segal, D., and Barbas, C. F., III (2001) Curr. Opin. Biotech. 12, 632–637[CrossRef][Medline] [Order article via Infotrieve]
  51. Huang, L. M., and Jeang, K. T. (1993) J. Virol. 67, 6937–6944[Abstract/Free Full Text]
  52. Kim, J. S., and Pabo, C. O. (1997) J. Biol. Chem. 272, 29795–29800[Abstract/Free Full Text]
  53. Ross, E. K., Buckler-White, A. J., Rabson, A. B., Englund, G., and Martin, M. A. (1991) J. Virol. 65, 4350–4358