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. 2020 Jul 30;94(16):e01002-20.
doi: 10.1128/JVI.01002-20. Print 2020 Jul 30.

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

Yuxia Bo #  1   2   3 , Shirley Qiu #  1   2   3 , Rory P Mulloy  1   2   3 , Marceline Côté  4   2   3
Affiliations

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

Yuxia Bo et al. J Virol. .

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), exposing a domain which binds to the filoviral receptor, the cholesterol transporter 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 clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we demonstrated 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 of 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 ones 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 method 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 showed that EBOV entry depends on both the homotypic fusion and protein sorting (HOPS) tethering 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.

Keywords: Ebola virus; filovirus; vesicular trafficking; virus entry.

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Figures

FIG 1
FIG 1
Inducible CRISPR/Cas9 system to investigate host trafficking factors required for viral entry. (A) Schematic of doxycycline (Dox)-inducible CRISPR/Cas9 system using dual gRNAs in HT1080 cells. (B) Time course of Flag-Cas9 expression in CRISPR cells after Dox induction. (C) (Left) Surveyor nuclease assay showing the region of NPC1 targeted by one of the dual gRNAs. (Right) Expression of Flag-Cas9 and NPC1 in NPC1 CRISPR cells after 4 days of Dox induction. (D) NPC1 CRISPR cells were induced for 4 days in Dox, followed by infection with βlam-VLPs harboring EBOV GP, MARV GP, VSV G, or JUNV GPC. Entry was detected by measuring the percentage of cells with cleaved CCF2, normalized to uninduced cells. Results are representative of 3 independent experiments. Asterisks indicate significant differences in entry compared to uninduced cells. ***, P < 0.001.
FIG 2
FIG 2
The C-Vps core is required for filovirus entry. VPS11 (A), VPS16 (B), VPS18 (C), and VPS33a (D) CRISPR cells were infected by a panel of βlam-VLPs harboring EBOV GP, MARV GP, VSV G, or JUNV GPC following 4 days of Dox induction. Entry was detected by measuring the percentage of cells with cleaved CCF2, normalized to uninduced cells. Results are representative of 3 independent experiments. Asterisks indicate significant differences in entry compared to uninduced cells. Surveyor nuclease assay results for one gRNA of each targeted gene are shown on the right. **, P < 0.01; ***, P < 0.001.
FIG 3
FIG 3
A HOPS-specific, but not CORVET-specific, subunit is required for filovirus entry. VPS3 (A), VPS8 (B), and VPS39 (C) CRISPR cells were infected by a panel of βlam-VLPs harboring EBOV GP, MARV GP, VSV G, or JUNV GPC following 4 days of Dox induction. Entry was detected by measuring the percentage of cells with cleaved CCF2, normalized to uninduced cells. Results are representative of 3 independent experiments. Asterisks indicate significant differences in entry compared to uninduced cells. Surveyor nuclease results for one gRNA of each targeted gene are shown on the right. *, P < 0.05; ***, P < 0.001.
FIG 4
FIG 4
UVRAG expression is required for filovirus entry. (A) UVRAG KO and WT-add-back CRISPR cells were induced in Dox for 4 days, followed by infection with βlam-VLPs harboring EBOV GP or VSV G. Entry was detected by measuring the percentage of cells with cleaved CCF2, normalized to uninduced cells. Results are representative of 3 independent experiments. Asterisks indicate significant differences in entry compared to uninduced cells. Surveyor nuclease results for UVRAG gRNA2 are shown on the right. (B) Infection of UVRAG KO or WT-add-back CRISPR cells by a panel of VLPs bearing different filoviral glycoproteins or VSV G following 4 days of Dox induction. Asterisks indicate significant differences in entry compared to uninduced cells. Results are representative of 3 independent experiments. (C) UVRAG KO and WT-add-back CRISPR cells were induced and infected with VLPs as for panel A. Entry in the low (lo), medium (med), and high (hi) mCherry-UVRAG-expressing cells was determined by mCherry expression (dot plot [inset]). Percent entry was significantly increased for both EBOV and VSV with increasing mCherry expression (one-way analysis of variance [ANOVA], Tukey’s test; P < 0.05). **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
EBOV trafficking to NPC1+ compartments is impaired in UVRAG-deficient cells. (A) UVRAG CRISPR KO and WT-add-back cells were induced in Dox for 4 days, followed by reseeding onto coverslips. Cells were then infected with GFP-VLPs harboring fusion-deficient EBOV ΔM GPF535R for 3 h. Cells were fixed, permeabilized, and stained with an NPC1 antibody and Hoechst. Cells were imaged on an LSM800 confocal microscope (Zeiss). The number of internalized VLPs (B) and colocalization between VLPs and NPC1 (C) were analyzed using Imaris software (Bitplane). Results shown are combined normalized data from 3 experiments. Asterisks indicate significant differences in colocalization compared to uninduced cells, as determined by an unpaired t test with Welch’s correction, and statistical significance determined using the Holm-Šídák method. ***, P < 0.001.
FIG 6
FIG 6
Deletion of UVRAG domains required for HOPS association impairs EBOV entry. (A) Schematic of mCherry-tagged UVRAG deletion constructs. (B) Immunoblot of mCherry-UVRAG in UVRAG CRISPR cells transduced with the deletion constructs. (C) Expression of LC3 in UVRAG CRISPR cells transduced with the deletion constructs was detected by immunoblotting following 4 days of induction in Dox. (D) UVRAG CRISPR KO, WT-add-back, or deletion construct-add-back cells were induced in Dox for 4 days, followed by infection with βlam-VLPs harboring EBOV GP or VSV G. Entry was detected by measuring the percentage of cells with cleaved CCF2, normalized to uninduced cells. Asterisks indicate significant differences in entry compared to uninduced cells. Results are representative of 3 independent experiments. ***, P < 0.001.
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