HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

DNA topoisomerases from rat liver: physiological variations.

Besides the nicking-closing (topoisomerase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei. The enzyme, partially purified, is able to catenate in vitro closed DNA circles in a magnesium-dependent, ATP-dependent, histone H1-dependent reaction, and to decatenate in vitro kinetoplast DNA networks to yield free minicircles in a magnesium-dependent and ATP-dependent reaction. It is largely similar to other eukaryotic type II topoisomerases in its requirements, and presumably belongs to this class of enzymes. Type I and type II activities were measured in rat liver nuclei as a function of regenerating time after partial hepatectomy: type I activity was not significantly changed during this process. In contrast, type II activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication

Nucleocapsid protein: A desirable target for future therapies against HIV-1

8noneThe currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.noneMori, Mattia; Kovalenko, Lesia; Lyonnais, Sébastien; Antaki, Danny; Torbett, Bruce E.; Botta, Maurizio; Mirambeau, Gilles; Mély, YvesMori, Mattia; Kovalenko, Lesia; Lyonnais, Sébastien; Antaki, Danny; Torbett, Bruce E.; Botta, Maurizio; Mirambeau, Gilles; Mély, Yve