SAMHD1: HIV-1 restriction, post-translational regulation and function

Despite enormous efforts to develop curative treatments or effective vaccination strategies against human immunodeficiency virus 1 (HIV-1), the virus still is a substantial global health thread and a social and economic burden. Understanding host-HIV-1 interactions will be essential to advance treatment options. Numerous pro- and anti-viral host factors are known to date, the latter are referred to as restriction factors. Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for HIV-1, active in myeloid and resting CD4+ T cells, which both are important HIV-1 target cells. To complete its obligate reverse transcription step, HIV-1 requires deoxyribonucleoside triphosphate (dNTPs) as a substrate for DNA synthesis. SAMHD1 is a dNTP triphosphate triphosphohydrolase (dNTPase). Therefore, SAMHD1 was proposed to limit cellular dNTP levels, which is supported by correlative data. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on its dNTPase activity is still under debate. This might be in part due to limitations in the myeloid models and genetic tools, which are available to genetically study the relationship between SAMHD1 T592 phosphorylation, anti-viral restriction and enzymatic functions of SAMHD1. Also, additional cellular functions of SAMHD1, as well as additional post-translational modifications could impact anti-viral restriction. To overcome technical limitations and the lack of knowledge, we used BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1, strongly inhibiting HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1 replication. We developed a pipeline to introduce specific mutations into the genomic SAMHD1 locus via CRISPR/Cas9-mediated homologous recombination and were able to generate homozygous phosphomimetic SAMHD1 T592E and phosphoablative SAMHD1 T592A mutants. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. In stark contrast however, T592E KI cells retain wild type dNTP levels and dNTP pool composition. Thus, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled. To further understand if SAMHD1 dNTPase activity contributes to HIV-1 restriction, we developed an advanced overexpression system to reliably screen mutants of SAMHD1 for their restrictive potential in macrophage-like BlaER1 cells. Indeed, we could identify mutants, which lack dNTPase activity, but seemed to maintain their restrictive potential. In line with this, we were unable to show an additional T592 phospho-regulation independent SAMHD1 function. SAMHD1 was recently proposed to play a role in the resolution of R-loops, tertiary DNA:RNA structures, which occur at sites of transcription-replication conflicts. As a surrogate model for the role of SAMHD1 in R-loop biology, we tested the effect of SAMHD1 depletion on CRISPR/Cas9 induced R-loops. We did not detect alterations of CRISPR/Cas9 KO and KI efficiency in absence of SAMHD1. Nevertheless, modulation of R-loop stability might still contribute to cellular and anti-viral functions of SAMHD1. The lack of experimental correlation between SAMHD1 dNTPase activity, HIV-1 restriction and T592 phospho-status might also be due to an incomplete understanding of the complexity of SAMHD1 regulation. To obtain a more sophisticated image of SAMHD1 phosphorylation sites, we performed a targeted mass spectrometric analysis of endogenous SAMHD1 phosphopeptides in restrictive versus non-restrictive myeloid cells. We were able to identify several differentially phosphorylated residues in addition to T592. The N-terminal phospho-hub consistent of T21, T25 and S33 seemed not to influence SAMHD1 localization, expression, T592 phosphorylation or HIV-1 replication. In contrast, T579 emerged as a potential phosphorylation site regulating HIV-1 restriction. However, mutants of T579 might affect SAMHD1 stability and further experiments will be necessary to confirm the role of pT579. We think that BlaER1 as a novel cell model for SAMHD1-mediated HIV-1 restriction and CRISPR/Cas9-mediated KI, will be helpful to unravel the interplay between SAMHD1-mediated HIV-1 restriction, SAMHD1 post-translational regulation and cellular functions of SAMHD1. This knowledge might be essential for the future development of curative or preventive strategies against HIV-1

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes

Soft X-ray imaging spectroscopy with micrometer resolution

Soft x-ray spectroscopy is invaluable for gaining insight into quantum materials. However, it is typically conducted in a spatially averaging way, making it blind to inhomogeneity in samples. Here, we demonstrate how we couple imaging to x-ray absorption spectroscopy and resonant inelastic x-ray scattering. Accordingly, we use a 2D detector and an off-axis Fresnel zone plate that images the sample in one spatial dimension and provides spectroscopic information in the other dimension. With our setup, we envision to enable a more detailed understanding of how the behavior of microscopic domains determines the functionality of quantum materials

Performance of the Large-Sized Telescope prototype of the Cherenkov Telescope Array

The next-generation ground-based gamma-ray Cherenkov Telescope Array Observatory (CTAO) will consist of imaging atmospheric Cherenkov telescopes (IACTs) of three different sizes distributed in two sites. The Large-Sized Telescopes will cover the low-energy end of the CTA energy range, starting at about 20 GeV. After its first years of operation at the CTA northern site, the Large-Sized Telescope prototype (LST-1) is in the final stage of its commissioning phase, having collected a significant amount of scientific data to date.In this contribution, we present the physics performance of the telescope using low-zenith Crab Nebula observations and Monte Carlo simulations fine-tuned accordingly. We show performance figures of merit such as the energy threshold, effective area, energy and angular resolution, and sensitivity based on the standard Hillas-parameters approach and following the source-independent and dependent analysis methods. The analysis threshold is estimated at 30 GeV. The energy resolution is around 30%, and the angular resolution is 0.3 degrees at 100 GeV.The best integral sensitivity of LST-1 is about 1.1% of the Crab Nebula flux above 250 GeV for 50 hours of observations. We also show the spectral energy distribution and light curve from Crab Nebula observations, which agree with results from other IACTs and link smoothly with Fermi-LAT when considering statistical and systematic uncertainties near the energy threshold