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The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

['Ruben M. Markosyan', 'Rush University Medical Center', 'Department Of Physiology', 'Biophysics', 'Chicago', 'Illinois', 'United States Of America', 'Mariana Marin', 'Department Of Pediatrics', 'Emory University']

Date: 2021-09

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins–a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion–the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.

Pathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa GPC-mediated fusion was augmented by the human LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

The molecular mechanism for arenavirus GPC-induced membrane fusion is not well understood. Progress in delineating the mechanism of arenavirus entry/fusion in late endosomes has been impaired by lack of knowledge regarding the precise lumenal pH or composition of intracellular compartments harboring the virus. Here, we use a cell-cell fusion model to examine the pH-, receptor- and lipid-dependence of LASV GPC-mediated fusion and characterize key intermediates of this process. We find that GPC-mediated cell-cell fusion is augmented by human LAMP1 expression and that GPC-LAMP1 binding shifts the pH optimum for fusion to a less acidic pH. We show that, similar to membrane fusion mediated by other viral proteins (e.g., [ 35 – 42 ]), LASV GPC-induced fusion progresses through a hemifusion intermediate and that fusion can be efficiently arrested upstream of hemifusion by incorporation of positive curvature-imposing lipids. Of note, irrespective of human LAMP1 expression, GPC-mediated fusion with the plasma membrane of a target cell appears sub-optimal, as this process tends to get stalled at a dead-end hemifusion intermediate that does not progress to a fusion pore. This finding indicates that LASV fusion may be augmented by a cofactor enriched in late endosomes. Indeed, we find that GPC-mediated membrane fusion is specifically enhanced by a late endosome-resident lipid, bis(monoacylglycero)phosphate (BMP). These findings reveal BMP as a cofactor that drives efficient LASV fusion with late endosomes.

Interestingly, arenaviruses tend to rely on more than one host factor for entry. The Lassa Fever virus (LASV) switches from α-DG to the LAMP1 receptor in acidic endosomes [ 24 – 26 ]. Similarly, a distant OW Lujo virus (LUJV) engages NRP-2 and CD63 at the plasma membrane and in endosomes, respectively [ 27 ]. Efficient entry of the NW Junin virus has been reported to require TfR1 and a voltage-gated calcium channel [ 28 ]. However, some arenaviruses can infect cells lacking the known receptors, albeit less efficiently [ 29 – 32 ]. Although LAMP1 promotes LASV entry/fusion, it is not strictly required for the GPC fusion activity [ 31 – 33 ]. Several lines of evidence indicate that acidic pH alone is sufficient to trigger LASV GPC conformational changes/functional inactivation [ 33 ], including GP1 dissociation from the GP2 [ 26 ] and fusion [ 31 , 33 ] (but see [ 34 ] reporting LASV GPC resistance to acid treatment).

Fusion of arenavirus with host cells is mediated by the GP glycoprotein complex (GPC), a class I viral fusion protein [ 2 , 3 , 12 – 17 ]. Like many viral glycoproteins, arenavirus GPC is synthesized as an inactive GPC precursor that is cleaved at two sites, one by a signal peptidase and the other by SKI-1/S1P protease. This generates a stable signal peptide (SSP) and non-covalently associated GP1 (surface) and GP2 (transmembrane) subunits. A unique feature of the arenavirus GP complex is that the SSP remains associated with the GP2 subunit after GPC cleavage. The ~58-residue long SSP plays critical roles in GPC cleavage and transport to the plasma membrane and controls the initiation of GP conformational changes upon exposure to low pH [ 10 , 18 – 23 ].

Old World (OW) and New World (NW) arenaviruses cause a range of diseases in humans, including severe hemorrhagic fever with high fatality rates of 15–35%. There are currently no FDA-approved vaccines or drugs to battle arenavirus infection. OW and NW arenaviruses infect a wide range of cells types in vitro, owing to their use of the ubiquitously expressed α-dystroglycan (α-DG) and transferrin receptor 1 (TfR1), respectively, for cell attachment and entry (reviewed in [ 1 – 3 ]). Binding of pathogenic Clade B NW arenaviruses to TfR1 initiates entry via clathrin-mediated endocytosis [ 3 – 5 ], whereas α-DG-driven OW arenavirus uptake occurs through a poorly characterized macropinocytosis-like pathway that is independent of clathrin, caveolin, dynamin-2, Rab5 and Rab7 [ 3 , 5 – 9 ]. Regardless of the specific receptor usage, NW and OW arenaviruses are thought to enter cells by undergoing low pH-triggered fusion with multivesicular bodies or late endosomes [ 2 , 6 , 8 , 10 , 11 ].

Results

GPC-LAMP1 interaction allows LASV fusion at higher pH We assessed the pH-dependence of LASV fusion by exposing the effector/HEK293T cell complexes to buffers of different acidity. A pH threshold of LASV GPC-mediated cell fusion was ~6.2, with maximal fusion at pH 4.8 (Fig 3A), in general agreement with previous studies [10,31]. Ectopic expression of LAMP1mut in target cells increased the overall efficiency of fusion without significantly affecting the shape of the curve (Fig 3A). The modest reduction in fusion efficiency at pH<4.8 may be caused by acid-mediated GPC inactivation known to occur at pH ≤ 4.0 in the absence of a target cell (S2B Fig and [26,33]). Parallel experiments with avian QT6 cells lacking human LAMP1 revealed that ectopic expression of human LAMP1mut increased GPC-mediated fusion (Fig 3B). The LAMP1mut effect was the most pronounced at mildly acidic pH, consistent with reports that LAMP1 binding shifts the pH optimum of GPC-mediated fusion to higher values [25,31,33]. A quasi-linear pH-dependence of fusion with both QT6 and QT6/LAMP1mut cells at pH below ~6.0 suggests that fusion is predominantly acid-dependent, but receptor-independent in this pH range. The difference between the pH-dependence of fusion with HEK/293T and QT6 cells is surprising and might indicate that cell type- or species-specific factors other than LAMP1 may promote LASV GPC refolding at acidic pH. PPT PowerPoint slide

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TIFF original image Download: Fig 3. pH-dependence of LASV GPC-mediated cell-cell fusion. (A) Fusion between COS7 cells transfected with LASV GPC and HEK293T cells mock-transfected or transfected with LAMP1mut was triggered by exposure to buffers of different acidity for 20 min at 37°C (optimal trigger). The results are means and SEM from three independent experiments. (B) pH-dependence of fusion, using the protocol described in panel A, was measured between LASV GPC-expressing COS7 cells and plain QT6 cells or QT6 cells transfected with LAMP1mut. The results are means and SEM from three independent experiments. *, p<0.05; **, p<0.01, ***, p<0.001. https://doi.org/10.1371/journal.ppat.1009488.g003

LASV fusion is reversibly arrested by positive-curvature lipids Diverse membrane fusion reactions can be blocked by exogenous lyso-lipids that confer positive curvature to the contacting membrane leaflets and thereby disfavor the formation of a net negative curvature stalk structure (Fig 4A and [50–52]). This lipid-arrested stage (LAS) is largely reversible upon washing away lysolipids and commences at neutral pH [44,53], implying that the viral proteins are arrested in a “committed” stage that is downstream of low pH-dependent steps. Like other protein-mediated fusion reactions, LASV GPC-mediated fusion was blocked when lyso-PC was present before and during a low pH pulse, but fusion ensued upon removal (at neutral pH) of this lipid (Fig 4B). This result implies that LASV fusion progresses through curved lipid intermediates of net negative curvature, likely a stalk and a hemifusion diaphragm. PPT PowerPoint slide

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TIFF original image Download: Fig 4. LASV GPC-mediated fusion progresses through a hemifusion intermediate that is blocked by lyso-lipids. (A) Illustration of cold-arrested (CAS, hemifusion) and lyso-lipid-arrested (LAS, pre-hemifusion) intermediates and the effect of chlorpromazine (CPZ). (B) Lipid-arrested stage of LASV fusion. GPC-expressing COS7 cells were bound to QT6 cells transfected with LAMP1mut in the presence of 285 μM stearoyl lyso-PC for 30 min at room temperature, followed by exposure to pH 5.0 at room temperature in the presence of lyso-PC. Cells were either washed with delipidated BSA (2 mg/ml) to remove lyso-PC (right bar) or left with lyso-PC (middle bar) and incubated for 30 min at 37°C. The results are means and SEM from three independent experiments. (C) The effect of CPZ on fusion between GPC-expressing COS7 cells and HEK293T cells mock-transfected or transfected with LAMP1 or LAMP1mut. Cell fusion was triggered under sub-optimal conditions (pH 6.2, 10 min at room temperature). After an additional 1 h incubation at 37°C, cells were treated with CPZ (0.5 mM) for 1 min or left untreated. The results are means and SEM from three independent experiments. (D) CPZ enhances LASV and Junin GPC-mediated cell fusion. Fusion between LASV or Junin GPC-expressing COS7 cells and HEK293T cells was triggered by exposure to pH 6.2 (for LASV) or pH 5.5 (JUNV). After a 20 min incubation at 37°C, cells were treated for 1 min with 0.5 mM CPZ at room temperature (n = 3). (E) Cold-arrested intermediate (CAS) of LASV GPC fusion. GPC-expressing COS7 cells were incubated with HEK293T cells (transfected or not with LAMP1 or LAMP1mut) for 30 min at room temperature and exposed to pH 6.2 on ice for 10 min. Cells were either immediately treated with cold 0.5 mM CPZ for 1 min or additionally incubated for 1 h at 37°C, neutral pH. The results are means and SEM from three independent experiments. (F) ST-193 captures LASV fusion at a hemifusion intermediate. COS7 cells expressing LASV GPC were mixed with HEK293T cells, attached to glass slides and allowed to adhere and form contacts for 30 min at room temperature. Fusion was initiated by exposure to pH 5.0 for 15 min at 37°C, in the presence or absence of 150 μM ST-193. Cells were either washed to remove the inhibitor or kept in the presence of ST-193, and either immediately treated with 0.5 mM CPZ for 1 min or further incubated at neutral pH for 1 h, 37°C. The results are means and SEM from three independent experiments. https://doi.org/10.1371/journal.ppat.1009488.g004

LASV fusion progresses through a hemifusion intermediate that can be arrested at low temperature Membrane fusion mediated by diverse viral glycoproteins progresses through a common hemifusion intermediate (reviewed in [54–56]) (Fig 4A). This intermediate is formed through merger of the contacting leaflets of two membranes, while distal leaflets form a new bilayer referred to as hemifusion diaphragm [57,58] (Fig 4A). It has been shown that low pH-triggered viral fusion can be arrested at a local hemifusion stage in the cold (referred to as cold-arrested stage, CAS) [46,53,59–61] (Fig 4A). After creating CAS, fusion can be recovered at neutral pH by raising temperature. In other words, steps of fusion downstream of CAS are temperature-dependent but no longer require low pH. The formation of a hemifusion intermediate, including the one formed at CAS, can be indirectly inferred by treating cells with chlorpromazine (CPZ) at neutral pH, which selectively destabilizes a hemifusion diaphragm and promotes full fusion [57] (Fig 4A). We first asked if LASV GPC-mediated fusion exhibits a natural tendency to get stuck at a hemifusion stage, as is the case for membrane fusion mediated by other low pH-dependent viruses, especially when suboptimal triggers for fusion (insufficiently low pH and/or reduced temperature) are employed [44,53,61]. We found that GPC-mediated fusion triggered under suboptimal conditions (pH 6.2, room temperature) was markedly enhanced by a brief exposure to CPZ after returning to neutral pH (Fig 4C). This CPZ-mediated fusion enhancement was much more pronounced for plain HEK293T cells (>6-fold), as compared to cells expressing wild-type or mutant LAMP1 (~2-fold, Fig 4C). Notably, over-expression of LAMP1 markedly decreased the fraction of dead-end hemifusion structures that did not naturally progress to full fusion. Higher levels of ectopically expressed LAMP1 on the cell surface did not considerably increase the overall efficiency of formation of hemifusion sites (i.e., CPZ-sensitive lipid intermediates), but markedly enhanced the probability of transition to full fusion at 37°C. A similar dramatic increase in cell-cell fusion was observed after treatment of Junin virus GPC-mediated cell fusion products with CPZ, whereas this treatment was without effect in control experiments using cells expressing a fusion-incompetent GPC mutant [20] (Fig 4D). Thus, under suboptimal conditions, GPC tends to create hemifusion structures that do not resolve into full fusion, and the formation of such dead-end intermediates is independent of the LAMP1 expression level; these dead-end hemifusion structures can be converted to full fusion by CPZ treatment. Next, we tested the possibility of capturing LASV fusion at a cold-arrested stage (CAS, Fig 4A) by exposing effector/target cell pairs to low pH in the cold. A reversible arrest of GPC-mediated cell-cell fusion at CAS was evident by the commencement of fusion after shifting to 37°C at neutral pH (Fig 4E). As observed for uninterrupted LASV GPC-mediated cell-cell fusion (Fig 2), ectopic expression of LAMP1 or LAMP1mut markedly enhanced the extent of fusion after shifting cells captured at CAS to 37°C (Fig 4E). Importantly, CPZ treatment of cells arrested at CAS (after returning to neutral pH, still at low temperature) promoted efficient fusion (Fig 4E), consistent with the formation of local hemifusion structures at CAS. In control experiments, CPZ treatment did not promote fusion of effector/target cell pairs that were not exposed to low pH or cells expressing a fusion-defective GPC mutant after exposure to low pH (e.g., Fig 4D). CPZ treatment induced more fusion between cells captured at CAS than a shift to 37°C and this difference was more apparent for fusion with suboptimal target cells (Fig 4E). These results show that GPC-LAMP1 interactions increase the probability of conversion of hemifusion to full fusion.

Arenavirus fusion inhibitor captures LASV GPC-induced fusion at a hemifusion stage The small-molecule arenavirus fusion inhibitor ST-193 has been shown to block pH-induced conformational changes in GPC, including the shedding of the GP1 subunit, but the fusion block can be overcome at sufficiently acidic pH [62]. ST-193 is thought to act by binding to the interface between the GP2 subunit transmembrane domain and the stable signal peptide [62], thereby disfavoring the early steps of GPC refolding at low pH. Since, the compound appears to interfere with pH-induced GPC refolding, we asked whether ST-193 blocks LASV fusion at early steps prior to membrane merger. Effector-target cell complexes were exposed to pH 5.0 at 37°C in the presence of a high concentration of ST-193 that almost completely inhibits cell-cell fusion (Figs 4F and S2A). Subsequent removal of the inhibitor at neutral pH resulted in only a partial recovery of fusion (Fig 4F), indicating that a large fraction of GPC proteins underwent irreversible conformational changes and inactivated at low pH in the presence of ST-193. Importantly, CPZ application immediately after removal of ST-193 and in control samples not exposed to ST-193 induced efficient fusion that exceeded the level of uninterrupted fusion at 37°C (Fig 4F). These findings show that, in the presence of ST-193, GPC undergoes low pH-dependent conformational changes that promote dead-end hemifusion. Similar levels of CPZ-mediated fusion for control and ST-193 treated samples (Fig 4F) suggest that the initial acid-dependent steps of GPC-mediated membrane fusion are not noticeably affected by this inhibitor when cells are subjected to an optimal trigger. Dead-end hemifusion structures formed in the presence of ST-193 are effectively converted to full fusion by CPZ treatment, while less than 30% of hemifusion structures naturally resolve into full fusion at 37°C (Fig 4F).

Late steps of LASV GPC-mediated fusion are enhanced by a late endosome-resident lipid LASV is thought to fuse with multivesicular bodies/late endosomes [2,3,6,8]. However, LASV GPC can mediate fusion with LAMP1-expressing cells at mildly acidic pH typical for early endosomes (Fig 3 and [31–33]). We therefore asked if LASV entry from early endosomes may be delayed due to the need for an additional host factor localized to late endosomes. The unusual anionic lipid, bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is greatly enriched in late endosomes (around 15% of the total lipids [63,64]). It has been reported that preincubation of cells with anti-BMP antibodies diminishes LASV infection [6]. We have now found that preincubation with anti-BMP antibody modestly inhibited LASVpp fusion and, to a lesser degree, IAVpp fusion, whereas pretreatment with control isotype antibodies was without effect (S3 Fig). The modest effect on IAVpp fusion may be related to disruption of biogenesis of multivesicular bodies and/or of cholesterol transport [65–68]: prolonged incubation with anti-BMP antibodies is known to disrupt several essential cellular processes, including cholesterol transport and biogenesis of multivesicular bodies [65–68]. These pleotropic effects may indirectly disfavor LASV fusion by disrupting virus uptake and/or transport to permissive intracellular compartments. In this regard, cell-cell fusion appears well-suited for exploring lipid-dependence of LASV GPC-mediated fusion. To directly investigate the effect of lipids on LASV fusion, the effector/target cell complexes were pretreated with BMP or zwitterionic lipid DOPC (control) prior to exposure to low pH. We observed specific promotion of LASV GPC-mediated cell fusion by exogenous BMP, but not by DOPC (Fig 5A). This enhancing effect of BMP was observed across the range of LAMP1 expression, using plain HEK293T cells and cells transfected with LAMP1 or LAMP1mut (Fig 5A). Also, BMP, but not another anionic lipid, DOPS, markedly enhanced GPC-mediated fusion with suboptimal QT6 cells (Fig 5B). This finding shows that non-specific electrostatic interactions are not responsible for the observed enhancement of LASV fusion by BMP. BMP also enhanced the Junin virus GPC-mediated cell fusion by ~4-fold, whereas DOPC and DOPS were without effect (Fig 5C). In contrast, influenza HA-mediated fusion was not affected by either BMP or DOPC but was modestly promoted by DOPS. In agreement with the published work on the lipid-dependence of Vesicular Stomatitis Virus (VSV) fusion [69], VSV G protein-mediated cell fusion was modestly augmented by BMP, but not by DOPC or DOPS treatment (Fig 5C). Note that failure of exogenous DOPS to promote cell-cell fusion mediated by GPC was not due to the lack of incorporation into the plasma membrane, as revealed by staining cells with Annexin V (S4A Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Lipid-dependence of LASV GPC-mediated cell fusion. (A) COS7 cells transiently expressing LASV GPC were co-incubated with mock-transfected or LAMP1 or LAMP1mut transected 293T cells in the presence of 10 μg/ml DOPC or BMP dissolved in BSA (1 mg/ml) for 20 min at room temperature. Cell fusion was triggered by exposure to pH 6.2 at room temperature in the absence of exogenous lipids and was measured after an additional incubation for 1 h at 37°C. The results are means and SEM from three independent experiments. (B) GPC-expressing COS7 cells were incubated with QT6 cells in the presence of 10 μg/ml DOPC, DOPS or BMP dissolved in BSA (1 mg/ml) for 20 min at room temperature. Cell fusion was triggered by exposure to pH 5.0 at 37°C for 20 min in the absence of exogenous lipids. The results are means and SEM from three independent experiments. (C) COS7 cells expressing LASV or Junin Virus GPC or IAV HA were brought in contact with HEK293T cells, incubated with 10 μg/ml DOPC, DOPS or BMP dissolved in BSA for 20 min at room temperature and exposed to pH 5.0 for 20 min at 37°C. The results are means and SEM from four independent experiments. (D) BMP facilitates transition from hemifusion to fusion. COS7 cells expressing LASV GPC were incubated with HEK293T cells in the presence of 10 μg/ml DOPC or BMP dissolved in BSA (1 mg/ml) for 20 min at room temperature. Cell fusion was initiated by exposure to pH 6.2 for 10 min at room temperature in the absence of lipids followed by incubation at 37°C for 1 h. followed by a brief (1 min) exposure to 0.5 mM CPZ or to PBS (control). The results are means and SEM from three independent experiments. (E) Lipid-dependence of transition from cold-arrested stage (CAS) to full fusion. Fusion between LASV GPC-expressing COS7 cells and QT6 cells was initiated by exposure to pH 5.0 for 10 min at 4°C followed by treatment with 10 μg/ml of indicated lipids in BSA for 10 min at room temperature, at neutral pH, and an additional 30 min-incubation at 37°C. https://doi.org/10.1371/journal.ppat.1009488.g005

BMP promotes the formation and growth of GPC-mediated fusion pores To further delineate the role of BMP in GPC-mediated fusion, we asked whether its effect is dependent on incorporation of this lipid into a target vs. GPC-expressing cells. Effector or target cells detached from culture dishes were separately treated with DOPC or BMP, mixed and exposed to low pH. In control experiments, a mixture of effector and target cells was pretreated with the indicated lipids. As shown in S4B Fig, addition of BMP increased the efficiency of GPC-mediated cell fusion by ~2.5-fold compared to DOPC control, irrespective of whether BMP was incorporated into effector or target cells. Promotion of LASV fusion upon BMP incorporation into effector cells is expected if it affects late, post-hemifusion steps of fusion; merged contacting leaflets of two membranes will allow BMP redistribution from one membrane to another at the potential fusion site. To determine whether BMP augments late stages of GPC-mediated fusion downstream of pH-independent steps, we employed two strategies. First, the products of GPC-mediated fusion of cells pretreated with BMP or DOPC or left untreated (negative controls) were briefly exposed to CPZ to fully fuse cells arrested at hemifusion. The increase in fusion was dramatic for untreated or DOPC-pretreated cells, but modest for BMP-pretreated cells which had largely fused prior to addition of CPZ (Fig 5D). The extent of fusion after CPZ addition was independent of pretreatment. These results suggest that the efficiency of formation of CPZ-sensitive hemifusion structures is independent of BMP, whereas the probability of conversion of these intermediates to full fusion is dramatically and specifically increased by BMP. The second strategy was to capture cell-cell fusion at CAS, add various lipids, at neutral pH, and then raise temperature. Fusion was potently promoted by the addition of BMP at CAS, but not by the addition of DOPC or DOPS (Fig 5E). Finally, we examined the effect of exogenously added BMP on the size of GPC-mediated fusion pores and their propensity to enlarge. Toward this goal, we performed time-lapse imaging of the redistribution of a small cytoplasmic marker, calcein, between effector and target cells (Fig 6A and 6B) and deduced the pore permeability from the rate of dye redistribution, as described previously [70,71]. Interestingly, GPC-mediated redistribution of calcein between the dye donor and acceptor cell pairs was much slower than for cell-cell fusion mediated by other viral glycoproteins [43,70]). Moreover, calcein redistribution often stalled after a few minutes so that the dye did not fully equilibrate between the two cells (Fig 6A and 6B). This result suggests that any GPC-mediated fusion pores that do form remain small under our experimental conditions and even tend to close. The average pore permeability profile confirmed the failure of GPC-mediated fusion pores to grow (Fig 6E). In stark contrast, fusion pores formed between BMP-pretreated cells grew efficiently, as evidenced by a quick and complete redistribution of calcein (Fig 6C, 6D and 6E). Control experiments showed no significant effect of DOPC on the initial size or enlargement of GPC-mediated fusion pores (Fig 6E). We also measured the kinetics of fusion pore formation based on the onset of calcein redistribution and found that BMP pretreatment markedly accelerated the rate of cell-cell fusion (Fig 6F). Collectively, the above results show that BMP selectively enhances post-hemifusion steps of LASV fusion, including the formation and dilation of fusion pores. PPT PowerPoint slide

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TIFF original image Download: Fig 6. BMP markedly promotes the formation and enlargement of LASV GPC-mediated fusion pores. Effector COS7 cells expressing GPC were loaded with calcein (green), mixed with unlabeled 293T cells and adhered to poly-lysine coated coverslips. Cells were then pre-treated with 20 μg/ml of BMP or DOPC for 10 min at room temperature or left untreated. Cell-cell fusion was triggered by transferring cells to a pH 5.0 buffer and quickly raising the temperature to 37°C through an IR temperature jump protocol (see Materials and Methods). (A, B) Snapshots and fluorescence intensities showing partial calcein redistribution between untreated effector and target (dye donor and acceptor) cells. (C, D) Same as in panels A, B, but for cells pretreated with BMP. (E) Ensemble average of permeabilities of six fusion pores for untreated and DOPC- or BMP-treated cells. Individual pore permeability traces calculated as described in Materials and Methods were aligned so that t = 0 represents a time point immediately before dye redistribution was detected. Inset: Initial permeability profiles of fusion pores. Error bars are SEM. (F) Kinetics of fusion pore formation for control and BMP-treated cells. The data points represent time after raising the temperature until the onset of calcein transfer. https://doi.org/10.1371/journal.ppat.1009488.g006

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