The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4FANCP deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.