Biochemical functions and structure of Caenorhabditis elegans ZK177.8 protein: Aicardi-Goutières syndrome SAMHD1 dNTPase ortholog.

Mutations in sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) are found in a neurodevelopmental disorder, Aicardi-Goutières syndrome, and cancers, and SAMHD1, which is a deoxynucleoside triphosphate (dNTP) triphosphorylase, was identified as a myeloid-specific HIV-1 restriction factor. Here, we characterized the enzymology and structure of an SAMHD1 ortholog of Caenorhabditis elegans, ZK177.8, which also reportedly induces developmental defects upon gene knockdown. We found ZK177.8 protein is a dNTPase allosterically regulated by dGTP. The active site of ZK177.8 recognizes both 2' OH and triphosphate moieties of dNTPs but not base moiety. The dGTP activator induces the formation of the enzymatically active ZK177.8 tetramers, and ZK177.8 protein lowers cellular dNTP levels in a human monocytic cell line. Finally, ZK177.8 tetramers display very similar X-ray crystal structure with human and mouse SAMHD1s except that its lack of the canonical sterile alpha motif domain. This striking conservation in structure, function, and allosteric regulatory mechanism for the hydrolysis of the DNA building blocks supports their host developmental roles.

Maedi-visna virus Vif protein uses motifs distinct from HIV-1 Vif to bind zinc and the cofactor required for A3 degradation.

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin-RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi-visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.

The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that depletes cellular dNTPs in noncycling cells to promote genome stability and to inhibit retroviral and herpes viral replication. In addition to being substrates, cellular nucleotides also allosterically regulate SAMHD1 activity. Recently, it was shown that high expression levels of SAMHD1 are also correlated with significantly worse patient responses to nucleotide analog drugs important for treating a variety of cancers, including acute myeloid leukemia (AML). In this study, we used biochemical, structural, and cellular methods to examine the interactions of various cancer drugs with SAMHD1. We found that both the catalytic and the allosteric sites of SAMHD1 are sensitive to sugar modifications of the nucleotide analogs, with the allosteric site being significantly more restrictive. We crystallized cladribine-TP, clofarabine-TP, fludarabine-TP, vidarabine-TP, cytarabine-TP, and gemcitabine-TP in the catalytic pocket of SAMHD1. We found that all of these drugs are substrates of SAMHD1 and that the efficacy of most of these drugs is affected by SAMHD1 activity. Of the nucleotide analogs tested, only cladribine-TP with a deoxyribose sugar efficiently induced the catalytically active SAMHD1 tetramer. Together, these results establish a detailed framework for understanding the substrate specificity and allosteric activation of SAMHD1 with regard to nucleotide analogs, which can be used to improve current cancer and antiviral therapies.

SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways.

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation.

The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.

SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates 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 nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which 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 long terminal repeat (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 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of 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 [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 the 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 potentially regulates viral latency in CD4+ T cells.IMPORTANCE A 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 nondividing 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.

Structural basis for recruitment of host CypA and E3 ubiquitin ligase by maedi-visna virus Vif.

Lentiviral Vif molecules target the host antiviral APOBEC3 proteins for destruction in cellular ubiquitin-proteasome pathways. Different lentiviral Vifs have evolved to use the same canonical E3 ubiquitin ligase complexes, along with distinct noncanonical host cofactors for their activities. Unlike primate lentiviral Vif, which recruits CBFß as the noncanonical cofactor, nonprimate lentiviral Vif proteins have developed different cofactor recruitment mechanisms. Maedi-visna virus (MVV) sequesters CypA as the noncanonical cofactor for the Vif-mediated ubiquitination of ovine APOBEC3s. Here, we report the cryo-electron microscopy structure of MVV Vif in complex with CypA and E3 ligase components. The structure, along with our biochemical and functional analysis, reveals both conserved and unique structural elements of MVV Vif and its common and distinct interaction modes with various cognate cellular proteins, providing a further understanding of the evolutionary relationship between lentiviral Vifs and the molecular mechanisms by which they capture different host cofactors for immune evasion activities.

Multifaceted HIV-1 Vif interactions with human E3 ubiquitin ligase and APOBEC3s.

APOBEC3 (A3) proteins are a family of host antiviral restriction factors that potently inhibit various retroviral infections, including human immunodeficiency virus (HIV)-1. To overcome this restriction, HIV-1 virion infectivity factor (Vif) recruits the cellular cofactor CBFß to assist in targeting A3 proteins to a host E3 ligase complex for polyubiquitination and subsequent proteasomal degradation. Intervention of the Vif-A3 interactions could be a promising therapeutic strategy to facilitate A3-mediated suppression of HIV-1 in patients. In this structural snapshot, we review the structural features of the recently determined structure of human A3F in complex with HIV-1 Vif and its cofactor CBFß, discuss insights into the molecular principles of Vif-A3 interplay during the arms race between the virus and host, and highlight the therapeutic implications.

A bacterial riboswitch class for the thiamin precursor HMP-PP employs a terminator-embedded aptamer.

We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the 'thiS motif' because of its frequent association with a gene coding for the ThiS protein, which delivers sulfur to form the thiazole moiety of the thiamin precursor HET-P. In the current report, we describe biochemical and genetic data demonstrating that thiS motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.

Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML.

Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.

The SAM domain of mouse SAMHD1 is critical for its activation and regulation.

Human SAMHD1 (hSAMHD1) is a retroviral restriction factor that blocks HIV-1 infection by depleting the cellular nucleotides required for viral reverse transcription. SAMHD1 is allosterically activated by nucleotides that induce assembly of the active tetramer. Although the catalytic core of hSAMHD1 has been studied extensively, previous structures have not captured the regulatory SAM domain. Here we report the crystal structure of full-length SAMHD1 by capturing mouse SAMHD1 (mSAMHD1) structures in three different nucleotide bound states. Although mSAMHD1 and hSAMHD1 are highly similar in sequence and function, we find that mSAMHD1 possesses a more complex nucleotide-induced activation process, highlighting the regulatory role of the SAM domain. Our results provide insights into the regulation of SAMHD1 activity, thereby facilitating the improvement of HIV mouse models and the development of new therapies for certain cancers and autoimmune diseases.

A threonine zipper that mediates protein-protein interactions: Structure and prediction.

We present the structure of an engineered protein-protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close-packing of the Thr residues, with only one intermonomer hydrogen bond (H-bond) between two of the Thr residues. This Thr-rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues. In previous work, we have shown that the side chain (χ1 ) dihedral angles of interface and core Thr residues can be predicted with high accuracy using a hard sphere plus stereochemical constraint (HS) model. Here, we demonstrate that in the Thr-rich local environment of the Thr zipper structure, we are able to predict the χ1 dihedral angles of most of the Thr residues. Some, however, are not well predicted by the HS model. We therefore employed explicitly solvated molecular dynamics (MD) simulations to further investigate the side chain conformations of these residues. The MD simulations illustrate the role that transient H-bonding to water, in combination with steric constraints, plays in determining the behavior of these Thr side chains. Broader Audience Statement: Protein-protein interactions are critical to life and the search for ways to disrupt adverse protein-protein interactions involved in disease is an ongoing area of drug discovery. We must better understand protein-protein interfaces, both to be able to disrupt existing ones and to engineer new ones for a variety of biotechnological applications. We have discovered and characterized an artificial Thr-rich protein-protein interface. This novel interface demonstrates a heretofore unknown property of Thr-rich surfaces: mediating protein-protein interactions.