TRAF6 and TAK1 Contribute to SAMHD1-Mediated Negative Regulation of NF-κB Signaling.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular deoxynucleoside triphosphate (dNTP) pool. SAMHD1 also suppresses the activation of NF-κB in response to viral infections and inflammatory stimuli. However, the mechanisms by which SAMHD1 negatively regulates this pathway remain unclear. Here, we show that SAMHD1-mediated suppression of NF-κB activation is modulated by two key mediators of NF-κB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor β-activated kinase 1 (TAK1). We compared NF-κB activation stimulated by interleukin (IL)-1β in monocytic THP-1 control and SAMHD1 knockout (KO) cells with and without partial TRAF6 knockdown (KD), or in cells treated with TAK1 inhibitors. Relative to control cells, IL-1β-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-κB (IκBα), an indication of pathway activation, and elevated levels of TNF-α mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IκBα phosphorylation and TNF-α mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF-α mRNA levels compared to control cells, and the effects were significantly reduced by TRAF6 KD or TAK1 inhibition. We further demonstrated that overexpressed SAMHD1 inhibited TRAF6-stimulated NF-κB reporter activity in HEK293T cells in a dose-dependent manner. SAMHD1 contains a nuclear localization signal (NLS), but an NLS-defective SAMHD1 exhibited a suppressive effect similar to the wild-type protein. Our data suggest that the TRAF6-TAK1 axis contributes to SAMHD1-mediated suppression of NF-κB activation and HIV-1 infection.