First Mariner Mos1 transposase inhibitors.

We described chemical inhibitors of Mos1 transposition. Some were already known to affect a related prokaryotic transposase (Tn5) or HIV-1 integrase, whereas the other were new compounds in this field. The new compounds were all organized around a bis-(heteroaryl)maleimides scaffold. Their mechanism of action depended on the chemical substitutions on the scaffold. The cross-activity, between HIV-1 integrase and Mos1 transposase, of the new group of inhibitors showed that Mos1 transposase could constitute an excellent surrogate HIV-1 inhibitor screen.

6-oxocytidine containing oligonucleotides inhibit the HIV-1 integrase in vitro.

Integration of the proviral DNA into the genome of infected cells is a key step of HIV-1 replication. Integration is catalyzed by the viral enzyme integrase (IN). 6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors of integrase in vitro. The inhibitory effect is sequence-specific and strictly requires the presence of the 6-oxocytidine base. It is due to the impairment of the integrase binding to its substrate and does not involve an auto-structure of the oligonucleotide.

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

Interactions of the C-terminus of viral protein R with nucleic acids are modulated by its N-terminus.

The basic viral protein R (Vpr) performs several functions during the human immunodeficiency virus HIV-1 retroviral cycle, including G2 mitosis arrest and nuclear import of the preintegration complex allowing lentivirus to replicate in nondividing cells. Accordingly, this protein was found in the nucleus of infected cells. In the virus, Vpr is incorporated through interaction with both nucleocapsid protein 7 (NCp7) and p6, two small proteins encoded by the C-terminal part of the Gag precursor. NCp7 is also involved in genomic RNA encapsidation during the budding process suggesting a possible interaction of Vpr with nucleic acids, either directly or via the NCp7 intermediate. Gel shift experiments were carried out with RNA and DNA using synthetic Vpr and peptide derivatives. The results show that Vpr binds to nucleic-acid inducing aggregates. This process, which requires the C-terminal basic domain of the protein (in particular the helical 70-80 domain), is regulated by the N-terminal region of Vpr. Moreover, NCp7 was shown to enhance RNA recognition by Vpr, a feature that could be required for Vpr encapsidation and during nuclear import of the preintegration complex.