Dataset Information

Dasatinib: effects on the macrophage phospho proteome with a focus on SAMHD1 and HIV-1 infection.

ABSTRACT: Macrophages are one of the main cellular targets of human immunodeficiency virus type 1 (HIV-1). Macrophage infection by HIV-1 is inefficient due to the presence of the viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1). Ex vivo human monocyte-derived macrophages (MDMs) express SAMHD1 in an equilibrium between active (unphosphorylated) and inactive (phosphorylated) states. We and others have shown that treatment of MDMs with the FDA-approved tyrosine kinase inhibitor, dasatinib, ablates SAMHD1 phosphorylation, thus skewing the balance towards a cellular state that is refractory to HIV-1 infection. We hypothesized that dasatinib inhibits a putative tyrosine kinase that is upstream of SAMHD1. In search for this tyrosine kinase, we probed several candidates and were unable to identify a single target that, when inhibited, was sufficient to explain the dephosphorylation of SAMHD1 we observe upon treatment with dasatinib. On the other hand, we probed the ability of dasatinib to directly inhibit the serine/threonine cyclin dependent kinases 1, 2, 4 and 6 and confirmed that dasatinib directly inhibits these kinases. Therefore, our results show that inhibition of the proximal CDKs 1, 2, 4 and 6 by dasatinib is clearly detectable, leads to blockade of infection by HIV-1, and may be sufficient to explain the activity of dasatinib against SAMHD1 phosphorylation.

SUBMITTER: Williams ESCP

PROVIDER: S-EPMC9802671 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dasatinib: effects on the macrophage phospho proteome with a focus on SAMHD1 and HIV-1 infection.

Williams Elizabeth S C P ESCP Szaniawski Matthew A MA Martins Laura J LJ Innis Emily A EA Alcamí José J Hanley Timothy M TM Spivak Adam M AM Coiras Mayte M Planelles Vicente V

Clinical research in HIV/AIDS 20221130 1

Macrophages are one of the main cellular targets of human immunodeficiency virus type 1 (HIV-1). Macrophage infection by HIV-1 is inefficient due to the presence of the viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1). <i>Ex vivo</i> human monocyte-derived macrophages (MDMs) express SAMHD1 in an equilibrium between active (unphosphorylated) and inactive (phosphorylated) states. We and others have shown that treatment of MDMs with the F ...[more]

PMID: 36589263

Similar Datasets

Project description:Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of additional SAMHD1-mediated anti-viral activity independent of T592 dephosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet a poor correlation between HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function, and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.IMPORTANCEWe introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

Project description: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.ImportanceIn 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.

Dataset Information

Dasatinib: effects on the macrophage phospho proteome with a focus on SAMHD1 and HIV-1 infection.

Publications

Dasatinib: effects on the macrophage phospho proteome with a focus on SAMHD1 and HIV-1 infection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets