St. Gelais, C. | bioRxiv Science

bioRxiv2020

TRAF6 and TAK1 contribute to SAMHD1-mediated negative regulation of NF-kB signaling

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular dNTP pool. SAMHD1 also suppresses the activation of NF-{kappa}B 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-{kappa}B activation is modulated by two key mediators of NF-{kappa}B signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor-{beta}-activated kinase-1 (TAK1). We compared NF-{kappa}B activation stimulated by interleukin (IL)-1{beta} 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-1{beta}-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-{kappa}B (I{kappa}B), an indication of pathway activation, and elevated levels of TNF- mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced I{kappa}B phosphorylation and TNF- mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF- 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-{kappa}B 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-{kappa}B activation and HIV-1 infection. ImportanceCells respond to pathogen infection by activating a complex innate immune signaling pathway, which culminates in the activation of transcription factors and secretion of a family of functionally and genetically related cytokines. However, excessive immune activation may cause tissue damage and detrimental effects on the host. Therefore, in order to maintain host homeostasis, the innate immune response is tightly regulated during viral infection. We have reported SAMHD1 as a novel negative regulator of the innate immune response. Here, we provide new insights into SAMHD1-mediated negative regulation of the NF-{kappa}B pathway at the TRAF6-TAK1 checkpoint. We show that SAMHD1 inhibits TAK1 activation and TRAF6 signaling in response to proinflammatory stimuli. Interestingly, TRAF6 knockdown in SAMHD1-deficient cells significantly inhibited HIV-1 infection and activation of NF-{kappa}B induced by virus infection. Our research reveals a new negative regulatory mechanism by which SAMHD1 participates in the maintenance of cellular homeostasis during HIV-1 infection and inflammation.

microbiology↗

bioRxiv2018

SAMHD1 Impairs HIV-1 Gene Expression and Reactivation of Viral Latency in CD4+ T-cells

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in non-dividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T-cells that are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 LTR activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T-cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 long terminal repeat (LTR)-driven gene expression at the level of transcription. SAMHD1 overexpression also suppressed LTR activity from human T-cell leukemia virus type 1 (HTLV-1), but not from murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D/R207N, or HD/RN) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro. These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression contributes to regulation of viral latency in CD4+ T-cells.\n\nIMPORTANCEA critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T-cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in non-dividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T-cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T-cells.

microbiology↗