Journal of Molecular Biology
A Novel Short Peptide is a Specific Inhibitor of the Human Immunodeficiency Virus Type 1 Integrase
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)
- et al.
Retroviral integrase inhibitors year 2000: update and perpectives
Antiviral Res.
(2000) - et al.
Nuclear import of HIV-1 preintegration complexes
Advan. Virus Res.
(1999) - et al.
Molecular mechanisms in retrovirus DNA integration
Antiviral Res.
(1997) - et al.
An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer
J. Biol. Chem.
(1995) - et al.
Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro
J. Biol. Chem.
(1993) - et al.
Recombining the structures of HIV integrase, RuvC and RNase H
Structure
(1995) In vitro assays for activities of retroviral integrase
Methods
(1997)- et al.
HIV-1 reverse transcription. A termination step at the center of the genome
J. Mol. Biol.
(1994) - et al.
An inhibitory monoclonal antibody binds at the turn of the helix-turn-helix motif in the N-terminal domain of HIV-1 integrase
J. Biol. Chem.
(2000) - et al.
The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase
FEBS Letters
(2000)