A Novel Short Peptide is a Specific Inhibitor of the Human Immunodeficiency Virus Type 1 Integrase

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Abstract

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3′ end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an α-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.

Introduction

HIV-1 integrase (IN) catalyzes the insertion of proviral DNA into the host genome and as such, it is essential for the viral replication cycle.1 Since IN has no cellular counterpart and is necessary for productive infection, identifying specific potent inhibitors of this enzyme should provide novel anti-HIV therapeutic strategies. In contrast to reverse transcriptase (RT) and protease (PR), the other two HIV-1 enzymes currently used as targets in the multi-therapy strategy, few inhibitors against IN have been described and none of them seems to behave as potential therapeutic agents.2 This lack of IN inhibitors is partly due to the difficulties encountered in structural studies of the whole protein and to insufficient information concerning the biochemical mechanism of proviral integration.

Retroviral integration proceeds in two steps: (i) 3′ end processing in which two nucleotides are removed from the 3′ end of each strand of the linear proviral DNA; (ii) DNA strand transfer, a concerted cleavage–ligation reaction, in which the recessed 3′ ends of the viral DNA are covalently joined to the host DNA. The two unpaired nucleotides at the 5′ ends of the viral DNA are then removed and the gaps between the viral and the target DNA are repaired. The nature of the DNA polymerase involved in the latter step remains to be established.

In vitro analyses have shown that only two elements are necessary for integration: the HIV-1 IN and the cis acting DNA sequences at the end of the proviral DNA long terminal repeats (LTRs). Although purified recombinant HIV-1 IN performs all the steps known to be required for end processing and strand transfer on model DNA substrates in vitro, such reactions differ from authentic integration because coordinate joining of the two viral ends remains inefficient. In vivo, the enzyme may attain the expected efficiency by interacting with viral or cellular proteins present in the pre-integration complex (PIC).3

In vivo and in vitro complementation studies suggest that the active HIV-1 IN is a multimer.4., 5. This enzyme displays three independent structural and functional domains which are able to form dimers themselves.6 (i) The amino-terminal domain (residues 1–50) contains the conserved HHCC motif and binds one atom of zinc.7 The structure of the amino-terminal domain has been solved by NMR spectroscopy.8 This region is involved in protein–protein interaction and contributes to the specific recognition of viral DNA ends.9., 10. (ii) The core or catalytic domain (residues 50–212) contains the highly conserved D,D(35)E motif present in retroviral integrases and retrotransposons. Mutations of any of the three acidic residues Asp64, Asp116 and Glu152 abolish the HIV-1 IN activity.11., 12., 13. (iii) The carboxy-terminal domain (residues 213–288) is the least conserved. This region exhibits similarity with an SH3 domain and is involved in non-specific DNA binding and multimerization.14., 15. Very recently, the introduction of five point mutations led to the determination of the crystal structure of the IN domain expressing the catalytic core and the carboxy-terminal domain.16 However, the three domains are required for in vitro 3′ end processing and DNA strand transfer.17 Despite several efforts to determine the structure of the entire protein, the low solubility of the native integrase has not allowed its determination.

Since HIV-1 integrase is essential to reach a productive infection, this enzyme is a key target for antiviral compounds. In an attempt to identify inhibitors against this enzyme, we used the two-hybrid system. This technique has been developed in order to allow identification of potential inhibitors of protein function in the form of small peptides (aptamers) that specifically recognize a protein of interest.18 These high affinity peptides may interfere with the activity of the target protein, either by inhibiting directly the enzyme activity or by blocking its interactions with other proteins or substrates. Peptides interfering selectively with a protein offer several advantages for studying protein functions, since they can be used to target not only specific proteins but also the specific functions of a given protein. In addition, these specific peptides could be expressed in living cells or vectorized into a given cellular compartment.

Here we report the identification and characterization of a 33-mer-peptide (I33) able to inhibit the HIV-1 IN activities in vitro. Its inhibitory potential was optimized by using a 12-mer peptide (EBR28) corresponding to the amino-terminal part of I33. This EBR28 peptide was shown to interact with the catalytic core of IN. In the EBR28 NMR structure, all the hydrophobic residues are located on one side of the α-helix and the more hydrophilic ones on the other side. This arrangement is probably important for its interaction with integrase. Various novel EBR28 derivatives were synthesized and assayed in vitro against HIV-1 IN, as well as on human HIV-1 infected cells.

Section snippets

Inhibition of in vitro HIV-1 integrase activity

In our search for novel inhibitors we used the yeast two-hybrid system to identify peptides able to strongly bind to HIV-1 IN. This screening led us to identify several clones containing IN-interacting inserts. Sequence analyses revealed that 15 inserts belonged to a non-coding region of the mitochondrial genome. They corresponded to two short peptides of different lengths: a 33-mer (I33) and a 29-mer (I29). They were identical except for the four extra amino acid residues in the N-terminal

Discussion

HIV-1 IN is a potential target for therapeutic intervention, given the essential role of proviral integration in the retroviral life cycle. The lack of any known human enzyme activity analogous to retroviral IN raises the hope that integration inhibitors might be relatively non-cytotoxic. Progress made in the understanding of the integration process has led to the discovery of compounds able to inhibit IN activity in vitro. Inhibitors of this enzyme have been described to act either on the

Yeast

JSC310, a yeast strain deficient for several proteases was used for the expression and purification of IN.48 The yeast strains used in the two-hybrid screening were HF7c and Y187 (Clontech). For the yeast lethal assay the AB2 strain was used.25

Bacteria

The E. coli strain DH5α was used for plasmid amplification, and BL21(DE3) for expression of His-tagged IN. Luria Beriani medium containing 50 mg/l of ampicillin was used for E. coli strains DH5α and BL21(DE3). Kanamycin (10 mg/l) was added as required.

Peptide synthesis

Acknowledgements

We thank S.H. Hughes (NCI, Frederick, Maryland, USA) for kindly providing HIV-1 IN antibodies, J.F. Mouscadet (UMR-CNRS 8532, Villejuif, France) for the generous gift of the histidine-tagged HIV-1 IN constructions and P. Durrens for his help with the two-hybrid assay. We acknowledge M.L. Andreola (UMR-CNRS 5097, Bordeaux, France) for fruitful discussions and pertinent suggestions and L. Tarrago-Litvak (UMR-CNRS 5097, Bordeaux, France) for critical comments on the manuscript. The excellent

References (63)

  • S. Maignan et al.

    Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases

    J. Mol. Biol.

    (1998)
  • Z.W. Lu et al.

    Conformational analysis of COOH-terminal segments of human C3a. Evidence of ordered conformation in an active 21-residue peptide

    J. Biol. Chem.

    (1984)
  • M.C. Morris et al.

    A new potent HIV-1 reverse transcriptase inhibitor

    J. Biol. Chem.

    (1999)
  • A. Caumont et al.

    High affinity interaction of HIV-1 integrase with specific and non-specific single-stranded short oligonucleotides

    FEBS Letters

    (1999)
  • T. Mosmann

    Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

    J. Immunol. Methods

    (1983)
  • F. Kippert

    A rapid permeabilization procedure for accurate quantitative determination of beta-galactosidase activity in yeast cells

    FEMS Microbiol. Letters

    (1995)
  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • D. Marion et al.

    Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurement of 1H–1H spin–spin coupling constants in proteins

    Biochem. Biophys. Res. Commun.

    (1983)
  • R. Hawkes et al.

    A dot-immunobinding assay for monoclonal and other antibodies

    Anal. Biochem.

    (1982)
  • J.M. Coffin et al.
    (1997)
  • T.M.R. Fletcher et al.

    Complementation of integrase function in HIV-1 virions

    EMBO J.

    (1997)
  • A. Engelman et al.

    Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex

    EMBO J.

    (1993)
  • R. Zheng et al.

    Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity

    Proc. Natl Acad. Sci. USA

    (1996)
  • M. Cai et al.

    Solution structure of the N-terminal zinc binding domain of HIV-1 integrase

    Nature Struct. Biol.

    (1997)
  • T.S. Heuer et al.

    Photo-cross-linking studies suggest a model for the architecture of an active HIV-1 integrase–DNA complex

    Biochemistry

    (1998)
  • J. Kulkosky et al.

    Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases

    Mol. Cell Biol.

    (1992)
  • P.M. Cannon et al.

    HIV-1 integrase: effect on viral replication of mutations at highly conserved residues

    J. Virol.

    (1994)
  • A.P. Eijkelenboom et al.

    The DNA-binding domain of HIV-1 integrase has an SH3-like fold

    Nature Struct. Biol.

    (1995)
  • R.A. Lutzke et al.

    Structure-based mutational analysis of the C-terminal DNA-binding domain of HIV-1 integrase: critical residues for protein oligomerization and DNA binding

    J. Virol.

    (1998)
  • J.C. Chen et al.

    Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding

    Proc. Natl Acad. Sci. USA

    (2000)
  • C. Vink et al.

    Identification of the catalytic and DNA-binding region of the HIV-1 integrase protein

    Nucl. Acid Res.

    (1993)
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