Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

HIV-1 replication is tightly regulated in host cells, and various restriction factors have important roles in inhibiting viral replication. SAMHD1, a well-known restriction factor, suppresses HIV-1 replication by hydrolyzing intracellular dNTPs, thereby limiting the synthesis of viral cDNA in quiescent cells. In this study, we revealed an additional and distinct mechanism of SAMHD1 inhibition during the postviral cDNA synthesis stage. Using immunoprecipitation and mass spectrometry analysis, we demonstrated the interaction between SAMHD1 and MX2/MxB, an interferon-induced antiviral factor that inhibits HIV-1 cDNA nuclear import. The disruption of endogenous MX2 expression significantly weakened the ability of SAMHD1 to inhibit HIV-1. The crucial region within SAMHD1 that binds to MX2 has been identified. Notably, we found that SAMHD1 can act as a sensor that recognizes and binds to the incoming HIV-1 core, subsequently delivering it to the molecular trap formed by MX2, thereby blocking the nuclear entry of the HIV-1 core structure. SAMHD1 mutants unable to recognize the HIV-1 core showed a substantial decrease in antiviral activity. Certain mutations in HIV-1 capsids confer resistance to MX2 inhibition while maintaining susceptibility to suppression by the SAMHD1-MX2 axis. Overall, our study identifies an intriguing antiviral pattern wherein two distinct restriction factors, SAMHD1 and MX2, collaborate to establish an alternative mechanism deviating from their actions. These findings provide valuable insight into the complex immune defense networks against exogenous viral infections and have implications for the development of targeted anti-HIV therapeutics.
OBJECTIVE: In contrast to most restriction factors that directly bind to viral components to exert their antiviral effects, SAMHD1, the only known deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, indirectly inhibits viral replication in quiescent cells by reducing the pool of dNTP substrates available for viral cDNA synthesis. Our study provides a novel perspective on the antiviral functions of SAMHD1. In addition to its role in dNTP hydrolysis, SAMHD1 cooperates with MX2 to inhibit HIV-1 nuclear import. In this process, SAMHD1 acts as a sensor for incoming HIV-1 cores, detecting and binding to them, before subsequently delivering the complex to the molecular trap formed by MX2, thereby immobilizing the virus. This study not only reveals a new antiviral pathway for SAMHD1 but also identifies a unique collaboration and interaction between two distinct restriction factors, establishing a novel line of defense against HIV-1 infection, which challenges the traditional view of restriction factors acting independently. Overall, our findings further indicate the intricate complexity of the host immune defense network and provide potential targets for promoting host antiviral immune defense.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1\'s ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SAMHD1 regulates cellular nucleotide homeostasis, controlling dNTP levels by catalysing their hydrolysis into 2\'-deoxynucleosides and triphosphate. In differentiated CD4+ macrophage and resting T-cells SAMHD1 activity results in the inhibition of HIV-1 infection through a dNTP blockade. In cancer, SAMHD1 desensitizes cells to nucleoside-analogue chemotherapies. Here we employ time-resolved cryogenic-EM imaging and single-particle analysis to visualise assembly, allostery and catalysis by this multi-subunit enzyme. Our observations reveal how dynamic conformational changes in the SAMHD1 quaternary structure drive the catalytic cycle. We capture five states at high-resolution in a live catalytic reaction, revealing how allosteric activators support assembly of a stable SAMHD1 tetrameric core and how catalysis is driven by the opening and closing of active sites through pairwise coupling of active sites and order-disorder transitions in regulatory domains. This direct visualisation of enzyme catalysis dynamics within an allostery-stabilised platform sets a precedent for mechanistic studies into the regulation of multi-subunit enzymes.

Resting CD4 T cells resist productive HIV-1 infection. The HIV-2/simian immunodeficiency virus protein viral accessory protein X (Vpx) renders these cells permissive to infection, presumably by alleviating blocks at cytoplasmic reverse transcription and subsequent nuclear import of reverse-transcription/pre-integration complexes (RTC/PICs). Here, spatial analyses using quantitative virus imaging techniques reveal that HIV-1 capsids containing RTC/PICs are readily imported into the nucleus, recruit the host dependency factor CPSF6, and translocate to nuclear speckles in resting CD4 T cells. Reverse transcription, however, remains incomplete, impeding proviral integration and viral gene expression. Vpx or pharmacological inhibition of the deoxynucleotide triphosphohydrolase (dNTPase) activity of the restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) increases levels of nuclear reverse-transcribed cDNA and facilitates HIV-1 integration. Nuclear import and intranuclear transport of viral complexes therefore do not pose important blocks to HIV-1 in resting CD4 T cells, and the limitation to reverse transcription by SAMHD1\'s dNTPase activity constitutes the main pre-integration block to infection.

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis.

OBJECTIVE: Aicardi Goutières Syndrome (AGS) is a genetic interferonopathy associated with multisystemic heterogeneous disease and neurologic dysfunction. AGS includes a broad phenotypic spectrum which is only partially explained by genotype. To better characterize this variability, we will perform a systematic analysis of phenotypic variability in familial cases of AGS.
METHODS: Among thirteen families, twenty-six siblings diagnosed with AGS were identified from the Myelin Disorders and Biorepository Project (MDBP) at the Children\'s Hospital of Philadelphia. Data were collected on the age of onset, genotype, neurologic impairment, and systemic complications. Neurologic impairment was assessed by a disease-specific scale (AGS Severity Scale) at the last available clinical encounter (range: 0-11 representing severe - attenuated phenotypes). The concordance of clinical severity within sibling pairs was categorized based on the difference in AGS Scale (discordant defined as >2-unit difference). The severity classifications were compared between sibling sets and by genotype.
RESULTS: Five genotypes were represented: TREX1 (n = 4 subjects), RNASEH2B (n = 8), SAMHD1 (n = 8) ADAR1 (n = 4), and IFIH1 (n = 2). The older sibling was diagnosed later relative to the younger affected sibling (median age 7.32 years [IQR = 14.1] compared to 1.54 years [IQR = 10.3]). Common presenting neurologic symptoms were tone abnormalities (n = 10/26) and gross motor dysfunction (n = 9/26). Common early systemic complications included dysphagia and chilblains. The overall cohort median AGS severity score at the last encounter was 8, while subjects presenting with symptoms before one year had a median score of 5. The TREX1 cohort presented at the youngest age and with the most severe phenotype on average. AGS scores were discordant for 5 of 13 sibling pairs, most commonly in the SAMHD1 pairs. Microcephaly, feeding tube placement, seizures and earlier onset sibling were associated with lower AGS scores (respectively, Wilcoxon rank sum: p = 0.0001, p < 0.0001, p = 0.0426, and Wilcoxon signed rank: p = 0.0239).
CONCLUSIONS: In this systematic analysis of phenotypic variability in familial cases, we found discordance between siblings affected by AGS. Our results underscore the heterogeneity of AGS and suggest factors beyond AGS genotype may affect phenotype. Understanding the critical variables associated with disease onset and severity can guide future therapeutic interventions and clinical monitoring. This report reinforces the need for further studies to uncover potential factors to better understand this phenotypic variability, and consequently identify potential targets for interventions in attempt to change the natural history of the disease.

Dysfunctional mutations in SAMHD1 cause Aicardi-Goutières Syndrome, an autoinflammatory encephalopathy with elevated interferon-α levels in the cerebrospinal fluid. Whether loss of function mutations in SAMHD1 trigger the expression of other cytokines apart from type I interferons in Aicardi-Goutières Syndrome is largely unclear. This study aimed to explore whether SAMHD1 dysfunction regulated the expression of IL-34, a key cytokine controlling the development and maintenance of microglia, in SH-SY5Y neural cells. We found that downregulation of SAMHD1 in SH-SY5Y cells resulted in the upregulation of IL-34 expression. The protein and mRNA levels of NF-κB p65, the transactivating subunit of a transcription factor NF-κB, were also upregulated in SAMHD1-knockdown SH-SY5Y cells. It was further found SAMHD1 knockdown in SH-SY5Y cells induced an upregulation of IL-34 expression through the canonical NF-κB-dependent pathway in which NF-κB p65, IKKα/β and the NF-κB inhibitor IκBα were phosphorylated. Moreover, knockdown of SAMHD1 in SH-SY5Y cells led to the translocation of NF-κB p65 into the nucleus and promoted NF-κB transcriptional activity. In conclusion, we found SAMHD1 dysfunction induced IL-34 expression via NF-κB p65 in neuronal SH-SY5Y cells. This finding could lay the foundation for exploring the role of IL-34-targeting microglia in the pathogenesis of Aicardi-Goutières Syndrome.

Influenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV.
To investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication.
In this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1\'s ability to restrict H5N1 is related to phosphorylation at 592-tyrosine.
In conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.