SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1 cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1 cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1 cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1 cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is an enzyme with deoxynucleotide triphosphohydrolase (dNTPase) activity. The ability to regulate the pool of cytosolic dNTPs allows SAMHD1 to restrict replication of viruses that depend on cellular dNTPs for genome replication, such as DNA viruses (1Hollenbaugh J.A. Gee P. Baker J. Daly M.B. Amie S.M. Tate J. et al.Host factor SAMHD1 restricts DNA viruses in non-dividing myeloid cells.PLoS Pathog. 2013; 9e1003481Crossref PubMed Scopus (132) Google Scholar, 2Sommer A.F. Riviere L. Qu B. Schott K. Riess M. Ni Y. et al.Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle.Sci. Rep. 2016; 626616Crossref Scopus (53) Google Scholar, 3Zhang K. Lv D.W. Li R. Conserved herpesvirus protein kinases target SAMHD1 to facilitate virus replication.Cell Rep. 2019; 28: 449-459.e445Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and retroviruses (4Goldstone D.C. Ennis-Adeniran V. Hedden J.J. Groom H.C. Rice G.I. Christodoulou E. et al.HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.Nature. 2011; 480: 379-382Crossref PubMed Scopus (627) Google Scholar, 5Powell R.D. Holland P.J. Hollis T. Perrino F.W. Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase.J. Biol. Chem. 2011; 286: 43596-43600Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Of note, SAMHD1 restricts human immunodeficiency virus type 1 (HIV-1) in noncycling cells, such as macrophages, dendritic cells, and resting CD4+ T cells (6Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. et al.Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (931) Google Scholar, 7Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Segeral E. et al.SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.Nature. 2011; 474: 654-657Crossref PubMed Scopus (1144) Google Scholar, 8St Gelais C. de Silva S. Amie S.M. Coleman C.M. Hoy H. Hollenbaugh J.A. et al.SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated by interferons.Retrovirology. 2012; 9: 105Crossref PubMed Scopus (152) Google Scholar, 9Baldauf H.M. Pan X. Erikson E. Schmidt S. Daddacha W. Burggraf M. et al.SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells.Nat. Med. 2012; 18: 1682-1687Crossref PubMed Scopus (460) Google Scholar). However, SAMHD1 mutations that reduce the dNTP pool without restricting HIV-1 infection have been described, suggesting a dNTPase-independent HIV-1 restriction mechanism (10Welbourn S. Dutta S.M. Semmes O.J. Strebel K. Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1.J. Virol. 2013; 87: 11516-11524Crossref PubMed Scopus (126) Google Scholar, 11Welbourn S. Strebel K. Low dNTP levels are necessary but may not be sufficient for lentiviral restriction by SAMHD1.Virology. 2016; 488: 271-277Crossref PubMed Scopus (43) Google Scholar). Germline mutations in the SAMHD1 gene are associated with development of Aicardi-Goutières syndrome, an autoimmune disease characterized by a type I interferon (IFN-I) dysregulation (12de Jesus A.A. Hou Y. Brooks S. Malle L. Biancotto A. Huang Y. et al.Distinct interferon signatures and cytokine patterns define additional systemic autoinflammatory diseases.J. Clin. Invest. 2020; 130: 1669-1682Crossref PubMed Scopus (110) Google Scholar, 13Ramantani G. Kohlhase J. Hertzberg C. Innes A.M. Engel K. Hunger S. et al.Expanding the phenotypic spectrum of lupus erythematosus in Aicardi-Goutieres syndrome.Arthritis Rheum. 2010; 62: 1469-1477Crossref PubMed Scopus (159) Google Scholar, 14Crow Y.J. Chase D.S. Lowenstein Schmidt J. Szynkiewicz M. Forte G.M. Gornall H.L. et al.Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.Am. J. Med. Genet. A. 2015; 167A: 296-312Crossref PubMed Scopus (387) Google Scholar). Furthermore, spontaneous induction of IFN-I in SAMHD1-deficient human monocytes has been reported (15Martinez-Lopez A. Martin-Fernandez M. Buta S. Kim B. Bogunovic D. Diaz-Griffero F. SAMHD1 deficient human monocytes autonomously trigger type I interferon.Mol. Immunol. 2018; 101: 450-460Crossref PubMed Scopus (16) Google Scholar), suggesting a physiological role of SAMHD1 in regulating the IFN-I pathway. We have demonstrated that SAMHD1 suppresses nuclear factor kappa-B and IFN-I signaling pathways in response to pro-inflammatory stimuli and virus infections (16Chen S. Bonifati S. Qin Z. St Gelais C. Kodigepalli K.M. Barrett B.S. et al.SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3798-E3807Crossref PubMed Scopus (78) Google Scholar, 17Qin Z. Bonifati S. St Gelais C. Li T.W. Kim S.H. Antonucci J.M. et al.The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.J. Biol. Chem. 2020; 295: 1575-1586Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 18Espada C.E. St Gelais C. Bonifati S. Maksimova V.V. Cahill M.P. Kim S.H. et al.TRAF6 and TAK1 contribute to SAMHD1-mediated negative regulation of NF-kappaB signaling.J. Virol. 2021; 95: e01970-e02020Crossref PubMed Scopus (13) Google Scholar). SAMHD1 interacts with inhibitor of nuclear factor kappa-B kinase epsilon (IKKε) and IFN regulatory factor 7 (IRF7) leading to inhibition of phosphorylation of IRF7 by IKKε. Moreover, SAMHD1 inhibited the activity of an IFN-sensitive response element (ISRE) reporter induced by IRF7, but not IRF3 overexpression (16Chen S. Bonifati S. Qin Z. St Gelais C. Kodigepalli K.M. Barrett B.S. et al.SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3798-E3807Crossref PubMed Scopus (78) Google Scholar). IFN-I are involved in limiting the replication of pathogens (19Barnes B. Lubyova B. Pitha P.M. On the role of IRF in host defense.J. Interferon Cytokine Res. 2002; 22: 59-71Crossref PubMed Scopus (276) Google Scholar, 20Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. et al.Triggering the interferon response: the role of IRF-3 transcription factor.J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (198) Google Scholar), modulating immune cell homeostasis and function (21Gough D.J. Messina N.L. Clarke C.J. Johnstone R.W. Levy D.E. Constitutive type I interferon modulates homeostatic balance through tonic signaling.Immunity. 2012; 36: 166-174Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and activating the adaptive immune response (22Tough D.F. Modulation of T-cell function by type I interferon.Immunol. Cell Biol. 2012; 90: 492-497Crossref PubMed Scopus (90) Google Scholar). Thus, understanding the mechanism by which SAMHD1 antagonizes IFN-I is of crucial importance. The IFN-I response in virus-infected cells establishes an antiviral state in neighboring cells to limit virus spread (23McNab F. Mayer-Barber K. Sher A. Wack A. O'Garra A. Type I interferons in infectious disease.Nat. Rev. Immunol. 2015; 15: 87-103Crossref PubMed Scopus (1432) Google Scholar). Upon virus infection, pathogen-associated molecular patterns, such as viral dsRNA, are recognized by the cytoplasmic sensors retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) (24Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5992) Google Scholar). Signal propagation occurs through a common adapter protein called mitochondrial antiviral-signaling protein (MAVS) (25Seth R.B. Sun L. Ea C.K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2548) Google Scholar, 26Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. et al.IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2047) Google Scholar, 27Brubaker S.W. Gauthier A.E. Mills E.W. Ingolia N.T. Kagan J.C. A bicistronic MAVS transcript highlights a class of truncated variants in antiviral immunity.Cell. 2014; 156: 800-811Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Upon engagement of pathogen-associated molecular patterns, conformational changes in RIG-I and MDA-5 facilitate activation of MAVS by phosphorylation and polymerization (28Liu B. Zhang M. Chu H. Zhang H. Wu H. Song G. et al.The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.Nat. Immunol. 2017; 18: 214-224Crossref PubMed Scopus (0) Google Scholar, 29Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. et al.Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347aaa2630Crossref Scopus (1028) Google Scholar, 30Guo W. Wei J. Zhong X. Zang R. Lian H. Hu M.M. et al.SNX8 modulates the innate immune response to RNA viruses by regulating the aggregation of VISA.Cell. Mol. Immunol. 2020; 17: 1126-1135Crossref PubMed Scopus (14) Google Scholar) followed by recruitment and activation of IKK-related kinases, TBK1, and IKKε (31Fang R. Jiang Q. Zhou X. Wang C. Guan Y. Tao J. et al.MAVS activates TBK1 and IKKepsilon through TRAFs in NEMO dependent and independent manner.PLoS Pathog. 2017; 13e1006720Crossref Scopus (114) Google Scholar, 32Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. et al.IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2119) Google Scholar). These kinases phosphorylate IRF3/7 transcription factors, leading to their dimerization and nuclear translocation where they promote IFN-α/β transcription (33Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation.Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (764) Google Scholar, 34Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1.Mol. Cell. Biol. 2000; 20: 4159-4168Crossref PubMed Scopus (178) Google Scholar). In most cell types, IRF3 is constitutively expressed, providing a fast antiviral response through the transcription of IFN-β and certain IFN-α genes. In contrast, IRF7 is highly induced by IFNs and activates the transcription of a larger set of IFN-α genes, leading to amplification of the IFN-α/β response (35Levy D.E. Marie I. Prakash A. Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity.Curr. Opin. Immunol. 2003; 15: 52-58Crossref PubMed Scopus (118) Google Scholar). In this study, we provide new insights into the molecular mechanisms by which SAMHD1 antagonizes IFN-I induction signaling. Our results show the capacity of SAMHD1 to suppress IFN-I responses relies on its ability to directly interact with key proteins in the RIG-I–like receptors (RLR) pathway, such as MAVS, IKKε, and IRF7. We show that SAMHD1 interacts with MAVS and inhibits MAVS aggregation and activation. SAMHD1 interaction with IKKε disrupts IRF7 binding to IKKε, which explains the ability of SAMHD1 to block IRF7 phosphorylation by IKKε. We demonstrate that SAMHD1 suppression of the IRF7-mediated antiviral response depends on its interaction with the inhibitory domain of IRF7 (IRF7-ID). We also identify three residues in IRF7-ID, essential for transactivation activity and SAMHD1 binding. Furthermore, we showed that IRF7 is required for efficient HIV-1 infection and viral transcription in THP-1 cells. Our findings revealed new mechanisms by which SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis in the context of viral infection, which help better understand the role of SAMHD1 in innate immunity. We have reported that SAMHD1 inhibits IFN-I signaling induced by the overexpression of IRF7, but not IRF3 (16Chen S. Bonifati S. Qin Z. St Gelais C. Kodigepalli K.M. Barrett B.S. et al.SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3798-E3807Crossref PubMed Scopus (78) Google Scholar). To better understand the underlying mechanisms, we first asked whether SAMHD1 inhibits IFN-I signaling upstream of IRF7 by components of the RLR pathway. HEK293T cells were cotransfected to express a luciferase reporter under control of the IFN-β promoter, MAVS, and increasing amounts of SAMHD1 WT or a dNTPase-defective HD/RN mutant (H206R and D207N) (36Ji X. Wu Y. Yan J. Mehrens J. Yang H. DeLucia M. et al.Mechanism of allosteric activation of SAMHD1 by dGTP.Nat. Struct. Mol. Biol. 2013; 20: 1304-1309Crossref PubMed Scopus (111) Google Scholar). Overexpression of SAMHD1 WT or HD/RN inhibited MAVS-induced IFN-β promoter activity in a dose-dependent manner (Fig. 1A). The highest level of SAMHD1 WT or HD/RN expression suppressed MAVS-induced IFN-β luciferase reporter by approximately 50% (Fig. 1A), suggesting that SAMHD1 suppresses MAVS-mediated IFN-I signaling independently of its dNTPase activity. We then utilized co-immunoprecipitation (Co-IP) to determine whether SAMHD1 interacts with MAVS. We found that SAMHD1 WT and HD/RN similarly interacted with full-length (FL) MAVS (Fig. 1B), indicating that the binding is independent of the dNTPase activity of SAMHD1. MAVS contains three functional domains, including the caspase-recruitment domain (CARD), a proline-rich region (Pro-rich), and a transmembrane domain (TM) (28Liu B. Zhang M. Chu H. Zhang H. Wu H. Song G. et al.The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.Nat. Immunol. 2017; 18: 214-224Crossref PubMed Scopus (0) Google Scholar). To identify the domain of MAVS required for the interaction with SAMHD1, we generated four truncation mutants of MAVS (Fig. 1C). HEK293T cells were cotransfected with plasmids expressing WT SAMHD1 and FL MAVS or individual MAVS mutants (Fig. 1C). WT SAMHD1 co-immunoprecipitated with FL MAVS; however, deletion of the CARD of MAVS (ΔCARD) or two MAVS mutants lacking the CARD and Pro-rich domains (ΔN and ΔN/ΔTM) abrogated their interactions with SAMHD1 (Fig. 1D). We then generated and tested the MAVS mutant (N) expressing CARD and Pro-rich domains, which efficiently bound to WT SAMHD1 (Fig. 1D). Together, these data suggest that SAMHD1 interacts with the CARD of MAVS. Viral infection of cells activates MAVS (28Liu B. Zhang M. Chu H. Zhang H. Wu H. Song G. et al.The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.Nat. Immunol. 2017; 18: 214-224Crossref PubMed Scopus (0) Google Scholar, 29Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. et al.Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347aaa2630Crossref Scopus (1028) Google Scholar, 37Qi N. Shi Y. Zhang R. Zhu W. Yuan B. Li X. et al.Multiple truncated isoforms of MAVS prevent its spontaneous aggregation in antiviral innate immune signalling.Nat. Commun. 2017; 815676Crossref Scopus (43) Google Scholar) and may affect its interactions with other cellular proteins. To examine whether endogenous SAMHD1 and MAVS interact in cells during viral infection, we infected monocytic THP-1 cells with Sendai virus (SeV) for 6 h and then performed IP using SAMHD1 antibodies. We found that endogenous SAMHD1 and MAVS interacted in THP-1 cells regardless of SeV infection (Fig. 2A). Of note, THP-1 cells express FL and a shorter splicing variant of SAMHD1 (38de Silva S. Hoy H. Hake T.S. Wong H.K. Porcu P. Wu L. Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.J. Biol. Chem. 2013; 288: 9284-9292Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) as evident in immunoblot (IB) analysis (Fig. 2A). Expression of SeV nucleoprotein (NP) served as a marker of infected cells (Fig. 2A). MAVS specifically localizes to the outer mitochondrial membrane and this localization is required for aggregation of MAVS and subsequent downstream signaling (25Seth R.B. Sun L. Ea C.K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2548) Google Scholar, 28Liu B. Zhang M. Chu H. Zhang H. Wu H. Song G. et al.The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination.Nat. Immunol. 2017; 18: 214-224Crossref PubMed Scopus (0) Google Scholar, 39Hou F. Sun L. Zheng H. Skaug B. Jiang Q.X. Chen Z.J. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.Cell. 2011; 146: 448-461Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). SAMHD1 shuttles between the cytosol and the nucleus (40Du J. Peng Y. Wang S. Hou J. Wang Y. Sun T. et al.Nucleocytoplasmic shuttling of SAMHD1 is important for LINE-1 suppression.Biochem. Biophys. Res. Commun. 2019; 510: 551-557Crossref PubMed Scopus (11) Google Scholar); however, whether it may also associate with mitochondria has not been investigated. To examine whether SAMHD1 is associated with mitochondria and whether MAVS expression is required for this localization, we performed cytosol and mitochondria fractionation in THP-1 control (Ctrl) and MAVS KO cells. Efficient KO of endogenous MAVS and SAMHD1 expression in these cell lines was confirmed by IB analysis (Fig. 2B). THP-1 cells were mock-infected or infected with SeV for 6 h and then subjected to cytosol and mitochondria fractionation and IB analysis. To demonstrate the purity of both fractions, the cytosolic protein tubulin and the mitochondrial protein voltage-dependent anion channel (VDAC) were used as markers (Fig. 2C). Interestingly, endogenous SAMHD1 was detected in both the cytosolic and mitochondrial fractions regardless of SeV infection (Fig. 2C), indicating that SAMHD1 is associated with mitochondria in THP-1 cells. Moreover, no difference in SAMHD1 association with mitochondria was observed between THP-1 Ctrl and MAVS KO cells (Fig. 2C), suggesting that MAVS is not necessary for SAMHD1 association with mitochondria. To examine whether SAMHD1 mitochondrial localization is specific to THP-1 cells only, we also performed the cytosol and mitochondria fractionation using HEK293T cells. Consistently, we found that endogenous SAMHD1 localized in both the cytosol and mitochondria in HEK293T cells regardless of SeV infection (Fig. 2D). To further examine whether SAMHD1 and MAVS directly interact, we performed an in vitro pull-down assay using purified, recombinant FL SAMHD1 and MAVS without the TM domain (aa 1–513). Our results demonstrated direct binding of recombinant FL SAMHD1 to MAVS without the TM domain, confirming that the TM domain of MAVS is not required for the binding (Fig. 2E). Together, these data suggest that SAMHD1 is a mitochondrion-associated protein likely through its interaction with MAVS. We hypothesized that SAMHD1 targets MAVS to regulate the IFN-I signaling pathway. To examine whether SAMHD1 inhibits MAVS aggregation, THP-1 Ctrl and SAMHD1 KO cells were mock-infected or infected with SeV. At 8 h postinfection, crude mitochondria of cells were isolated for analysis. Interestingly, semidenaturing detergent agarose gel electrophoresis (SDD-AGE) analysis showed that SAMHD1 KO THP-1 cells displayed increased MAVS aggregation, and SeV infection further enhanced MAVS aggregation compared to control cells (Fig. 3A, SDD-AGE panel). Concomitant to increased MAVS aggregation, SAMHD1 KO THP-1 cells showed a 12-fold and 2-fold increase in phosphorylation of TBK1 (S172) and IRF3 (S396) upon SeV infection, respectively, compared to control cells (Fig. 3A, SDS-PAGE panel). Detection of SeV NP served as a marker of virus infection and replication (Fig. 3A). Immediately downstream of MAVS aggregation, the kinases IKKε and TBK1 are recruited to MAVS and activated by phosphorylation (31Fang R. Jiang Q. Zhou X. Wang C. Guan Y. Tao J. et al.MAVS activates TBK1 and IKKepsilon through TRAFs in NEMO dependent and independent manner.PLoS Pathog. 2017; 13e1006720Crossref Scopus (114) Google Scholar, 41Chau T.L. Gioia R. Gatot J.S. Patrascu F. Carpentier I. Chapelle J.P. et al.Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated?.Trends Biochem. Sci. 2008; 33: 171-180Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). To determine whether SAMHD1 can disrupt binding between MAVS and IKKε, Co-IP experiments were performed. FLAG-tagged MAVS was expressed in HEK293T cells alone (Fig. 3B, lane 1) or with myc-IKKε (lanes 2–5) in the absence (lane 2) or presence (lanes 3–5) of increasing amounts of HA-SAMHD1. MAVS was immunoprecipitated with an anti-FLAG antibody, and coprecipitation of IKKε was assessed by IB. As expected, IKKε interacted with MAVS, while the amount of coprecipitated IKKε decreased with increasing expression of SAMHD1 (Fig. 3B). Previous studies showed that MAVS can be phosphorylated by TBK1 and IKKs (29Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. et al.Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347aaa2630Crossref Scopus (1028) Google Scholar). Of note, MAVS IB exhibited two bands when IKKε was co-expressed and the upper band could be phosphorylated MAVS (Fig. 3B, lanes 2–5 of input samples). Furthermore, upon SeV infection, phospho-IKKε levels were significantly increased (3- to 123-fold) in THP-1 SAMHD1 KO cells compared with control cells (Fig. 3C). Thus, SAMHD1 suppresses MAVS activation in response to viral infection, impairs IKKε recruitment to MAVS, and inhibits IKKε phosphorylation upon virus infection. We have previously shown SAMHD1 interacts with IKKε, but not TBK1 (16Chen S. Bonifati S. Qin Z. St Gelais C. Kodigepalli K.M. Barrett B.S. et al.SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3798-E3807Crossref PubMed Scopus (78) Google Scholar, 17Qin Z. Bonifati S. St Gelais C. Li T.W. Kim S.H. Antonucci J.M. et al.The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.J. Biol. Chem. 2020; 295: 1575-1586Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). However, whether this interaction was direct remained unknown. To address this question, we performed an in vitro pull-down assay with purified recombinant FL SAMHD1 and IKKε. Our results indicated direct binding between these two recombinant proteins (Fig. 4A). To examine whether SAMHD1 inhibits IFN-I signaling induced by IKKε in cells, we performed an IFN-β promoter luciferase reporter assay. We observed that overexpression of IKKε in HEK293T cells activated the IFN-β promoter luciferase reporter and that co-expression of SAMHD1 inhibited the activation in a dose-dependent manner (Fig. 4B). To map the domains of IKKε responsible for SAMHD1 binding, we constructed a series of truncated mutants of IKKε based on the functional domains of the protein (Fig. 4C) and tested their interactions with FL SAMHD1. Myc-tagged FL IKKε and IKKε mutants Δ1-Δ4 (Fig. 4D), but not the IKKε mutant lacking the kinase domain (ΔKD) (Fig. 4E), coprecipitated with HA-SAMHD1, indicating that the KD of IKKε is required for the interaction. To determine whether IKKε catalytic activity is required for binding to SAMHD1, we used an IKKε mutant (K38A) that ablates its kinase activity (32Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. et al.IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2119) Google Scholar). We found that the IKKε K38A mutant bound to SAMHD1 similarly to WT IKKε (Fig. 4F), suggesting that IKKε catalytic activity is not required for the binding. IKKε amino-terminal KD is sufficient for IRF3/7 binding (42Prins K.C. Cardenas W.B. Basler C.F. Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1.J. Virol. 2009; 83: 3069-3077Crossref PubMed Scopus (195) Google Scholar), and we have previously reported that SAMHD1 inhibits IKKε-mediated IRF7 phosphorylation (16Chen S. Bonifati S. Qin Z. St Gelais C. Kodigepalli K.M. Barrett B.S. et al.SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-kappaB and interferon pathways.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3798-E3807Crossref PubMed Scopus (78) Google Scholar). Therefore, we sought to investigate whether SAMHD1 sterically prevents IKKε KD–IRF7 interaction. HEK293T cells were transfected to express myc-tagged IKKε KD, FLAG-IRF7, and increasing amounts of HA-SAMHD1 and then subjected to IP using myc antibodies (Fig. 4G). As expected, myc-IKKε KD coprecipitated with FLAG-IRF7. Interestingly, the amount of IRF7 immunoprecipitated with IKKε KD was reduced by SAMHD1 in a dose-dependent manner (Fig. 4G). These results suggest that SAMHD1 inhibits IRF7 and IKKε KD interaction, thereby reducing IKKε-mediated IRF7 phosphorylation. Among nine mammalian IRFs identified, IRF3 and IRF7 are the major transcription factors involved in IFN-I induc