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Abstract Background The role of type I IFNs in protecting against coronavirus (CoV) infections is not fully understood. While CoVs are poor inducers of type I IFNs in tissue culture, several studies have demonstrated the importance of the type I IFN response in controlling MHV infection in animals. The protective effectors against MHV infection are, however, still unknown. Results In order to get more insight into the antiviral gene expression induced in the brains of MHV-infected mice, we performed whole-genome expression profiling. Three different mouse strains, differing in their susceptibility to infection with MHV, were used. In BALB/c mice, which display high viral loads but are able to control the infection, 57 and 121 genes were significantly differentially expressed (≥ 1.5 fold change) upon infection at 2 and 5 days post infection, respectively. Functional association network analyses demonstrated a strong type I IFN response, with Irf1 and Irf7 as the central players. At 5 days post infection, a type II IFN response also becomes apparent. Both the type I and II IFN response, which were more pronounced in mice with a higher viral load, were not observed in 129SvEv mice, which are much less susceptible to infection with MHV. 129SvEv mice lacking the type I interferon receptor (IFNAR-/-), however, were not able to control the infection. Gene expression profiling of these mice identified type I IFN-independent responses to infection, with IFN-γ as the central player. As the BALB/c and the IFNAR-/- 129SvEv mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with MHV in order to identify type I IFN-dependent transcriptional responses. Many known IFN-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. We speculate that the additional type I IFN-dependent genes that we discovered may also be important for protection against MHV infection. Conclusion Transcriptional profiling of mice infected with MHV demonstrated the induction of a robust IFN response, which correlated with the viral load. Profiling of IFNAR-/- mice allowed us to identify type I IFN-independent and -dependent responses. Overall, this study broadens our present knowledge of the type I and II IFN-mediated effector responses during CoV infection in vivo.

Abstract Background Sensitivity and accuracy are key points when using microarrays to detect alterations in gene expression under different conditions. Critical to the acquisition of reliable results is the preparation of the RNA. In the field of virology, when analyzing the host cell's reaction to infection, the often high representation of viral RNA (vRNA) within total RNA preparations from infected cells is likely to interfere with microarray analysis. Yet, this effect has not been investigated despite the many reports that describe gene expression profiling of virus-infected cells using microarrays. Results In this study we used coronaviruses as a model to show that vRNA indeed interferes with microarray analysis, decreasing both sensitivity and accuracy. We also demonstrate that the removal of vRNA from total RNA samples, by means of virus-specific oligonucleotide capturing, significantly reduced the number of false-positive hits and increased the sensitivity of the method as tested on different array platforms. Conclusion We therefore recommend the specific removal of vRNA, or of any other abundant 'contaminating' RNAs, from total RNA samples to improve the quality and reliability of microarray analyses.

Abstract Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy.

Lysosomes catabolize lipids and other biological molecules, a function essential for cellular and organismal homeostasis. Key to lipid catabolism in the lysosome is bis(monoacylglycero)phosphate (BMP), a major lipid constituent of intralysosomal vesicles (ILVs) and a stimulator of lipid-degrading enzymes. BMP levels are altered in a broad spectrum of human conditions, including neurodegenerative diseases. Although BMP synthase was recently discovered, it has long been thought that BMP's unique stereochemistry confers resistance to acid phospholipases, a requirement for its role in the lysosome. Here, we demonstrate that PLA2G15, a major lysosomal phospholipase, efficiently hydrolyzes BMP with primary esters regardless of stereochemistry. Interestingly, we discover that BMP's unique esterification position is what confers resistance to hydrolysis. Purified PLA2G15 catabolizes most BMP species derived from cell and tissue lysosomes under acidic conditions. Furthermore, PLA2G15 catalytic activity against synthesized BMP stereoisomers with primary esters was comparable to its canonical substrates. Conversely, BMP with secondary esters is intrinsically stable in vitro and requires acyl migration for hydrolysis in lysosomes. Consistent with our biochemical data, PLA2G15-deficient tissues and cells accumulate multiple BMP species, a phenotype reversible by supplementing wildtype PLA2G15 but not its catalytically dead mutant. Increasing BMP levels by targeting PLA2G15 reverses the cholesterol accumulation phenotype in Niemann Pick Disease Type C (NPC1) patient fibroblasts and significantly ameliorate disease pathologies in NPC1-deficient mice leading to extended lifespan. Our findings establish the rules that govern the stability of BMP in the lysosome and identify PLA2G15 as a lysosomal BMP hydrolase and as a potential target for modulating BMP levels for therapeutic intervention.

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRING KO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRING KO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells 1 . The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells 2-5 . Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.

Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERV-K Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus., Competing Interests: The authors have declared that no competing interests exist.

In order to replicate, most pathogens need to enter their target cells. Many viruses enter the host cell through an endocytic pathway and hijack endosomes for their journey towards sites of replication. For delivery of their genome to the host cell cytoplasm and to avoid degradation, viruses have to escape this endosomal compartment without host detection. Viruses have developed complex mechanisms to penetrate the endosomal membrane and have evolved to co-opt several host factors to facilitate endosomal escape. Conversely, there is an extensive variety of cellular mechanisms to counteract or impede viral replication. At the level of cell entry, there are cellular defense mechanisms that recognize endosomal membrane damage caused by virus-induced membrane fusion and pore formation, as well as restriction factors that block these processes. In this Cell Science at a Glance article and accompanying poster, we describe the different mechanisms that viruses have evolved to escape the endosomal compartment, as well as the counteracting cellular protection mechanisms. We provide examples for enveloped and non-enveloped viruses, for which we discuss some unique and unexpected cellular responses to virus-entry-induced membrane damage., Competing Interests: Competing interestsT.R.B. is co-founder and shareholder of Haplogen GmbH and Scenic Biotech BV., (© 2018. Published by The Company of Biologists Ltd.)

Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins, such as type I interferons, interleukin-6 (IL-6), or tumor necrosis factor alpha (TNF-α). In the present study, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for the capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSVs) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication, not entry. Importantly, an antiviral interferon signature was completely absent in FGF16-treated cells. Nevertheless, the antiviral effect of FGF16 is broad, as it was evident on multiple cell types and also on infection by coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy. IMPORTANCE Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins, such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus, we tested 756 human secreted proteins for the capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this secretome screen on viral infection, we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable for enhancing oncolytic virus therapy., (Copyright © 2018 American Society for Microbiology.)

A controlled local inflammatory response is essential for adequate fracture healing. However, the current literature suggests that local and systemic hyper-inflammatory conditions after major trauma induce increased influx of neutrophils into the fracture hematoma (FH) and impair bone regeneration. Inhibiting neutrophil chemotaxis towards the FH without compromising the hosts' defense may therefore be a target of future therapies that prevent impairment of fracture healing after major trauma. We investigated whether chemotaxis of neutrophils towards the FH could be studied in vitro. Moreover, we determined whether chemotaxis of neutrophils towards the FH was mediated by the CXCR1, CXCR2, FPR, and C5aR receptors. Human FHs were isolated during an open reduction internal fixation (ORIF) procedure within 3 days after trauma and spun down to obtain the fracture hematoma serum. Neutrophil migration towards the FH was studied using Ibidi™ Chemotaxis 3D μ-Slides and image analysis of individual neutrophil tracks was performed. Our study showed that the human FH induces significant neutrophil chemotaxis, which was not affected by blocking CXCR1 and CXCR2. In contrast, neutrophil chemotaxis towards the FH was significantly inhibited by chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS), which blocks FPR and C5aR. Blocking only C5aR with CHIPSΔ1F also significantly inhibited neutrophil chemotaxis towards the FH. Our finding that neutrophil chemotaxis towards the human FH can be blocked in vitro using CHIPS may aid the development of therapies that prevent impairment of fracture healing after major trauma.

Human type A Enteroviruses (EV-As) cause diseases ranging from hand-foot-and-mouth disease to poliomyelitis-like disease. Although cellular receptors are identified for some EV-As, they remain elusive for the majority of EV-As. We identify the cell surface molecule KREMEN1 as an entry receptor for coxsackievirus A10 (CV-A10). Whereas loss of KREMEN1 renders cells resistant to CV-A10 infection, KREMEN1 overexpression enhances CV-A10 binding to the cell surface and increases susceptibility to infection, indicating that KREMEN1 is a rate-limiting factor for CV-A10 infection. Furthermore, the extracellular domain of KREMEN1 binds CV-A10 and functions as a neutralizing agent during infection. Kremen-deficient mice are resistant to CV-A10-induced lethal paralysis, emphasizing the relevance of Kremen for infection in vivo. KREMEN1 is also essential for infection by a phylogenetic and pathogenic related group of EV-As. Collectively these findings highlight the importance of KREMEN1 for these emerging pathogens and its potential as an antiviral therapeutic target., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Proximal femur fractures are a common injury after low energy trauma in the elderly. Most rehabilitation programs are based on restoring mobility and early resumption of weight-bearing. However, therapy compliance is low in patients following lower extremity fractures. Moreover, little is known about the relevance of gait parameters and how to steer the rehabilitation after proximal femur fractures in the elderly. Therefore, the aim of this prospective, randomized controlled trial is to gain insight in gait parameters and evaluate if real-time visual biofeedback can improve therapy compliance after proximal femur fractures in the elderly., Methods: This is a two-arm, parallel-design, prospective, randomized controlled trial. Inclusion criteria are age ≥ 60 years, a proximal femur fracture following low energy trauma, and unrestricted-weight bearing. Exclusion criteria are cognitive impairment and limited mobility before trauma. Participants are randomized into either the control group, which receives care as usual, or the intervention group, which receives real-time visual biofeedback about weight-bearing during gait in addition to care as usual. Spatiotemporal gait parameters will be measured in 94 participants per group during a 30-m walk with an ambulatory biofeedback system (SensiStep). The progress of rehabilitation will be evaluated by the primary outcome parameters maximum peak load and step duration in relation to the discharge date. Secondary outcome parameters include other spatiotemporal gait parameters in relation to discharge date. Furthermore, the gait parameters will be related to three validated clinical tests: Elderly Mobility Scale; Functional Ambulation Categories; and Visual Analogue Scale. The primary hypothesis is that participants in the intervention group will show improved and faster rehabilitation compared to the control group., Discussion: The first aim of this multicenter trial is to investigate the normal gait patterns after proximal femur fractures in the elderly. The use of biofeedback systems during rehabilitation after proximal femur fractures in the elderly is promising; therefore, the second aim is to investigate the effect of real-time visual biofeedback on gait after proximal femur fractures in the elderly. This could lead to improved outcome. In addition, analysis of the population may indicate characteristics of subgroups that benefit from feedback, making a differentiated approach in rehabilitation strategy possible., Trial Registration: TrialRegister.nl, NTR6794 . Registered on 31 October 2017.

Background: Previous studies have shown limited therapy compliance in weight-bearing in patients following total hip arthroplasty., Research Question: The purpose of this pilot RCT is to determine the immediate and late effect of real-time, visual biofeedback on weight-bearing during rehabilitation after THA in elderly., Methods: 24 participants who underwent THA were randomized to either the control or the intervention group. The intervention group received real-time, visual biofeedback on weight-bearing during training with the physical therapist during hospitalization and at twelve weeks follow up., Results: Without biofeedback, therapy compliance was limited. Significant improvement in peak load was found in the intervention group in the early postoperative phase. In contrast to the control group, the peak load at twelve weeks was significantly higher in the intervention group compared to the pre-operative peak load, indicating a lasting effect of early biofeedback. Other gait parameters were not significantly different in the early postoperative phase. In the intervention group a longer walking distance was observed and the use of walking aids was reduced at twelve weeks., Significance: Biofeedback systems could be promising to improve outcomes and reduce costs in future rehabilitation programs after THA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Individuals with lower extremity fractures are often instructed on how much weight to bear on the affected extremity. Previous studies have shown limited therapy compliance in weight bearing during rehabilitation. In this study we investigated the effect of real-time visual biofeedback on weight bearing in individuals with lower extremity fractures in two conditions: full weight bearing and touch-down weight bearing., Methods: 11 participants with full weight bearing and 12 participants with touch-down weight bearing after lower extremity fractures have been measured with an ambulatory biofeedback system. The participants first walked 15m and the biofeedback system was only used to register the weight bearing. The same protocol was then repeated with real-time visual feedback during weight bearing. The participants could thereby adapt their loading to the desired level and improve therapy compliance., Results: In participants with full weight bearing, real-time visual biofeedback resulted in a significant increase in loading from 50.9±7.51% bodyweight (BW) without feedback to 63.2±6.74%BW with feedback (P=0.0016). In participants with touch-down weight bearing, the exerted lower extremity load decreased from 16.7±9.77kg without feedback to 10.27±4.56kg with feedback (P=0.0718). More important, the variance between individual steps significantly decreased after feedback (P=0.018)., Conclusions: Ambulatory monitoring weight bearing after lower extremity fractures showed that therapy compliance is low, both in full and touch-down weight bearing. Real-time visual biofeedback resulted in significantly higher peak loads in full weight bearing and increased accuracy of individual steps in touch-down weight bearing. Real-time visual biofeedback therefore results in improved therapy compliance after lower extremity fractures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: The cellular demand for cholesterol requires control of its biosynthesis by the mevalonate pathway. Regulation of HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), a rate-limiting enzyme in this pathway and the target of statins, is a key control point herein. Accordingly, HMGCR is subject to negative and positive regulation. In particular, the ability of oxysterols and intermediates of the mevalonate pathway to stimulate its proteasomal degradation is an exquisite example of metabolically controlled feedback regulation. To define the genetic determinants that govern this process, we conducted an unbiased haploid mammalian genetic screen., Approach and Results: We generated human haploid cells with mNeon fused to endogenous HMGCR using CRISPR/Cas9 and used these cells to interrogate regulation of HMGCR abundance in live cells. This resulted in identification of known and new regulators of HMGCR, and among the latter, UBXD8 (ubiquitin regulatory X domain-containing protein 8), a gene that has not been previously implicated in this process. We demonstrate that UBXD8 is an essential determinant of metabolically stimulated degradation of HMGCR and of cholesterol biosynthesis in multiple cell types. Accordingly, UBXD8 ablation leads to aberrant cholesterol synthesis due to loss of feedback control. Mechanistically, we show that UBXD8 is necessary for sterol-stimulated dislocation of ubiquitylated HMGCR from the endoplasmic reticulum membrane en route to proteasomal degradation, a function dependent on its UBX domain., Conclusions: We establish UBXD8 as a previously unrecognized determinant that couples flux across the mevalonate pathway to control of cholesterol synthesis and demonstrate the feasibility of applying mammalian haploid genetics to study metabolic traits., (© 2017 The Authors.)

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gβγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.

Background: A variety of techniques for measuring lower limb loading exists, each with their own limitations. A new ambulatory biofeedback system was developed to overcome these limitations. In this study, we described the technical aspects and validated the accuracy of this system., Methods: A bench press was used to validate the system in the static situation. Ten healthy volunteers were measured by the new biofeedback system and a dual-belt instrumented treadmill to validate the system in the dynamic situation., Results: Bench press results showed that the sensor accurately measured peak loads up to 1000 N in the static situation. In the healthy volunteers, the load curves measured by the biofeedback system were similar to the treadmill. However, the peak loads and loading rates were lower in the biofeedback system in all participants at all speeds., Conclusions: Advanced sensor technologies used in the new biofeedback system resulted in highly accurate measurements in the static situation. The position of the sensor and the design of the biofeedback system should be optimized to improve results in the dynamic situation.