The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4 + T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1. dNTP induced oligomerization | enzyme catalysis | innate immunity T he steady-state composition and concentration of deoxynucleotide triphosphate pools in mammalian cells are highly regulated because of the mutagenic consequences of dNTP imbalances in dividing cells (1, 2) as well as the important antiviral effects of dNTP pool depletion in quiescent cells (3,4). In all cell types, the ultimate pool balance is determined by dNTP-dependent regulatory pathways that affect the activities of enzymes involved in both synthesis and degradation of dNTPs (5-7). The most important highly up-regulated synthetic enzyme during S phase of dividing cells is the R1/R2 isoform of ribonucleotide triphosphate reductase, which ensures that dNTP precursors are plentiful for DNA synthesis (8). However, in quiescent cells of the immune system (resting CD4 + T cells, macrophages, and dendritic cells), where dNTP pools are ∼10-fold lower than dividing cells, the ultimate pool levels are likely determined by a balance between the activities of the R1/p53R2 isoform of ribonucleotide triphosphate reductase and the degradative dNTP triphosphohydrolase sterile α-motif/histidineaspartate domain-containing protein 1 (SAMHD1) (9). The highly dynamic nature of dNTP pools demands finely tuned mechanisms for feedback regulation of these enzymes by dNTPs as well as coarse regulatory mechanisms (posttranslational modifications, transcriptional regulation, and proteasomal targeting) that serve to turn these activities on and off at appropriate stages of the cell cycle and in specific cell types (10, 11). dNTP triphosphohydrolase enzymes, such as SAMHD1,...

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.DOI: http://dx.doi.org/10.7554/eLife.18447.001

We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein−protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.