PMID- 33177202 OWN - NLM STAT- MEDLINE DCOM- 20210315 LR - 20210714 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 95 IP - 3 DP - 2021 Jan 13 TI - TRAF6 and TAK1 Contribute to SAMHD1-Mediated Negative Regulation of NF-kappaB Signaling. LID - 10.1128/JVI.01970-20 [doi] LID - e01970-20 AB - 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-kappaB 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-kappaB activation is modulated by two key mediators of NF-kappaB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor beta-activated kinase 1 (TAK1). We compared NF-kappaB activation stimulated by interleukin (IL)-1beta 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-1beta-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-kappaB (IkappaBalpha), an indication of pathway activation, and elevated levels of TNF-alpha mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IkappaBalpha phosphorylation and TNF-alpha mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF-alpha 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-kappaB 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-kappaB activation and HIV-1 infection.IMPORTANCE Cells 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-kappaB 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-kappaB 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. CI - Copyright (c) 2021 Espada et al. FAU - Espada, Constanza E AU - Espada CE AD - Department of Microbiology and Immunology, Carver College of Medicine, The University of Iowa, Iowa City, Iowa, USA. FAU - St Gelais, Corine AU - St Gelais C AD - Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA. FAU - Bonifati, Serena AU - Bonifati S AD - Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA. FAU - Maksimova, Victoria V AU - Maksimova VV AD - Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA. FAU - Cahill, Michael P AU - Cahill MP AD - Department of Microbiology and Immunology, Carver College of Medicine, The University of Iowa, Iowa City, Iowa, USA. FAU - Kim, Sun Hee AU - Kim SH AD - Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA. FAU - Wu, Li AU - Wu L AUID- ORCID: 0000-0002-5468-2487 AD - Department of Microbiology and Immunology, Carver College of Medicine, The University of Iowa, Iowa City, Iowa, USA li-wu@uiowa.edu. LA - eng GR - P30 CA086862/CA/NCI NIH HHS/United States GR - R01 AI141495/AI/NIAID NIH HHS/United States GR - R01 AI150343/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20210113 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (NF-kappa B) RN - 0 (Tifab protein, human) RN - EC 2.7.11.25 (MAP Kinase Kinase Kinases) RN - EC 2.7.11.25 (MAP kinase kinase kinase 7) RN - EC 3.1.5.- (SAM Domain and HD Domain-Containing Protein 1) RN - EC 3.1.5.- (SAMHD1 protein, human) SB - IM MH - *Gene Expression Regulation MH - HEK293 Cells MH - HIV Infections/*immunology/metabolism/virology MH - HIV-1/physiology MH - Humans MH - Immunity, Innate/*immunology MH - Intracellular Signaling Peptides and Proteins/genetics/*metabolism MH - MAP Kinase Kinase Kinases/genetics/*metabolism MH - NF-kappa B/genetics/*metabolism MH - SAM Domain and HD Domain-Containing Protein 1/genetics/*metabolism MH - Signal Transduction PMC - PMC7925110 OTO - NOTNLM OT - HIV-1 OT - NF-kappaB activation OT - SAMHD1 OT - TAK1 OT - TRAF6 OT - inflammation OT - regulation EDAT- 2020/11/13 06:00 MHDA- 2021/03/16 06:00 PMCR- 2021/07/13 CRDT- 2020/11/12 05:34 PHST- 2020/10/05 00:00 [received] PHST- 2020/11/04 00:00 [accepted] PHST- 2020/11/13 06:00 [pubmed] PHST- 2021/03/16 06:00 [medline] PHST- 2020/11/12 05:34 [entrez] PHST- 2021/07/13 00:00 [pmc-release] AID - JVI.01970-20 [pii] AID - 01970-20 [pii] AID - 10.1128/JVI.01970-20 [doi] PST - epublish SO - J Virol. 2021 Jan 13;95(3):e01970-20. doi: 10.1128/JVI.01970-20. Print 2021 Jan 13.