Glucosylceramidase Maintains Influenza Virus Infection by Regulating Endocytosis.

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.

Sphingosine 1-Phosphate Lyase Enhances the Activation of IKKε To Promote Type I IFN-Mediated Innate Immune Responses to Influenza A Virus Infection.

Sphingosine 1-phosphate (S1P) lyase (SPL) is an intracellular enzyme that mediates the irreversible degradation of the bioactive lipid S1P. We have previously reported that overexpressed SPL displays anti-influenza viral activity; however, the underlying mechanism is incompletely understood. In this study, we demonstrate that SPL functions as a positive regulator of IKKε to propel type I IFN-mediated innate immune responses against viral infection. Exogenous SPL expression inhibited influenza A virus replication, which correlated with an increase in type I IFN production and IFN-stimulated gene accumulation upon infection. In contrast, the lack of SPL expression led to an elevated cellular susceptibility to influenza A virus infection. In support of this, SPL-deficient cells were defective in mounting an effective IFN response when stimulated by influenza viral RNAs. SPL augmented the activation status of IKKε and enhanced the kinase-induced phosphorylation of IRF3 and the synthesis of type I IFNs. However, the S1P degradation-incompetent form of SPL also enhanced IFN responses, suggesting that SPL's pro-IFN function is independent of S1P. Biochemical analyses revealed that SPL, as well as the mutant form of SPL, interacts with IKKε. Importantly, when endogenous IKKε was downregulated using a small interfering RNA approach, SPL's anti-influenza viral activity was markedly suppressed. This indicates that IKKε is crucial for SPL-mediated inhibition of influenza virus replication. Thus, the results illustrate the functional significance of the SPL-IKKε-IFN axis during host innate immunity against viral infection.

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Glucosylceramide synthase maintains influenza virus entry and infection.

Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.

Inhibition of Ebola Virus by a Molecularly Engineered Banana Lectin.

Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.

Novel Sphingolipid-Based Cancer Therapeutics in the Personalized Medicine Era.

Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, including cell death and proliferation. The myriad of pro-death and pro-survival cellular pathways involving sphingolipids provide a plethora of opportunities for dysregulation in cancers. In recent years, modulation of these sphingolipid metabolic pathways has been in the forefront of drug discovery for cancer therapeutics. About two decades ago, researchers first showed that standard of care treatments, e.g., chemotherapeutics and radiation, modulate sphingolipid metabolism to increase endogenous ceramides, which kill cancer cells. Strikingly, resistance to these treatments has also been linked to altered sphingolipid metabolism, favoring lipid species that ultimately lead to cell survival. To this end, many inhibitors of sphingolipid metabolism have been developed to further define not only our understanding of these pathways but also to potentially serve as therapeutic interventions. Therefore, understanding how to better use these new drugs that target sphingolipid metabolism, either alone or in combination with current cancer treatments, holds great potential for cancer control. While sphingolipids in cancer have been reviewed previously (Hannun & Obeid, 2018; Lee & Kolesnick, 2017; Morad & Cabot, 2013; Newton, Lima, Maceyka, & Spiegel, 2015; Ogretmen, 2018; Ryland, Fox, Liu, Loughran, & Kester, 2011) in this chapter, we present a comprehensive review on how standard of care therapeutics affects sphingolipid metabolism, the current landscape of sphingolipid inhibitors, and the clinical utility of sphingolipid-based cancer therapeutics.

Targeting cancer cells in the tumor microenvironment: opportunities and challenges in combinatorial nanomedicine.

Cancer therapies of the future will rely on synergy between drugs delivered in combination to achieve both maximum efficacy and decreased toxicity. Nanoscale drug delivery vehicles composed of highly tunable nanomaterials ('nanocarriers') represent the most promising approach to achieve simultaneous, cell-selective delivery of synergistic ratios of combinations of drugs within solid tumors. Nanocarriers are currently being used to co-encapsulate and deliver synergistic ratios of multiple anticancer drugs to target cells within solid tumors. Investigators exploit the unique environment associated with solid tumors, termed the tumor microenvironment (TME), to make 'smart' nanocarriers. These sophisticated nanocarriers exploit the pathological conditions in the TME, thereby creating highly targeted nanocarriers that release their drug payload in a spatially and temporally controlled manner. The translational and commercial potential of nanocarrier-based combinatorial nanomedicines in cancer therapy is now a reality as several companies have initiated human clinical trials.