Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells

Ten quick tips for getting the most scientific value out of numerical data.

Most studies in the life sciences and other disciplines involve generating and analyzing numerical data of some type as the foundation for scientific findings. Working with numerical data involves multiple challenges. These include reproducible data acquisition, appropriate data storage, computationally correct data analysis, appropriate reporting and presentation of the results, and suitable data interpretation. Finding and correcting mistakes when analyzing and interpreting data can be frustrating and time-consuming. Presenting or publishing incorrect results is embarrassing but not uncommon. Particular sources of errors are inappropriate use of statistical methods and incorrect interpretation of data by software. To detect mistakes as early as possible, one should frequently check intermediate and final results for plausibility. Clearly documenting how quantities and results were obtained facilitates correcting mistakes. Properly understanding data is indispensable for reaching well-founded conclusions from experimental results. Units are needed to make sense of numbers, and uncertainty should be estimated to know how meaningful results are. Descriptive statistics and significance testing are useful tools for interpreting numerical results if applied correctly. However, blindly trusting in computed numbers can also be misleading, so it is worth thinking about how data should be summarized quantitatively to properly answer the question at hand. Finally, a suitable form of presentation is needed so that the data can properly support the interpretation and findings. By additionally sharing the relevant data, others can access, understand, and ultimately make use of the results. These quick tips are intended to provide guidelines for correctly interpreting, efficiently analyzing, and presenting numerical data in a useful way

Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs

Table_1.XLS

<p>Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.</p

Data_Sheet_1.PDF

<p>Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.</p

Different G based nucleotides can be accommodated in the SAMHD1 allosteric site.

<p>(<b>A</b>) The contents and conformation of the allosteric sites for structures of SAMHD1(115–583)-ddGTP (top), SAMHD1(115–583[R164A])-dGTP (middle) and SAMHD1(115–626)-GTP (bottom) are shown. Nucleotides are shown in stick representation, SAMHD1 residues making contacts with the nucleotides are labelled. (<b>B</b>) SEC-MALLS analysis of SAMHD1(115–626) incubated with ddGTP/dATP(blue), dGTP/dATP (green) or GTP/dATP (orange). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks.</p

Model for phospho-regulation of SAMHD1 restriction.

<p>In the absence of dNTPs Apo-SAMHD1 is found in a monomer-dimer equilibrium regardless of the phosphorylation state (<b>1</b>). At high dNTP levels, typically in cycling cells, constitutively abundant GTP combines with dNTPs to fill allosteric sites. In both phosphorylated and un-phosphorylated SAMHD1 this results in the formation of an activated tetramer (<b>2</b>) that in the non-phosphorylated protein also includes additional intra-tetramer CtD interactions forming a stable activated tetramer (<b>3</b>). Under these conditions, both activated and stable activated tetramers hydrolyse the dNTP pool at comparable rates. At lower dNTP levels, the stabilisation afforded by the CtD interactions maintains enzyme activity in non-phosphorylated SAMHD1 by preventing the loss of dNTP-activator from the allosteric site. However, in phospho-SAMHD1, activating dNTPs dissociate from the allosteric site (<b>4</b>) resulting in disassembly of the tetramer and down-regulation of triphosphohydrolase activity. At very low levels, such as in differentiated myeloid cells, CtD-stabilised tetramers still retain activating dNTPs in the allosteric site (<b>5</b>) and SAMHD1 remains catalytically competent. It can therefore rapidly respond to any increase in intracellular dNTPs to maintain the dNTP levels below the threshold required for HIV-1 replication.</p

Solution oligomeric state and steady state kinetics of SAMHD1.

<p>(<b>A</b>) SEC-MALLS analysis of SAMHD1 monomer-dimer-tetramer equilibrium for SAMHD1(115–583) (red) and SAMHD1(115–626) (black). The chromatograms are the output from the differential refractometer. The points are the weight-averaged molar masses determined at 1-second intervals throughout elution of chromatographic peaks. Dashed line chromatograms are apo-protein, solid lines are upon addition of 0.5 mM dGTP (<b>B</b>) Tetramer stability. SAMHD1(115–626) was incubated with 0.1 mM GTP and 0.5 mM dATP for the specified time intervals and then the oligomeric state analysed by SEC-MALLS. (<b>C</b>) Analysis of dATP hydrolysis during the time course shown in <b>B</b>. The chromatogram (black) is the recorded UV absorbance at 260 nm from the SEC-MALLS column for the five-minute incubation time point. Reference chromatograms (dashed lines) for substrate dATP (red) and product dA (blue) are overlaid. (<b>D</b>) Steady-state kinetic analysis of GTP stimulated hydrolysis of TTP by SAMHD1. The dependence of the rate of on substrate concentration for SAMHD1(115–626) (black) and SAMHD1(115–583) (red) are plotted. Solid lines are the best fit to the data using the Michaelis-Menten expression. Error bars represent the standard error of the mean (SEM) of three independent measurements. The derived constants K_M and k_cat for the reaction are displayed inset.</p

X-ray data collection and structure refinement statistics.

<p>*Values in parentheses are for highest-resolution shell</p><p>X-ray data collection and structure refinement statistics.</p