p66 Trp24 and Phe61 are essential for accurate association of HIV-1 reverse transcriptase with primer/template.

Preventing dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) constitutes an alternative strategy to abolish virus proliferation. We have previously demonstrated that a short peptide derived from the Trp cluster of the connection domain disrupts the RT dimer by interacting with Trp24 and Phe61 in a cleft located between the fingers and the connection domains of p51. Both Trp24 and Phe61 of p51 are essential for the stability of the RT dimer. Here, in order to understand the requirement of Trp24 and Phe61 in the p66 subunit, we have investigated their implication in the formation of RT-primer/template (p/t) complexes and in RT processivity by combining pre-steady-state and steady-state kinetics with site-directed mutagenesis. We demonstrate that both residues are essential for proper binding of the p/t and control conformational changes required for RT ordered mechanism. Trp24 and Phe61 act on p/t binding and remodeling of the catalytic site. Phe61G mutation increases the binding "on" rate of both p/t and mismatched p/t, yielding an unfavorable RT-p/t for polymerase catalysis, unable to pursue mispair extension. Considering the structure of unliganded RT, Phe61 seems to be involved in the dynamics of p66 thumb-finger interactions and in stabilization of the p/t in the catalytic site. In contrast, the p66 Trp24G mutation alters the overall kinetics of p/t binding and is essentially involved in stabilizing the RT-p/t complex by contacting the 5' overhang of the template strand. Mutation of both Trp24 and Phe61 alters mispair extension efficiency, suggesting that disruption of the tight contacts between the fingers domain and the 5' overhang of the template strand increases RT fidelity and reduces RT processivity. Taken together, these studies infer that mutations altering the aromatic nature of Phe61 or Trp24 that may occur to counteract peptide inhibitors targeting this region will generate an unstable RT exhibiting low polymerase activity and higher fidelity. As such, our work suggests that the combined application of peptide-based RT dimerization inhibitors is likely to be highly efficient.

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy.

Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNA(Lys,2) to two distinct primer binding sites (PBS) at the genome 5' and 3' ends. Only the 5' PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.

A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction.

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.

Insight into the mechanism of a peptide inhibitor of HIV reverse transcriptase dimerization.

The biologically active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase (RT) found in infectious virions are heterodimers. We have previously shown that the dimeric nature of reverse transcriptase represents an important target for the design of a new class of antiviral agents and have designed a short peptide (Pep-7) derived from the tryptophan-rich motif of the connection subdomain that blocks dimerization of reverse transcriptase in vitro and abolishes viral infection. In the present work, we have investigated the mechanism through which this peptide inhibits RT dimerization and consequently viral propagation. We demonstrate that Pep-7 interacts preferentially with the p51 subunit within the heterodimeric reverse transcriptase, which destabilizes reverse transcriptase dimer conformation, thereby triggering dissociation. We have identified two residues Trp(24) and Phe(61), located on the fingers subdomain of p51, required for Pep-7 binding. Selective mutation of these residues on p51 to a glycine dramatically alters the stability of the RT-heterodimer suggesting that the fingers subdomain of p51 is also involved in stabilization of reverse transcriptase. We propose that the binding site of Pep-7 is located in a cleft between the fingers and the connection subdomains of p51 that contains the two highly conserved residues Phe(61) and Trp(24).