Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its transmembrane domain or interaction with monocarboxylate transporter 1.

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

CRISPR knockout (KO) screens have identified host factors regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Here, we conducted a meta-analysis of these screens, which showed a high level of cell-type specificity of the identified hits, highlighting the necessity of additional models to uncover the full landscape of host factors. Thus, we performed genome-wide KO and activation screens in Calu-3 lung cells and KO screens in Caco-2 colorectal cells, followed by secondary screens in four human cell lines. This revealed host-dependency factors, including AP1G1 adaptin and ATP8B1 flippase, as well as inhibitors, including mucins. Interestingly, some of the identified genes also modulate Middle East respiratory syndrome coronavirus (MERS-CoV) and seasonal human coronavirus (HCoV) (HCoV-NL63 and HCoV-229E) replication. Moreover, most genes had an impact on viral entry, with AP1G1 likely regulating TMPRSS2 activity at the plasma membrane. These results demonstrate the value of multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential targets for therapeutic interventions.

Identification of Copper Transporter 1 as a Receptor for Feline Endogenous Retrovirus ERV-DC14.

Vertebrates harbor hundreds of endogenous retroviral (ERV) sequences in their genomes, which are considered signs of past infections that occurred during evolution. On rare occasions, ERV genes like env are maintained and coopted by hosts for physiological functions, but they also participate in recombination events with exogenous retroviruses to generate rearranged viruses with novel tropisms. In domestic cats, feline leukemia virus type D (FeLV-D) has been described as a recombinant virus between the infectious FeLV-A and likely the ERV-DC14 env gene that resulted in an extended tropism due to the usage of a new uncharacterized retroviral receptor. Here, we report the identification of SLC31A1 encoding the copper transporter 1 (CTR1) as a susceptibility gene for ERV-DC14 infection. Expression of human CTR1 into nonpermissive cells was sufficient to confer sensitivity to ERV-DC14 pseudotype infection and to increase the binding of an ERV-DC14 Env ligand. Moreover, inactivation of CTR1 by genome editing or cell surface downmodulation of CTR1 by a high dose of copper dramatically decreased ERV-DC14 infection and binding, while magnesium treatment had no effect. We also investigated the role of CTR1 in the nonpermissivity of feline and hamster cells. While feline CTR1 was fully functional for ERV-DC14, we found that binding was strongly reduced upon treatment with conditioned medium of feline cells, suggesting that the observed resistance to infection was a consequence of CTR1 saturation. In contrast, hamster CTR1 was inactive due to the presence of a N-linked glycosylation site at position 27, which is absent in the human ortholog. These results provide evidence that CTR1 is a receptor for ERV-DC14. Along with chimpanzee endogenous retrovirus type 2, ERV-DC14 is the second family of endogenous retrovirus known to have used CTR1 during past infections of vertebrates. IMPORTANCE Receptor usage is an important determinant of diseases induced by pathogenic retroviruses. In the case of feline leukemia viruses, three subgroups (A, B, and C) based on their ability to recognize different cell host receptors, respectively, the thiamine transporter THTR1, the phosphate transporter PiT1, and the heme exporter FLVCR1, are associated with distinct feline diseases. FeLV-A is horizontally transmitted and found in all naturally infected cats, while FeLV-B and FeLV-C have emerged from FeLV-A, respectively, by recombination with endogenous retroviral env sequences or by mutations in the FeLV-A env gene, both leading to a switch in receptor usage and in subsequent in vivo tropism. Here, we set up a genetic screen to identify the retroviral receptor of ERV-DC14, a feline endogenous provirus whose env gene has been captured by infectious FeLV-A to give rise to FeLV-D in a process similar to FeLV-B. Our results reveal that the copper transporter CTR1 was such a receptor and provide new insights into the acquisition of an expanded tropism by FeLV-D.

Characterization of XPR1/SLC53A1 variants located outside of the SPX domain in patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurological disease characterized by deposits of calcium phosphate in the basal ganglia and other regions of the brain. Pathogenic variants in the XPR1/SLC53A1 gene, which encodes the only known inorganic phosphate exporter, cause an autosomal dominant form of PFBC. These variants are typically located in the SPX N-terminal domain of the protein. Here, we characterize three XPR1 variants outside of SPX in three PFBC patients with an apparently sporadic presentation: c.1375C > T p.(R459C), c.1855A > G p.(N619D) and c.1886T > G p.(I629S), with the latter identified as the first XPR1/SLC53A1 de novo mutation to occur in a PFBC proband. When tested in an in vitro physiological complementation assay, the three XPR1 variants were impaired in phosphate export function, although they were normally expressed at the cell surface and could serve as functional receptors for retrovirus entry. Moreover, peripheral blood cells from the p.N619D patient could be assayed ex vivo and displayed significantly impaired phosphate export. Our results establish for the first time the clinical and molecular characteristics of XPR1 variants located outside the SPX domain and assert a direct link between these variants, deficient phosphate export, and PFBC. Moreover, we unveiled new structural features in XPR1 C-terminal domain that play a role in phosphate export and disease.

E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis.

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.

XPR1 mutations are a rare cause of primary familial brain calcification.

Mutations in XPR1, a gene encoding an inorganic phosphate exporter, have recently been identified in patients with primary familial brain calcification (PFBC). Using Sanger sequencing, we screened XPR1 in 18 unrelated patients with PFBC and no SLC20A2, PDGFB, or PDGFRB mutation. XPR1 variants were tested in an in vitro physiological complementation assay and patient blood cells were assessed ex vivo for phosphate export. We identified a novel c.260T > C, p.(Leu87Pro) XPR1 variant in a 41-year-old man complaining of micrographia and dysarthria and demonstrating mild parkinsonism, cerebellar ataxia and executive dysfunction. Brain (123)I-Ioflupane scintigraphy showed marked dopaminergic neuron loss. Peripheral blood cells from the patient exhibited decreased phosphate export. XPR1 in which we introduced the mutation was not detectable at the cell surface and did not lead to phosphate export. These results confirm that loss of XPR1-mediated phosphate export function causes PFBC, occurring in less than 8 % of cases negative for the other genes, and may be responsible for parkinsonism.

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.

Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.

Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

Profiling of Nutrient Transporter Expression in Human Stem Cell-Derived Cardiomyocytes Exposed to Tyrosine Kinase Inhibitor Anticancer Drugs Using RBD Ligands.

We applied a novel profiling approach using receptor binding domain (RBD) ligands to cell surface domains of a panel of nutrient transporters to characterize the impact of a number of tyrosine kinase inhibitor anticancer drugs on human stem cell-derived cardiomyocytes. High-content screening and flow cytometry analysis showed diagnostic changes in nutrient transporter expression correlating with glycolysis and oxidative phosphorylation-based cell metabolism in glucose and galactose media. Cluster analysis of RBD binding signatures of drug-treated cells cultured in glucose medium showed good correlation with sensitization of mitochondrial toxicity in cells undergoing oxidative phosphorylation in galactose medium. These data demonstrate the potential for RBD ligands as profiling tools to improve the clinical predictivity of in vitro cell assays for drug toxicity.

Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans.

Inorganic phosphate uptake is a universal function accomplished by transporters that are present across the living world. In contrast, no phosphate exporter has ever been identified in metazoans. Here, we show that depletion of XPR1, a multipass membrane molecule initially identified as the cell-surface receptor for xenotropic and polytropic murine leukemia retroviruses (X- and P-MLV), induced a decrease in phosphate export and that reintroduction of various XPR1 proteins, from fruit fly to human, rescued this defect. Inhibition of phosphate export was also obtained with a soluble ligand generated from the envelope-receptor-binding domain of X-MLV in all human cell lines tested, as well as in diverse stem cells and epithelial cells derived from renal proximal tubules, the main site of phosphate homeostasis regulation. These results provide new insights on phosphate export in metazoans and the role of Xpr1 in this function.

Metabolic adaptation of neutrophils in cystic fibrosis airways involves distinct shifts in nutrient transporter expression.

Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters.

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Regional characterization of energy metabolism in the brain of normal and MPTP-intoxicated mice using new markers of glucose and phosphate transport.

The gibbon ape leukemia virus (GALV), the amphotropic murine leukemia virus (AMLV) and the human T-cell leukemia virus (HTLV) are retroviruses that specifically bind nutrient transporters with their envelope glycoproteins (Env) when entering host cells. Here, we used tagged ligands derived from GALV, AMLV, and HTLV Env to monitor the distribution of their cognate receptors, the inorganic phosphate transporters PiT1 and PiT2, and the glucose transporter GLUT1, respectively, in basal conditions and after acute energy deficiency. For this purpose, we monitored changes in the distribution of PiT1, PiT2 and GLUT1 in the cerebellum, the frontal cortex, the corpus callosum, the striatum and the substantia nigra (SN) of C57/BL6 mice after administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridinium (MPTP), a mitochondrial complex I inhibitor which induces neuronal degeneration in the striato-nigral network.The PiT1 ligand stained oligodendrocytes in the corpus callosum and showed a reticular pattern in the SN. The PiT2 ligand stained particularly the cerebellar Purkinje cells, while GLUT1 labelling was mainly observed throughout the cortex, basal ganglia and cerebellar gray matter. Interestingly, unlike GLUT1 and PiT2 distributions which did not appear to be modified by MPTP intoxication, PiT1 immunostaining seemed to be more extended in the SN. The plausible reasons for this change following acute energy stress are discussed.These new ligands therefore constitute new metabolic markers which should help to unravel cellular adaptations to a wide variety of normal and pathologic conditions and to determine the role of specific nutrient transporters in tissue homeostasis.