Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses1-3. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H+ -ATPase 4-8. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator/RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, while loss of SLC38A9 expression impaired amino acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid-sensing machinery that controls the activation of mTOR.

Summary Background School closures have occurred globally during the COVID-19 pandemic. However, empiric data on transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children and in educational settings are scarce. In Australia, most schools have remained open during the first epidemic wave, albeit with reduced student physical attendance at the epidemic peak. We examined SARS-CoV-2 transmission among children and staff in schools and early childhood education and care (ECEC) settings in the Australian state of New South Wales (NSW). Methods Laboratory-confirmed paediatric (aged ≤18 years) and adult COVID-19 cases who attended a school or ECEC setting while considered infectious (defined as 24 h before symptom onset based on national guidelines during the study period) in NSW from Jan 25 to April 10, 2020, were investigated for onward transmission. All identified school and ECEC settings close contacts were required to home quarantine for 14 days, and were monitored and offered SARS-CoV-2 nucleic acid testing if symptomatic. Enhanced investigations in selected educational settings included nucleic acid testing and SARS-CoV-2 antibody testing in symptomatic and asymptomatic contacts. Secondary attack rates were calculated and compared with state-wide COVID-19 rates. Findings 15 schools and ten ECEC settings had children (n=12) or adults (n=15) attend while infectious, with 1448 contacts monitored. Of these, 633 (43·7%) of 1448 had nucleic acid testing, or antibody testing, or both, with 18 secondary cases identified (attack rate 1·2%). Five secondary cases (three children; two adults) were identified (attack rate 0·5%; 5/914) in three schools. No secondary transmission occurred in nine of ten ECEC settings among 497 contacts. However, one outbreak in an ECEC setting involved transmission to six adults and seven children (attack rate 35·1%; 13/37). Across all settings, five (28·0%) of 18 secondary infections were asymptomatic (three infants [all aged 1 year], one adolescent [age 15 years], and one adult). Interpretation SARS-CoV-2 transmission rates were low in NSW educational settings during the first COVID-19 epidemic wave, consistent with mild infrequent disease in the 1·8 million child population. With effective case-contact testing and epidemic management strategies and associated small numbers of attendances while infected, children and teachers did not contribute significantly to COVID-19 transmission via attendance in educational settings. These findings could be used to inform modelling and public health policy regarding school closures during the COVID-19 pandemic. Funding NSW Government Department of Health.

Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit the therapeutic applications. Here, we employed a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anti-cancer compound YM155 on SLC35F2, an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo.These findings suggest a novel route to targeted DNA damage by exploiting tumor and patientspecific import of YM155.

Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.HCV | Golgi fragmentation | autophagy | IRGM | membranous web H epatitis C virus (HCV) is a positive-sense RNA virus in the family Flaviviridae that is a major cause of chronic liver disease. All positive-strand RNA viruses studied until now, including HCV, replicate their genomes in association with cellular membrane rearrangements. In this process, viruses remodel intracellular membranes [e.g., of mitochondria, endoplasmic reticulum (ER), and plasma membrane] to generate membrane structures such as single-or double-membrane vesicles that contribute to viral replication complexes (VRCs). HCV replication takes place at a unique subcellular compartment, the membranous web (MW), which has been proposed to be derived from the ER (1, 2). The HCV MW has a complex morphology consisting of clusters of single-, double-, and multimembrane vesicles and probably includes autophagosomes and lipid droplets (1, 3, 4). Recent findings reveal that the MW is produced by distinct HCV nonstructural (NS) proteins acting through sequential interaction with several host factors, such as the virus-targeted phosphatidylinositol-4 kinase III α (PI4KIIIα) (2, 3), but the full spectrum of host components and precise membrane composition that supports HCV replication are not fully defined.Autophagy is an evolutionarily conserved cellular mechanism that involves intracellular membrane trafficking and degradation to maintain cell homeostasis. Viruses, including HCV, have been reported to exploit autophagy for replication purposes (4-6), but the mechanism by which this exploitation occurs is largely unknown. De novo synthesis of autophagosomes is a complex process that involves the formation of a phagophore membrane and its elongation. Initiation of autophagy is regulated by the mammalian target of rapamycin complex 1 (mTORC1), which neg...

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that SET domain bifurcated 2 (Setdb2) was the only protein lysine methyltransferase induced during influenza virus infection. Setdb2 expression depended on type-I interferon signaling and it repressed the expression of the neutrophil attractant Cxcl1 and other NF-κB target genes. This coincided with Setdb2 occupancy at the Cxcl1 promoter, which in the absence of Setdb2 displayed reduced H3K9 tri-methylation. Setdb2 hypomorphic gene-trap mice exhibited increased neutrophil infiltration in sterile lung inflammation and were less sensitive to bacterial superinfection upon influenza virus infection. This suggests that a Setdb2-mediated regulatory crosstalk between the type-I interferon and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

SummaryLipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

Solute Carriers (SLCs) represent the largest family of transmembrane transporters in humans and constitute major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR/Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the compounds screened suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.