Sphingomyelinases and Lysosomal Trafficking in Ebolavirus Entry
Miller, Mary 1974-
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Ebolavirus (EBOV) is a negative sensed, single-strand filovirus which causes up to 90% mortality. Despite increasing outbreaks over the past decade, no approved vaccines or therapies exist. One drug treatment strategy is to target virus entry mechanisms. EBOV enters the cell using a macropinocytosis-like mechanism, which requires actin, microtubules, and phosphatidylinositol 3-kinase (PI3K). Additionally, pH-dependent lysosomal enzymes, cathepsins B and L, are needed to proteolytically cleave the virus surface glycoprotein. This action facilitates fusion between the virus and cell membranes prior to infection. However, it is unclear where fusion occurs in the cell. Furthermore, although EBOV colocalizes with early endosomes and late endosomes, the role of lysosomes has yet to be elucidated. To determine whether EBOV colocalizes with lysosomes during entry, fluorescently-tagged virus-like particles (VLPs) were generated by overexpressing EBOV glycoprotein and virus protein 40 (VP40-GFP) in 293FT cells. EBOVLP colocalization with lysosomal marker, Lamp1, was visualized using an immunofluorescence assay. Interestingly, colocalization occurred in non-permeabilized cells, suggesting an interaction with EBOVLPs and lysosomes at the cell surface. By applying chemical and genetic inhibitors of lysosomal exocytosis to cells inoculated with replication-competent EBOV, it appeared that this vesicle trafficking pathway was required for infection. During lysosomal exocytosis, pH-dependent acid sphingomyelinase (ASM) is released to the plasma membrane‟s outer leaflet, resulting in ASM-dependent macropinosome formation. Because EBOV enters via a macropinocytosis-like mechanism, the role of ASM in infection was assessed. Specific ASM chemical inhibitors and RNA interference (RNAi) were used to show that ASM was important for EBOV-GFP infection. A mechanistic approach was then employed to isolate which step of viral entry – binding, internalization, trafficking, or fusion – was being blocked by ASM inhibition. Using a mixing contents assay, it was determined that ASM inhibitors target a fusion or pre-fusion step. It was also found that ASM colocalized with EBOVLPs at the plasma membrane shortly after binding, similar to Lamp1. Finally, pseudotyped viruses containing a common core virus encoated with either vii EBOV, Lassa virus (LASV), or Venezuelan Equine Encephalitis virus (VEEV) surface glycoproteins were incubated with cells treated with ASM inhibitors. EBOV GP pseudotyped viruses were more sensitive to ASM inhibition, suggesting a GP-specific role for ASM in entry. Next, because ASM was found to be important, the role of other sphingomyelinases in EBOV infection was assessed. A role for neutral sphingomyelinase 2 activation was also tested by applying the same assays as for ASM. It was determined that NSM inhibitors also block EBOV infection at a fusion or pre-fusion step of EBOV entry. Taken together, these findings suggest that EBOV binding stimulates lysosomal exocytosis, resulting in sphingomyelinase-dependent infection of cells. In conclusion, inhibitors of lysosomal exocytosis and sphingomyelinases may be useful as potential drug therapies to prevent EBOV infection.