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Human LAMP1 accelerates Lassa virus fusion and potently promotes fusion pore dilation upon forcing viral fusion with non-endosomal membrane [1]

['You Zhang', 'Department Of Pediatrics', 'Division Of Infectious Diseases Emory University School Of Medicine', 'Atlanta', 'Georgia', 'United States Of America', 'Juan Carlos De La Torre', 'Department Of Immunology', 'Microbiology', 'The Scripps Research Institute']

Date: 2022-11 

Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.

Lassa virus (LASV) enters cells via fusion with acidic endosomes mediated by the viral glycoprotein complex (GPC) interaction with the intracellular receptor LAMP1. However, the requirement for LAMP1 is not absolute, as LASV can infect avian cells expressing a LAMP1 ortholog that does not interact with GPC. To delineate the role of LAMP1 in LASV entry, we developed assays to monitor the formation of nascent fusion pores, as well as their initial and complete dilation to sizes that allow productive infection of avian cells by LASV GPC pseudoviruses. This novel approach provided unprecedented details regarding the dynamics of LASV fusion pores and revealed that ectopic expression of human LAMP1 in avian cells leads to a marked acceleration of fusion but modestly increases the likelihood of complete pore dilation and infection. In contrast, human LAMP1 expression dramatically enhanced the propensity of nascent pores to fully enlarge when LASV fusion with the plasma membrane was forced by exposure to low pH. Thus, whereas the role of LAMP1 in LASV fusion is confounded by an interplay between multiple endosomal factors, the plasma membrane is a suitable target for mechanistic dissection of the roles of host factors in LASV entry.

Copyright: © 2022 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we employed a combination of single LASV pseudovirus (LASVpp) tracking in live cells alongside bulk virus-cell fusion and infectivity assays to assess the effect of hLAMP1 on distinct steps of viral fusion. Pseudoviruses co-labeled with viral content marker and an internal pH sensor enable detection of single virus fusion events and monitoring the initial enlargement of fusion pores, whereas bulk fusion/infectivity assay report functional enlargement of fusion pores. We find that hLAMP1 overexpression in both human and avian cell lines moderately promotes fusion and infection through an endocytic entry pathway, whereas LASV GPC-mediated fusion at the cell surface forced by exposure to low pH was dramatically enhanced and accelerated in hLAMP1-expressing cells. Real-time imaging of forced LASVpp fusion with avian cells revealed a strong enhancement in fusion pore dilation by upon hLAMP1 expression. Our results thus provide new insights into the role of hLAMP1 in early and late stages of LASV fusion and reveal key differences in permissiveness of endosomes and the plasma membrane for LASV fusion.

Our recent study identified a novel co-factor required for completion of LASV fusion–the late-endosome-resident lipid, bis(monoacylglycero)phosphate (BMP) [ 12 ]. BMP specifically and potently promotes the late stages of LASV GPC-mediated fusion–formation and enlargement of fusion pores. Whereas hLAMP1 binding clearly augments low pH-dependent refolding of LASV GPC, whether this intracellular receptor also modulates late steps of virus fusion remains unclear. Using a cell-cell fusion model, we have shown that hLAMP1 overexpression facilitates transition from hemifusion (merger of contacting membrane leaflets without fusion pore formation) to full fusion [ 12 ]. However, the role of hLAMP1 in controlling distinct steps of virus-endosome fusion has not been elucidated.

Recent studies by others and our group have shown that hLAMP1, while promoting LASV fusion and infection, is not absolutely required for virus entry, since cells lacking human LAMP1 support basal levels of LASV fusion/infection [ 10 – 12 ]. The ability of LASV GPC to mediate membrane fusion in the absence of hLAMP1 is consistent with the reports that sufficiently acidic pH induces irreversible GPC conformational changes leading to shedding of the GP1 subunit [ 9 , 12 , 13 ]. Mechanistic studies revealed that hLAMP1 binding shifts the pH-optimum for GPC-mediated fusion to a higher pH [ 10 , 12 ]. Thus, LASV fusion with hLAMP1 expressing cells is likely initiated in less acidic maturing endosomal compartments, prior to virus delivery into late endosomes/lysosomes.

LASV is an Old World mammarenavirus that infects a broad host range of cells from different species. LASV cell entry is mediated by the viral surface glycoprotein complex (GPC), a trimeric class I fusion protein that consists of non-covalently associated surface (GP1) and transmembrane (GP2) subunits (reviewed in [ 1 , 2 ]). The GP1 and GP2 subunits are generated through cleavage of the GPC precursor by the host cell protease subtilisin kexin isozyme-1(SKI-1)/site 1 protease (S1P). GP1 is involved in receptor binding and GP2 in membrane fusion. A unique feature of arenavirus GPC proteins is that their stable signal peptide (SSP), which is cleaved off the GP precursor, remains associated with the GPC and plays an important regulatory role in low pH-induced conformational changes in GPC that lead to fusion of the viral and cell membranes [ 3 – 8 ]. LASV GPC attachment to the alpha-dystroglycan receptor on the cell surface leads to virus internalization and transport to acidic endosomes where low pH promotes virus dissociation from alpha-dystroglycan and attachment to the intracellular receptor, LAMP1. LAMP1, a marker for late endosomes/lysosomes, has been shown to serve as a specific receptor for LASV and not for other mammarenaviruses, such as lymphocytic choriomeningitis virus (LCMV) [ 9 ]. Human LAMP1 (hLAMP1), but not human LAMP2 or avian LAMP1, promotes LASV fusion with late endosomes [ 9 ].

Results

hLAMP1 expression dramatically promotes forced LASV pseudovirus fusion with the plasma membrane To probe the effects of hLAMP1 on LASVpp fusion with membranes that do not contain significant amounts of this intracellular receptor or other endosomal factors that may facilitate LASV entry and to ensure a more tractable system for viral fusion, we bypassed the internalization and endosomal trafficking steps by forcing LASVpp fusion with the plasma membrane through exposure to low pH. Ectopic expression of hLAMP1 dramatically enhanced the forced LASVpp fusion with both A549 and DF-1 cells, as measured by the BlaM assay (Figs 2C and S2B). Expression of LAMP1-WT or LAMP1-d384 in DF-1 cells caused a somewhat less dramatic (~30-fold) increase in LASVpp fusion compared to A549 cells in which ~120-160-fold increase in signal was detected. The much stronger effect of hLAMP1 expression on forced LASVpp fusion with A549 compared to DF-1 cells (Fig 2C) was somewhat unexpected, given that the former cells express endogenous hLAMP1, small amounts of which may be present at the cell surface. The striking increase in the efficiency of forced LASVpp fusion is in agreement with the marked enhancement of LASV GPC-mediated cell-cell fusion upon ectopic expression of hLAMP1 in DF-1 cells [12]. Consistent with the higher expression of the mutant LAMP1 on the cell surface (Fig 1), forced fusion with both A549 and DF-1 cells expressing LAMP1-d384 was significantly more efficient than fusion with cells expressing LAMP1-WT (Figs 2C and S2B). Thus, ectopic hLAMP1 expression greatly enhances the otherwise sub-optimal low pH-mediated LASVpp fusion with the plasma membrane, in contrast to fusion with endosomes which is less affected by hLAMP1 overexpression. Akin to the effect of hLAMP1 overexpression on the forced viral fusion (Fig 2C), forced LASVpp infection was also potently enhanced by ectopic expression of this receptor in both cell types (Figs 2E and S2D). hLAMP1 expression had a commensurate effect on viral fusion and infection in DF-1 cells, whereas an increase in fusion efficiency was much more pronounced compared to infection in A549 cells (Fig 2C and 2E). A discordance between fold-enhancement of forced fusion vs infection upon hLAMP1 expression in A549 cells might be due to less efficient post-fusion steps of infection following LASVpp entry at the plasma membrane compared to entry from endosomes in these but not DF-1 cells. Given the strikingly potent enhancing effect of ectopic hLAMP1 expression on the forced LASVpp fusion, we sought to determine if such enhancement may be caused by a global, non-specific effect of hLAMP1 on the plasma membrane. Toward this goal, we measured the fusion of control pseudoviruses bearing the unrelated VSV G protein (VSVpp) with the parental and hLAMP1 expressing cells. As expected, ectopic expression of hLAMP1 did not considerably enhance VSVpp fusion through an endosomal or forced pathway (S3A and S3B Fig). This finding rules out a non-specific effect of hLAMP1 on VSV G-mediate virus fusion with endosomes or the plasma membrane.

Single LASV pseudovirus fusion proceeds through a viral membrane permeabilization step, irrespective of cell type or hLAMP1 expression levels We next sought to determine which steps of viral fusion are facilitated by hLAMP1 expression by single virus tracking. LASVpp were labeled with the mCherry-2xCL-YFP-Vpr construct, which is incorporated into HIV-1 pseudoviruses and is cleaved by the viral protease upon virus maturation, producing free mCherry and YFP-Vpr [19]. Loss of mCherry signal from the viral particle entering a cell reflects mCherry release into the cytoplasm through a fusion pore, whereas YFP-Vpr is retained in the viral core for a considerable time and thus serves as a reference marker for reliable detection of single fusion events. Also, importantly, the pH-sensitive YFP fluorescence is quenched at low pH [20], thereby reporting changes in intraviral pH. Using this marker, we have previously shown that LASVpp fusion with endosomes is preceded by a drop in intraviral pH due to permeabilization of the viral membrane in acidic endosomes [20]. This loss of a barrier function of the viral membrane prior to fusion is manifested in YFP fluorescence (green) quenching, whereas subsequent virus fusion results in simultaneous loss of mCherry (red) and recovery of YFP signal due to re-neutralization of the viral interior connected to the cytoplasm through a fusion pore (Fig 4A). We refer to fusion events preceded by viral membrane permeabilization as type II fusion, whereas fusion occurring without a prior loss of the viral membrane barrier function are termed type I fusion events [20]. Our pilot results suggest that viral membrane permeabilization in type II fusion events is caused by conformational changes in LASV GPC and requires virus-cell contact prior to exposure to low pH. The above double labeling of pseudoviruses with the content marker, mCherry, and the viral core-associated pH sensor, YFP-Vpr, enables sensitive detection of nascent proton-permeable fusion pores, as well as their initial dilation that allows mCherry escape into the cytoplasm. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Single LASVpp fusion with DF-1 cells. (A) Illustration of fusion of mCherry-2xCL-YFP-Vpr labeled single LASVpp in an acidic endosome. An increase in the virus membrane permeability leads to quenching of the intraviral YFP signal (green) in acidic environment. Subsequent virus fusion with the endosomal membrane results in a loss of mCherry signal (red) through a fusion pore and concomitant re-neutralization of virus interior, as evidenced by YFP signal dequenching. (B) LASVpp fusion events (YFP dequenching) with instant mCherry release (quick fusion pore dilation). Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with DF-1-LAMP1-WT cell showing YFP quenching at 31.3 min and YFP dequenching/mCherry loss at 34.7 min corresponding to virus interior acidification and fusion, respectively (see S1 Movie). (C) LASVpp fusion events with delayed mCherry release relative to YFP dequenching. Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with a DF-1-LAMP1-WT cell showing YFP quenching at 39.7 min, YFP dequenching at 42.7 min and mCherry loss at 43.2 min (arrows), indicating virus interior acidification, small fusion pore formation and fusion pore dilation to a diameter exceeding the size of mCherry, respectively (see S2 Movie). (D) LASVpp fusion events (YFP dequenching) without mCherry release. Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with a DF-1-LAMP1-WT cell showing YFP quenching at 42.6 min and dequenching at 47.4 min without mCherry loss (see S3 Movie). https://doi.org/10.1371/journal.ppat.1010625.g004 Using the aforementioned virus labeling strategy, we imaged single LASVpp fusion with DF-1 endosomes of cells expressing or lacking hLAMP1. LASVpp undergoes type II fusion with A549 cells ([20]). We also found all type II fusion events with control DF-1 cells (S5 Fig). DF-1 cells were chosen over A549 cells for the subsequent imaging experiments for the following reasons: (1) low permissiveness to LASV fusion/infection in the absence of ectopically expressed hLAMP1; (2) consistent effects of hLAMP1 expression on fusion and infection across different virus platforms (LASVpp, LASV-VLP and LCMV-LASV GPC) (Figs 2 and 3 and S5 Fig); and (3) tolerance to prolonged exposure to low pH, which is essential for the 1 hour-long forced fusion imaging experiments described below. All single LASVpp fusion events in DF-1 cells were of type II phenotype, regardless of the hLAMP1 expression (Figs 4B and 4C and S5). Interestingly, a fraction of single LASVpp fusion events exhibited a delayed mCherry release relative to YFP dequenching, which marks the opening of a nascent fusion pore (Fig 4C). This lag in mCherry release ranged from several seconds to minutes (see below) and was most likely caused by delayed enlargement of nascent fusion pores to sizes (~4 nm) that allowed mCherry release. Thus, YFP dequenching in the context of type II virus-endosome fusion provides a highly sensitive means to detect very small fusion pores, whereas mCherry release is contingent on pore enlargement to a diameter exceeding ~4 nm. In addition to type II fusion events culminating in mCherry loss, we also observed YFP quenching and subsequent dequenching without mCherry release for as long as we tracked viral particles (Fig 4D). The lack of mCherry release could be due to a failure of nascent fusion pores to enlarge and allow mCherry release or due to a full fusion of immature viral particles in which the mCherry marker was not cleaved off the Vpr-based core marker by the HIV-1 protease [19]. Indeed, almost 20% of particles contained uncleaved mCherry-YFP-Vpr construct, as judged by the lack of mCherry release upon saponin lysis in vitro (S6 Fig). However, assuming that HIV-1 maturation does not affect GPC-mediated fusion, the fraction of “no-release” events should be constant across conditions. So, the greater fraction of “instantly” dilating pores, but not pores that failed to release mCherry upon ectopic hLAMP1 expression (see below), argues against the possibility that all “no-release” events correspond to fusion of immature particles. Another reason for YFP dequenching without mCherry release could be virus recycling to the cell surface. However, events that did not culminate in mCherry release were also observed upon low pH-forced virus fusion with the plasma membrane where low pH was maintained throughout the experiment (see below), suggesting that virus cycling to the cell surface is less likely to be responsible for YFP dequenching. We therefore conditionally refer to YFP dequenching without mCherry release as stalled fusion. Of note, all three types of fusion pores–quickly and slowly dilating and those that fail to release mCherry–were observed in A549 cells (S7A–S7D Fig). Regardless of the timing of mCherry release relative to YFP dequenching, LASVpp fusion was associated with a mixed diffusive and directional motion pattern (Fig 4, right graphs), which is typical for endosomal trafficking of internalized cargo and viruses (e.g., [21]).

hLAMP1 overexpression accelerates single LASV pseudovirus fusion with endosomes without affecting dilation of fusion pores Analysis of the kinetics of nascent fusion pore formation (YFP dequenching) revealed a ~2-fold increase in the fusion rate with DF-1 cells expressing LAMP1-WT or LAMP1-d384 compared to control cells (Fig 5A). The faster kinetic of single LASVpp fusion with hLAMP1-expressing DF-1 cells was not caused by more efficient or faster virus endocytosis and delivery into acidic endosomes. Neither the fraction of particles exhibiting YFP quenching (i.e., virus interior acidification in acidic endosomes irrespective of subsequent fusion) nor the kinetics of YFP quenching were dependent of hLAMP1 expression (S8A and S8B Fig). Notably, only ~15% of cell-bound particles exhibited YFP quenching in acidic endosomes of DF-1 cells expressing or lacking hLAMP1, suggesting that LASVpp uptake was slow/inefficient. Of note, the kinetics of nascent LASVpp pore formation in parental A549 cells was much faster than in DF-1 cells and as fast as the accelerated kinetics of hLAMP1 expressing DF-1 cells (compare Figs 5A to S7E), highlighting the cell type-dependence of the multi-step LASVpp entry process. Importantly, hLAMP1 expression markedly shortened the lag between virus interior acidification (YFP quenching) and small pore formation (YFP dequenching) from ~18 min to ~8 min, on average (Fig 5B). The larger lag to small fusion pore formation after virus entry into acidic compartments may thus be responsible for the slower LASVpp fusion in cells lacking hLAMP1 (Fig 5A). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Human LAMP1 expression promotes single LASVpp fusion with DF-1 cells. (A) Efficiencies of single LASVpp fusion with instant mCherry release, delayed mCherry release, and without mCherry release in pQCXIP, LAMP1-WT and LAMP1-d384 DF-1 cells. Data shown are means ± SD of 5 independent experiments. Asterisks inside the bars represent significance relative to the vector control. Differences between LAMP1-WT and LAMP1-d384 are not statistically significant for all three categories of fusion. Inset: normalized fractions of each category of fusion. (B) The distribution of lag times between small fusion pore formation (YFP dequenching) and pore enlargement (loss of mCherry) for LASVpp fusion with DF-1 pQCXIP, LAMP1-WT and LAMP1-d384 cells. (C) Kinetics of small pore formation (YFP dequenching) for single LASVpp fusion events in control and hLAMP1 expressing DF-1 cells. (D) The distribution of lag times between LASVpp membrane permeabilization (YFP quenching) and small fusion pore formation (YFP dequenching) for LASVpp fusion with DF-1 pQCXIP, LAMP1-WT and LAMP1-d384 cells. Normalized fractions of different single virus fusion events were analyzed by Fisher’s exact test using R Project. Data of lag time between YFP dequenching and mCherry release was analyzed by non-parametric Mann-Whitney test using GraphPad. Other results were analyzed by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; NS, not significant. https://doi.org/10.1371/journal.ppat.1010625.g005 In DF-1 cells, we observed three single LASVpp fusion phenotypes–instant and delayed pore enlargement and stalled fusion,–irrespective of ectopic hLAMP1 expression (Fig 5C). All three types of single fusion events were promoted ~4-6-fold upon hLAMP1 expression, in general agreement with the bulk LASVpp fusion results (Fig 2B). It should be stressed, however, that hLAMP1 expression did not significantly alter the relative weights of different types of fusion events, including the “instantly” dilating pores (Fig 5C, Inset). Another approach to evaluate the effect of hLAMP1 on the propensity of fusion pores to enlarge is to assess the time required for nascent pore (detected by YFP dequenching) to dilate to sizes that allow mCherry release. “Instant” pore enlargement was defined as simultaneous (within our temporal resolution of 6 sec) YFP dequenching and mCherry loss. The lag time to mCherry release was not significantly affected by hLAMP1 expression (Fig 5D), in excellent agreement with the finding that hLAMP1 did not significantly increase the fraction of “instant” mCherry release events (Fig 5C, Inset). These results show that ectopic expression of hLAMP1 does not noticeably promote the initial enlargement of fusion pores formed between LASVpp and endosomes.

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