Glucosylceramidase Maintains Influenza Virus Infection by Regulating Endocytosis

Abstract

Influenza virus is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza virus infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza virus infection following clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9)-mediated knockout (KO) of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both Western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) green fluorescent protein (GFP) reporter virus. We further showed that the reduction in infection correlated with impaired influenza virus trafficking to late endosomes and hence with fusion and entry. To examine whether GBA is required for other enveloped viruses, we compared the results seen with entry mediated by the glycoproteins of Ebola virus, influenza virus, vesicular stomatitis virus (VSV), and measles virus in GBA knockout cells. Entry inhibition was relatively robust for Ebola virus and influenza virus, modest for VSV, and mild for measles virus, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested that hypothesis and found that trafficking of epidermal growth factor (EGF) to late endosomes and degradation of its receptor were impaired in GBA knockout cells. Collectively, our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as of cellular cargos, including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize “late-penetrating” viruses, including influenza virus. IMPORTANCE Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.

Glucosylceramide synthase maintains influenza virus entry and infection

“…Previously, we found that deleting GBA1, the enzyme that converts GlcCer (the primary product of UGCG enzymatic activity) to ceramide (Fig 1) increases GlcCer levels and impairs endosome trafficking and influenza virus entry [25]. Here we found that knocking out UGCG decreases GlcCer levels (in both HEK 293 and A549 cells), impairing the entry of endosomeentering viruses.…”

“…Previous studies demonstrated that loss of expression or inhibition of several sphingolipidmetabolizing enzymes leads to reduced influenza virus infection (Fig 1). We recently discovered that glucosylceramidase (GBA) is required for optimal influenza virus entry [25]. We thus hypothesized that glucosylceramide synthase (UGCG), which converts ceramide to glucosylceramide, may also play a role in the influenza virus life cycle (green box).…”

“…Several studies have demonstrated that inhibition of distinct enzymes in the sphingolipid pathway results in alterations to influenza virus infection levels. Pharmacological inhibition of sphingomyelin synthesis (through serine palmitoyltransferase) and sphingosine kinase, as well as genetic ablation of sphingomyelin synthase and glucosylceramidase (red Xs) led to decreased influenza virus infection [23][24][25]34]. Conversely, reductions in ceramide synthesis through inhibition of ceramide synthase led to an increase in influenza virus replication [22].…”

“…Several studies have investigated the role of specific sphingolipids in influenza virus infection, including sphingomyelin [21], ceramide [22], and sphingosine-1-phosphate [23,24] (Fig 1). Most recently, we determined that deletion of glucosylceramidase (GBA) leads to reduced influenza virus entry and impaired cellular endocytosis [25]. Loss of GBA led to increases in the amount of the glycosphingolipid, glucosylceramide, and delayed trafficking of influenza virus to late endosomes/lysosomes.…”

Antiviral activity of Glucosylceramide synthase inhibitors against SARS-CoV-2 and other RNA virus infections

“…Human Cytomegalovirus (HCMV) induces elevation of ceramide and GM2-ganglioside (10), and Dengue virus induces elevation of ceramide and sphingomyelin (11). Additionally, Influenza virus was shown to induce Sphingomyelin and GlcCer elevation (12,13) and suppression of the biosynthesis of cellular sphingolipids results in the inhibition of the maturation of influenza virus particles in vitro (14,15). Moreover, iminosugars are known for their broad-spectrum antiviral activity, presumably because of their mechanism of action as endoplasmic reticulum (ER)-resident α-glucosidases I and II inhibitors (16).…”