BackgroundGenomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation.ResultsHere, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication.ConclusionsOur data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0153-9) contains supplementary material, which is available to authorized users.

]. DNA-damaging agents, such as ionizing radiation and chemical compounds generating replication-blocking lesions like cis-platinum, also cause DSBs. Retroviral integration involving a cut of the genomic DNA can also be considered a potentially mutagenic event. The cellular DNA repair machinery could thus be required either for cell defense against viral infection or, inversely, for the stable integration of the viral genome. Since these machineries are not completely defined at present, they require better characterization. Retroviral integration is catalyzed by the virally encoded integrase (IN) in concert with other viral and cellular factors forming the preintegration complex (PIC). As a final step, stable integration of retroviral DNA requires the repair of the DNA gaps remaining after the reaction catalyzed by IN. Several cellular repair mechanisms, including base excision repair (3, 14, 43, 44), homologous recombination (HR) (19), and nonhomologous end-joining (NHEJ) (9-11, 26), are thought to be involved in the integration step. Direct interactions between HIV-1 IN and human RAD18 have been reported (31), and cell-based studies have indicated that RAD18 and RAD52 can inhibit HIV-1 infection (22,27). These data suggest that cellular RAD DNA repair machineries can play a dual role by preventing or restricting retroviral viral replication/parasitism and/or participating directly in the stability of the integrated viral DNA, a crucial step of the infection process.Human RAD51 (hRAD51), belonging to the HR DNA repair RAD52 epistasis group, has been previously shown to interact functionally with HIV-1 IN both in vitro and in a yeast integration model (12). A downregulation of HIV-1 IN activity by hRAD51 was also reported in in vitro integration assays, but the inhibition mechanism remained unsolved. Since the enhancement of this inhibition could constitute a new antiviral approach as well as signify an original restriction pathway, we focused our work here on the molecular mechanism underlying this process both in vitro and in vivo. We especially studied whether this hRAD51-mediated IN inhibition could be promoted in HIV-1-infected cells.Using standard in vitro integration assays under conditions promoting or inhibiting hRAD51 activity (hRAD51 mutants, presence or absence of ATP, addition of a hRAD51 stimulatory compound), we show that the formation of an active hRAD51 presynaptic nucleofilament corresponding to the nucleocomplex formed on the two homologous DNA strands during HR is required for inhibition. An IN-DNA dissociation system was also set up in order to better determine the effect of the hRAD51 nucleofilament on the active integration complex. Using this system we show that this nucleofilament can dissociate IN from its substrate. Finally, the availability of RS-1, a chemical compound able to stimulate hRAD51 activity, allowed us to demonstrate that the stimulation of the hRAD51-mediated DNA repair process in HIV-1-infected cells can lead to the inhibition of retroviral replication by decreasing int...

The ability of retroviruses to integrate their genomes into host chromatin is a key step for the completion of their replication cycle. Selection of a suitable chromosomal integration site has been described as a hierarchical mechanism involving both cellular and viral proteins but the exact molecular determinants are still unclear. We recently showed that the spumaretrovirus prototype foamy virus (PFV) Gag protein is acting as a chromatin tether by interacting with the nucleosome acidic patch (Lesbats et al. PNAS 114(21)). Disruption of the nucleosome binding leads to a dramatic delocalization of both the viral particles and the integration sites accompanied with a reduction of integrated genes expression. These data show for the first time a direct interaction between retroviral structural proteins with the host chromosomes, and highlight their importance in the integration sites selection.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro . Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.