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, 332 (6029), 589-92

Conserved Eukaryotic Fusogens Can Fuse Viral Envelopes to Cells

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Conserved Eukaryotic Fusogens Can Fuse Viral Envelopes to Cells

Ori Avinoam et al. Science.

Abstract

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Figures

Fig. 1
Fig. 1
A family of eukaryotic cell-cell fusogens: FF orthologs from two phyla fuse mammalian BHK cells (A) Two trees produced using maximum parsimony analysis show phylogenic relationships of 25 taxa (left; based on the TGFβ-RI like domain, Fig. S1B) and 14 taxa (right; based on the full length extracellular domain), FF proteins are classified into three subgroups EFF-1-like (red), AFF-1-like (orange) and FF (green) (Table S1). Consistency, retention and composite indexes are in (11). (B%#x2013;D) Immunofluorescence with anti-Flag antibodies (green), and nuclei DAPI staining (blue) on BHK cells transfected with (B) empty vector, (C) aff-1, (D) Tsp-ff-1 and (E) Bfl-ff-1. Cotransfection marker (red). Scale bars are 20 μm. (F) Fusion index for BHKs expressing FF proteins and negative control (empty vector). Data are means ± SE. Empty vector, n=14, aff-1, n=14, Tsp-ff-1, n=8, Bfl-ff-1, n=9; n represents number of experiments.
Fig. 2
Fig. 2
AFF-1 can complement the infection of a fusion deficient VSVΔG (A) Titers of VSVΔG pseudoviruses. The type of protein on the viral membrane (Bald or AFF-1) and on the BHK cell membrane (Vector, AFF-1 or EFF-1) is indicated (Fig. S3). Anti-VSVG antibody (αG) was used to neutralize any residual VSVΔG-G virus (11) (Fig. S5). Titers are in infectious units (IU) representing the number of cells expressing GFP per microliter 24 hours after virus inoculation. Data are mean ± SE (n=3 experiments). The inset shows background infection. We found no significant difference between infection of BHKAFF-1 and BHK-EFF-1 (Two-tailed paired t test, P=0.5841). (B) Infection of BHKs monitored as GFP expression; phase contrast (top panels), fluorescence (bottom panels). VSVΔG-G served as positive control (Fig. S5). Scale bar is 50 μm.
Fig. 3
Fig. 3
BHK-AFF-1 can fuse with BHK-EFF-1 cells (A) Negative control. Mixed cells co-transfected with empty vector and RFPnes or CFPnls. Scale bar is 100 μm. (B) Red, BHK-AFF-1 and Cyan, BHK-EFF-1 cells were mixed. Hybrids express cyan nuclei and red cytoplasm (Arrows). (C) BHK cell with red cytoplasm surrounding two cyan nuclei appeared following AFF-1-EFF-1 expression and mixing of the cells. Scale bar is 10 μm. (D) Hybrid binucleate cell appeared following AFF-1 expression and mixing of the cells. (E) Quantification of content mixing experiments. Red, cyan and purple-colored pies represent the fraction of multinucleated cells (2 nuclei or higher). Results are mean of four independent experiments (n ≥ 1000 total cells). As indicated: (i) Empty vector transfected cells only. All multinucleated cells were bi-nucleated (red or blue, not purple); total binucleate cells = 4%, probably dividing cells. (ii) AFF-1 expressing cells (red) mixed with empty vector transfected cells (cyan). Elevation in multinucleation was only observed for AFF-1 expressing cells (red, 11%; cyan, 3%). One cell with a single nucleus expressing both markers (red and cyan) was observed. (iii) AFF-1 expressing cells (red) mixed with AFF-1 expressing cells (cyan) resulting in four cell populations – mononucleated white (64%), multinucleated red (13%), cyan (12%) and mixed (purple, 11%). (iv) AFF-1 expressing cells (red, 9%) mixed with EFF-1 expressing cells (cyan, 11%). AFF-1 and EFF-1 expressing cells fuse (purple, 18%).
Fig. 4
Fig. 4
Electron microscopy of VSVΔG-AFF-1 reveals specific bulky surface spikes (A–C) Negative stained particles of (A) VSVΔG; (B) VSVΔG-G; and (C) VSVΔG-AFF-1. (D–E) Anti-AFF-1 polyclonal antibodies followed by immunogold labeling and negative stain of (D) VSVΔG-G and (E) VSVΔG-AFF-1 (Fig. S7 and S8). (F–H) CryoEM of (F) VSVΔG; (G) VSVΔG-G; and (H) VSVΔG-AFF-1. (I–K) Top, center and bottom slice from VSVΔG-AFF-1 tomogram (movie S3). (L–M) Slices from cryoET of vesicles co-purified with VSVΔG-AFF-1 preparations displaying penta- or hexa- meric “flower” shaped assemblies (movie S5). Scale bars are 100 nm and 10 nm for the insets; Arrows: surface spike assemblies; Arrowheads: gold particles; White Square: indicating area shown magnified in inset.

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