Abstract The NF- κ B family of transcription factors and associated signalling pathways are abundant and ubiquitous in human immune responses. Activation of NF- κ B transcription factors by viral pathogen-associated molecular patterns, such as viral RNA and DNA, is fundamental to anti-viral innate immune defences and pro-inflammatory cytokine production that steers adaptive immune responses. Diverse non-viral stimuli, such as lipopolysaccharide and cytokines, also activate NF- κ B and the same anti-pathogen gene networks. Viruses adapted to human cells often encode multiple proteins aimed at varied NF- κ B pathway targeted to mitigate the anti-viral effects of NF- κ B-dependent host immunity. In this study we have demonstrated using numerous assays, in a number of different cell types, that plasmid-encoded or virus-delivered Simian Immunodeficiency Virus (SIV) accessory protein Vpx is a broad antagonist of NF- κ B signalling active against diverse innate NF- κ B agonists. Using targeted Vpx mutagenesis, we showed that this novel Vpx phenotype is independent of known Vpx cofactor DCAF1 and other cellular binding partners, including SAMHD1, STING and the HUSH complex. We found that Vpx co-immunoprecipitated with canonical NF- κ B transcription factor p65 and not NF- κ B transcription factor proteins p50 or p100, preventing nuclear translocation of p65, a novel mechanism of NF- κ B antagonism by lentiviruses. We found that broad antagonism of NF- κ B activation by Vpx was conserved across distantly related lentiviruses as well as for Vpr from SIV Mona monkey (SIVmon), which has Vpx-like SAMHD1-degradation activity. Importance Broad antagonism of NF- κ B activation has been described for HIV-1, but not for Vpx-encoding lentiviruses such as HIV-2. Here we extend our understanding of lentiviral antagonism by identifying an interaction between Vpx and transcription factor NF- κ B p65, leading to inhibition of its nuclear translocation and broad NF- κ B antagonism. This further evidences a requirement for lentiviruses to target universal regulators of immunity, including NF- κ B, to avoid the anti-viral sequelae of pro-inflammatory gene expression stimulated by both viral and extra-viral agonists, such as lipopolysaccharide translocation, during disruption of the gut microbiome barrier during HIV-1 infection. Further structural studies of p65 targeting by Vpx may yield translational insights in the form of novel pan-NF- κ B inhibitors for pathologies characterised by excessive NF- κ B activity. Our findings are also relevant to the gene therapy field where virus-like particle associated Vpx is routinely used to enhance vector transduction through antagonism of SAMHD1, and perhaps also through manipulation of other pathways such as NF- κ B.

HIV-2/SIV Vpx antagonises NF-κB activation by targeting p65