NRP2 and CD63 Are Host Factors for Lujo Virus Cell Entry

Abstract

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.

Keywords: CD63; LUJV; Lujo virus; NRP2; arenavirus; entry receptor; haploid genetics.

Figure 1. LUJV GP-mediated cell entry requires a distinct set of host factors

(A) Wild-type (WT) and α-DG knockout (DAG1ko) HAP1 cells were inoculated with the indicated VSVs (multiplicity of infection [MOI]=0.5). In parallel, HAP1 cells were mock-treated or pre-treated with TfR1 blocking antibodies for 1h before infection. Cells were fixed 8h post virus inoculation, stained with DAPI (to detect the nuclei) and infection (visible through virus-encoded eGFP expression) was detected by fluorescence microscopy. Scale bar: 10μm. (B) Data from the haploid screen with VSV-LUJV. The y-axis indicates the significance of enrichment of gene-trap insertions compared with unselected control cells. Circles represent individual genes and their size corresponds to the number of unique disruptive insertion sites identified in the virus-selected population. Genes with significance scores above 10 are labeled, colored and grouped according to function. (C) Wild-type HAP1 cells (WT), knockout clones (TMEM30Ako, CD63ko and NRP2ko) and reconstituted clones (+TMEM30A, +CD63 and +NRP2) were infected with VSV (control) or VSV-LUJV (MOI=3). Cells were fixed 6h post virus inoculation and infection was visualized by fluorescence microscopy. Scale bar: 10μm. (D) HAP1, HEK293T and BSR-T7 cells were transduced with empty (mock) retrovirus or with virus expressing NRP2-HA. Next, cells were inoculated with the indicated viruses (MOI=3). Cells were fixed 6h post inoculation and infection was measured by quantifying eGFP fluorescence. The data is presented as the percentage eGFP expression of each virus in cells overexpressing NRP2 compared to mock-transduced cells for each cell line. Significant differences (t-test; p-value <0.05) between each condition (n=3 experimental replicates) are indicated by the asterisks. (E) Western blot of total cell lysates from HAP1 and HUVEC cells. Membranes were probed with anti-NRP2, anti-α-DG or anti-TfR1 antibodies to detect endogenous expression in both cell types. An anti-CDK4 antibody was used as a loading control. (F) HUVEC cells were inoculated with the indicated viruses (MOI=1, as determined on HAP1 cells). Cells were fixed 6h post virus inoculation and infection was visualized by fluorescence microscopy. Cells were counter-stained with DAPI (shown as a separate panel). Scale bar: 10μm. (G) Quantification of the experiment shown in panel F. The data is presented as the percentage of infected (eGFP-positive) cells (n=3 experimental replicates).

Figure 2. LUJV GP binds the N-terminal domain of NRP2

(A) Flag-tagged LASV GP and LUJV GP was immobilized on beads and incubated with cell lysates from HEK293T cells (transduced with control [vector] or NRP2-HA retrovirus). Bound proteins (IP: immunoprecipitation) and input samples were subjected to immunoblot analysis. (B) Schematic representation of all the NRP2 constructs used in the biochemistry and infection experiments described in panel C and D. The design of the constructs was based on the reported NRP2 crystal structure (Appleton et al., 2007). (C) Flag-tagged LASV GP and LUJV GP was immobilized on beads and incubated with cell lysates from HEK293T cells expressing vector control, HA-tagged full-length NRP2 or the individual mutants depicted in panel B. Bound proteins (IP: immunoprecipitation) and input samples were subjected to immunoblot analysis. An anti-CDK4 antibody was used as a loading control. (D) Soluble Fc-tagged LUJV GP1 (sGP1Fc-LUJV) was immobilized on beads and incubated with total cell lysate from HEK293T cells expressing NRP2-HA. Empty beads served as a control. Bound proteins (pulldown and beads) as wells as the input samples were subjected to immunoblot analysis. (E) HAP1 cells expressing vector control, HA-tagged full-length NRP2 or the individual NRP2 mutants were inoculated with VSV (control) or VSV-LUJV (MOI=0.2) for 6h at 37°C. Cells were fixed and stained with DAPI after which infection was visualized by fluorescence microscopy. Scale bar: 10μm. (F) HAP1 wild-type or NRP2ko cells were co-inoculated with fluorescently labeled VSV-LUJV (in red) and VSV-LASV (in white) for 15min at 37°C. Cells were subsequently washed and stained with fluorescently labeled WGA (in cyan) to visualize the cell boundaries and the viral particles attached to it. Representative images are shown. (G) Quantification of panel F (combined data of two independent experiments). Shown is the percentage of cell-associated VSV-LUJV particles over VSV-LASV particles in HAP1 wild-type (WT) or NRP2ko cells (n=22 and 27 cells, respectively). The significant difference (unpaired t-test; p-value=0.0023) is indicated by the asterisk.

Figure 3. CD63 promotes LUJV GP-mediated membrane fusion under acidic conditions

(A) HEK293T cells overexpressing mCherry-tagged CD63 (in red) were inoculated with VSV-LUJV-MeGFP (MOI=150; in cyan) and inoculum was removed 1h post infection. Virus entry was monitored by live-cell imaging 105min post virus inoculation (see Movie S1). Shown is an image of a representative cell for which still-frames are depicted with 20sec intervals. (B) HAP1 cells were pre-treated with DMSO (control) or with 100nM Bafilomycin A1 (BafA1) for 30min at 37°C. Cells were subsequently inoculated with VSV (control) or VSV-LUJV (MOI=3) for 6h and fixed. Infection was visualized by fluorescence microscopy. Scale bar: 10μm. (C) HEK293T cells transduced with control (empty) retrovirus or with virus expressing NRP2-HA, mCherry-tagged wild-type CD63 or a mutant with a disrupted lysosomal targeting motif (mCherry-CD63_AA) were transfected with pCAGGS-LUJV GP-Flag and pCMV-eGFP. 48h post transfection, cells were washed with neutral or acidic culture medium for 5min at 37°C. Medium was replaced with normal medium and cells were recovered for 30min at 37°C. Subsequently, cells were fixed and syncytia (visible through extensive content [eGFP] mixing) were visualized by fluorescent microscopy (outlined in red). Scale bar: 10μm. (D) HUVEC cells were transduced with control (empty) lentivirus or with virus expressing a guide RNA targeting CD63. Cells were inoculated with VSV (control) or VSV-LUJV (MOI=3), fixed, stained with DAPI and CD63 antibodies (shown as separate panels) and analyzed for infection by fluorescence microscopy. White arrows indicate infected cells that are CD63-positive. Scale bar: 10μm. (E) Wild-type (WT) and CD63ko HAP1 cells were inoculated with the indicated viruses (MOI=3). Cells were fixed 6h post inoculation and infection was measured by quantifying eGFP fluorescence. The data is presented as the percentage eGFP expression of each virus in CD63ko cells compared to WT cells. A significant difference (Two-way ANOVA, p-value <0.05) between conditions is indicated by the asterisk (n=3 experimental replicates).

Figure 4. Wild-type LUJV requires NRP2 for infection

(A) HUVEC cells were transduced with control (empty) lentivirus or with virus expressing a guide RNA targeting NRP2. Next, cells were inoculated with VSV (control) or VSV-LUJV (MOI=3), fixed, stained with DAPI (shown as a separate panel) and analyzed for infection by fluorescence microscopy. Scale bar: 10μm. (B) Western blot analysis of the cells described in panel A. Membranes were probed with an anti-NRP2 antibody for detection of endogenous NRP2. An anti-CDK4 antibody was used as a loading control. (C) The pools of HUVECs mentioned in panel A were transduced with control (empty) retrovirus or with virus expressing NRP2-HA. Subsequently, cells were inoculated with VSV-LUJV and analyzed for infected cells as described above. Scale bar: 10μm. (D) Wild-type (WT), NRP2ko and NRP2-HA reconstituted HUVEC cells were infected with authentic LUJV (MOI=0.5). Cells were fixed 16h post virus inoculation, permeabilized and stained with DAPI. Infection was visualized by immunofluorescence microscopy using an α-NP antibody. Scale bar: 10μm. (E) In parallel to the immunofluorescence experiment in panel E, cells were inoculated with LUJV (MOI=0.2) and the production of viral progeny after 36h was monitored by plaque assays. The data is presented as plaque-forming units (PFU) per ml of culture medium. Significant differences (ANOVA test; p-value <0.05) between each condition are indicated by the asterisks (n=4 experimental replicates). (F) HUVECs were mock-treated or treated with serial dilutions of polyclonal NRP2 antibody. Following 30min incubation on ice, cells were exposed to LUJV Zambia R4356 (MOI=1) for 1h at 37°C in the presence or absence of NRP2 antibody and incubated at 37°C for an additional hour. Cells were then washed and incubated for 36h before fixation. Infected cells were visualized by immunostaining using an arenavirus NP-specific polyclonal guinea pig serum. The number of infected cells were quantified for all conditions and are expressed as percentage of inhibition (n=2 experimental replicates). The continuous line represents the nonlinear fit of all the data points (R²=0.93). Depicted are representative images of the control versus the highest NRP2 antibody concentration. All experiments involving wild-type LUJV infections were carried out under BSL4 conditions.