Stimulation of the Human RAD51 Nucleofilament Restricts HIV-1 Integration In Vitro and in Infected Cells

Abstract

]. 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...

Solution Conformations of Prototype Foamy Virus Integrase and Its Stable Synaptic Complex with U5 Viral DNA

“…For example, the cellular double-strand break repair proteins Rad51 (Cosnefroy et al, 2012; Desfarges et al, 2006), Ku (Zheng et al, 2011), and Rad18 (Mulder et al, 2002) have all been reported to interact with IN and may play a role in repairing the gaps in retroviral DNA integration intermediates to complete the creation of provirus (Yoder and Bushman, 2000). …”

Double-strand DNA breaks and repair response in human immature oocytes and their relevance to meiotic resumption

“…γH2AX is an effector that modifies and makes accessible the structure of chromatin domains at the lesion sites [11,12], ultimately acting as a catalyst for the recruitment of the necessary cell cycle checkpoint and DNA repair factors [13]. Rad51 is the central enzymatic component of the mechanism that ensures homologous recombination and error-free repair of DSBs [14]. Collectively, the present data reveal the occurrence of DSBs and the activation of a repair response in human oocytes, suggesting novel aspects of the context that dictate success or failure of meiotic resumption.…”

Poly(ADP-Ribose) Polymerase 1 Promotes Transcriptional Repression of Integrated Retroviruses

“…This event triggers a complex cellular response that is directed to preserve the integrity of the host genome, as well as its chromatin architecture. This complex cellular response includes DNA damage repair and chromatin remodeling (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”