Choline is an essential nutrient that is required for synthesis of the main eukaryote phospholipid, phosphatidylcholine (PC). Macrophages are innate immune cells that survey and respond to danger and damage signals. Although it is well known that energy metabolism can dictate macrophage function, little is known as to the importance of choline homeostasis in macrophage biology. We hypothesized that the uptake and metabolism of choline is important for macrophage inflammation. Polarization of primary bone marrow macrophages with LPS resulted in an increased rate of choline uptake and higher levels of PC synthesis. This was attributed to a substantial increase in the transcript and protein expression of the choline transporter-like protein-1 (CTL1) in polarized cells. We next sought to determine the importance of choline uptake and CTL1 for macrophage immune responsiveness. Chronic pharmacological or CTL1 antibody-mediated inhibition of choline uptake resulted in altered cytokine secretion in response to LPS, which was associated with increased the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC). These experiments establish a previously unappreciated link between choline phospholipid metabolism and macrophage immune responsiveness, highlighting a critical and regulatory role for macrophage choline uptake via the CTL1 transporter.

Nadya M Morrow, Amy C. Burke, Joshua P Samsoondar, Kyle E Seigel, Andrew Wang, Dawn E. Telford, Brian G. Sutherland, Conor O'Dwyer, Gregory R. Steinberg, Morgan D Fullerton, Murray W. Huff

Morgan D. Fullerton, Rebecca J. Ford, Chelsea P. McGregor, Nicholas D. LeBlond, Shayne A. Snider, Stephanie A. Stypa, Emily A. Day, Šárka Lhoták, Jonathan D. Schertzer, Richard C. Austin, Bruce E. Kemp, Gregory R. Steinberg

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps. Ebola virus (EBOV) and Marburg virus (MARV) are zoonotic pathogens that can cause severe hemorrhagic fevers in humans and non-human primates. They are members of the growing Filoviridae family that also includes three other species of Ebolaviruses known to be highly pathogenic in humans. While vaccines for EBOV are being deployed and showed high efficacy, pan-filoviral treatment is still lacking. To infect cells, EBOV requires the endosomal/lysosomal resident protein Niemann-Pick C1 (NPC1). Accordingly, viral particles require extensive trafficking within endosomal pathways for entry and delivery of the viral genome into the host cell cytoplasm. Here, we used chemical biology to reveal that EBOV triggers receptor tyrosine kinase (RTK)-dependent signaling to traffic to intracellular vesicles that contain the receptor and are conducive to entry. The characterization of host trafficking factors and signaling pathways that are potentially triggered by the virus are important as these could be targeted for antiviral therapies. In our study, we identified several RTK inhibitors, some of which are FDA-approved drugs, that potently block EBOV infection. Since all filoviruses known to date, even Měnglà virus that was recently discovered in bats in China, use NPC1 as their entry receptor, these inhibitors have the potential to be effective pan-filovirus antivirals.

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.