Filoviruses Use the HOPS Complex and UVRAG To Traffic to Niemann-Pick C1 Compartments during Viral Entry

Abstract

Ebola virus (EBOV) entry requires internalization into host cells and extensive trafficking through the endolysosomal network in order to reach late endosomal/lysosomal compartments that contain triggering factors for viral membrane fusion. These triggering factors include low-pH activated cellular cathepsin proteases, which cleave the EBOV glycoprotein (GP) to expose the binding domain of the filoviral receptor, Niemann-Pick C1 (NPC1). Here, we report that trafficking of EBOV to NPC1 requires expression of the homotypic fusion and protein sorting (HOPS) tethering complex as well as its regulator, UV radiation resistance associated gene (UVRAG). Using an inducible CRISPR/Cas9 system, we demonstrate that depletion of HOPS subunits as well as UVRAG impairs entry by all pathogenic filoviruses. UVRAG depletion resulted in reduced delivery of EBOV virions to NPC1+ cellular compartments. Furthermore, we show that deletion of a domain on UVRAG known to be required for interaction with the HOPS complex results in impaired EBOV entry. Taken together, our studies demonstrate that EBOV requires both expression and coordination between the HOPS complex and UVRAG in order to mediate efficient viral entry. IMPORTANCE Ebola viruses (EBOV) and other filoviruses cause sporadic and unpredictable outbreaks of highly lethal diseases. The lack of FDA-approved therapeutics, particularly those with panfiloviral specificity, highlights the need for continued research efforts to understand aspects of the viral life cycle that are common to all filoviruses. As such, viral entry is of particular interest as all filoviruses must reach cellular compartments containing the viral receptor, Niemann-Pick C1, to enter cells. Here we present an inducible CRISPR/Cas9 methodology to rapidly and efficiently generate knockout cells in order to interrogate the roles of a broad range of host factors in viral entry. Using this approach, we show that EBOV entry depends on both the homotypic fusion and protein sorting tethering (HOPS) complex in coordination with UV radiation resistance associated gene (UVRAG). Importantly, we demonstrate that the HOPS complex and UVRAG are required by all pathogenic filoviruses, representing potential targets for panfiloviral therapeutics.

Ebola virus triggers receptor tyrosine kinase-dependent signaling to promote the delivery of viral particles to entry-conducive intracellular compartments

“…To reach NPC1, filoviral particles must be internalized via macropinocytosis and then undergo extensive trafficking through the endosomal labyrinth whereby endosomes transition from Rab5+ to Rab7+ compartments [7][8][9]. Also involved in filovirus trafficking to NPC1 are the homotypic fusion and protein sorting (HOPS) and PIKfyve/ArPIKfyve/Sac3 (PAS) tethering and trafficking complexes [2,[10][11][12]. These complexes, along with the small GTPase Rab7, regulate vesicular fusion events required for specific trafficking of cargoes to late endosomes and lysosomes in cells [13,14].…”

Navigating the Complex Landscape of Ebola Infection Treatment: A Review of Emerging Pharmacological Approaches

“…In the low pH environment of the endolysosome, the GP 1 subunit is cleaved by host low-pH-dependent cathepsins B and L, thereby releasing the mucin-like subdomains and glycan caps and exposing the receptor-binding site. Once exposed, the GP 1 receptor-binding site engages the NPC1 receptor, an intracellular cholesterol transporter that is ubiquitously expressed and located on the interior membrane of late endosomes and lysosomes [ 10 , 23 , 82 , 83 ]. Cleavage of the glycan cap was shown to induce changes in GP 1 , which in turn enabled flexibility in GP 2 , which is essential for fusion.…”

Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells

“…Many host proteins have been identified to be important for endosomal trafficking of EBOV, such as the small GTPase Rab7, the homotypic fusion and protein sorting (HOPS) complex, the UV Radiation Resistance Associated (UVRAG) protein, and the PIKfyve-ArPIKfyve-Sac3 trafficking complex (10,15,16). In addition, studies have shown that EBOV particles can stimulate signaling cascades such as the Akt/PI3K pathway, the latter being required for EBOV trafficking to NPC1 (16)(17)(18)(19).…”