ABSTRACT
Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.
Subject(s)
Down-Regulation , HIV Infections/enzymology , HIV-1/physiology , Rad51 Recombinase/metabolism , Virus Integration , Cell Line , DNA Repair , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , HIV Infections/genetics , HIV Infections/virology , HIV Integrase/genetics , HIV Integrase/metabolism , HIV-1/enzymology , HIV-1/genetics , Humans , Protein Binding , Rad51 Recombinase/chemistry , Rad51 Recombinase/genetics , Recombination, GeneticABSTRACT
Intracellular transport of karyophilic cargos comprises translocation to the nuclear envelope and subsequent nuclear import. Small cargos such as isolated proteins can reach the nuclear envelope by diffusion but movement of larger structures depends on active translocation, typically using microtubules. Centripetal transport ends at the perinuclear microtubule organizing centre called the spindle pole body (SPB) in yeast. Previously, we found by two hybrids that the karyophilic lentiviral-encoded integrase (IN) interacts with two yeast microtubule-associated proteins, Dyn2p (dynein light chain protein) and Stu2p, a centrosomal protein (de Soultrait et al., 2002). Thus, to investigate the hinge between cytoplasmic retrograde transport and nuclear import, we decided to analyse HIV-1 IN trafficking in yeast as the model, since each of these biological mechanisms is evolutionarily conserved in eukaryotic cells. Here, we found an accumulation of IN at the SPB in yeast via Stu2p colocalization. Disruption of the microtubule network by nocodazole or IN expression in a dynein 2-deficient yeast strain prevented IN accumulation in the nuclear periphery and additionally inhibited IN transport into the nucleus. By mutagenesis, we showed that trafficking of IN towards the SPB requires the C-terminus of the molecule. Taking our findings together, we proposed a model in which IN nuclear import seems to depend on an essential intermediate step in the SPB. We found that Dyn2p and Stu2p play an important role in driving IN toward MTOC and could optimize nuclear entry of the retroviral enzyme. Our results suggest a new hypothesis in keeping with the current HIV-1 intracellular trafficking model.
Subject(s)
Cell Nucleus/metabolism , HIV Integrase/metabolism , Microtubules/metabolism , Models, Biological , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus/drug effects , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cytoplasm/genetics , Cytoplasm/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Dyneins , Gene Expression , HIV Integrase/chemistry , HIV Integrase/genetics , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/drug effects , Nocodazole/pharmacology , Protein Structure, Tertiary , Protein Transport/drug effects , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection.
