Fig 1.
Arenavirus membrane permeability increases prior to virus fusion.
(A) Illustration of mCherry-CL-YFP-Vpr labeled single LASVpp fusion. LASVpp is internalized and trafficked to acidic endosomes where the viral membrane is permeabilized. Increases in viral membrane permeability lead to acidification of the virus’ interior which is manifested in YFP signal quenching. LASVpp-endosome fusion results in mCherry release into the cytoplasm and concomitant re-neutralization of the virus’ interior, seen as recovery of YFP signal. (B) Single LASVpp fusion with A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 12.3 min and fusion (YFP dequenching and mCherry loss) at 25.9 min (see S1 Movie). (C) A single MACVpp fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 7.0 min and fusion (YFP dequenching and mCherry loss) at 29.0 min. (D) HIV-1 particles labeled with mCherry-CL-YFP-Vpr and pseudotyped with LASV, MACV GPc or IAV HA were attached to cells by spinoculation in the cold, and their entry/fusion was triggered by shifting to 37°C. Percentage of particles releasing mCherry is plotted. Data are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. Numbers on the top of bars are numbers of total particles analyzed. Asterisks and NS on the top of bars represent the significance relative to the LASVpp fusion efficiency in A549 cells. **, p<0.01; NS, not significant. (E) Kinetics of single GPc and IAV pseudoviruse fusion with different target cells.
Fig 2.
Arenavirus VLPs undergo membrane permeabilization prior to fusion.
(A) Illustration of single VLP fusion labeled with NP-DmCherry/NP-DYFP. VLP is internalized, trafficked to acidic endosomes where the viral membrane is permeabilized, leading to acidification of the viral interior and quenching of YFP fluorescence. Subsequent fusion of VLP and endosomal membrane neutralizes the VLP interior and results in recovery of YFP signal. (B) A single Candid-1 VLP fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show VLP interior acidification (YFP quenching) at 12.1 min and fusion (YFP dequenching) at 33.4 min. (see S3 Movie). (C) A single LASV VLP fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 34.0 min and fusion (YFP dequenching) at 57.9 min. (D) A single JUNV VLP fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show VLP interior acidification (YFP quenching) at 7.3 min and fusion (YFP dequenching) at 25.6 min. (E) A single MACV VLP fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 4.0 min and fusion (YFP dequenching) at 8.7 min.
Fig 3.
LASV pseudovirus interior acidification occurs shortly after entering acidic compartments.
(A) Illustration of internalization and trafficking of LASVpp colabeled with pHuji and Gag-EcpH. LASVpp is internalized and trafficked to acidic endosomes where the viral surface probe, pHuji, is quenched. The LASVpp membrane permeability increases in acidic compartments, leading to the viral interior acidification and quenching of the internal low pH probe, EcpH. (B) Single LASVpp entry into acidic endosome and viral membrane permeabilization in A549 cells. Time-lapse images (top) and fluorescence traces (bottom) show that, shortly after virus entry into the acidic endosomes (pHuji quenching at 107.6 min), membrane permeabilization occurs, resulting in virus’ interior acidification (EcpH quenching) at 108.2 min (see S4 Movie). (C) Single IAVpp entry into acidic endosome in A549 cells without membrane permeabilization. Time-lapse images (top) and fluorescence traces (bottom) show that IAVpp entry into the acidic endosome at 23.9 min leading to pHuji quenching with EcpH signal maintaining (see S5 Movie). (D) Bafilomycin A1 (BafA1) inhibits pHuji quenching. Data shown are means ± SD of 2 independent experiments. Results were analyzed by Student’s t-test. *, p<0.05. (E) Kinetics of single LASVpp exterior and interior acidification (pHuji and EcpH quenching, respectively). Results were analyzed by Student’s t-test. NS, not significant. (F) Distribution of lag times between pHuji and EcpH quenching for each single pseudovirus.
Fig 4.
Virus-cell contact promotes LASVpp membrane permeabilization at low pH.
(A, B) Illustration of the effects of LASVpp exposure to low pH on a coverslip (A) and on the cell surface (B). LASVpp were bound to the cell surface or to poly-L-lysine coated coverslips at 4°C. GPc conformational changes are triggered by applying membrane impermeable pH 5.0 citrate buffer. (C) Mean YFP intensity decay of all coverslip-attached particles in the image field or on cells. Dotted-lines are single- exponential decay fits of data shown by solid lines. Control YFP quenching profile upon application of a membrane-permeable pH 5.0 acetic buffer is shown. Exponential decay rates k are in 1/sec. Results were analyzed by Student’s t test, ***, p<0.001; NS, p>0.05. (D, E) Representative examples of four types of YFP quenching of single LASVpp on coverslip (D) or on cells (E). The point of adding a low pH buffer is marked with the red arrows. (F) Examples of the fast single virus YFP quenching events in A549 cell surface (#1-#4) overlaid onto the average YFP intensity profile for single LASVpp acidified on coverslip. The shaded area represents standard deviation of the mean YFP decay curve. (G, H) Quantification of the above four categories of YFP-Vpr quenching of single LASVpp on coverslip (G) or on DF-1 cells (H). Numbers within the bars are the total numbers of particles analyzed by Fisher’s exact test. ***, p<0.001; NS, not significant.
Fig 5.
Membrane permeability increases are observed for fusing but not non-fusing LASVpp.
(A) Normalized average single YFP-Vpr intensity profiles of LASVpp upon exposure to low pH on a coverslip or upon entry into A549 cells through a conventional endocytic pathway (fused vs. non-fused particles). Individual YFP intensity decays are aligned at the onset of quenching. Photobleaching-mediated YFP signal decay in PBS+/+ is shown as reference. Shadowed area is the 95% confidence interval. Data were analyzed by two-way ANOVA. **, p<0.01; ***, p<0.001; NS, not significant. (B) The YFP-Vpr intensity decay rates for fusing vs non-fusing particles, as well as particles on a coverslip obtained by single-exponential fitting are shown. The rate of YFP photobleaching is also shown. Data were analyzed by Student’s t-test. ***, p<0.001. (C) Quantification of different types of YFP-Vpr quenching of fused or non-fused single LASVpp in live cell analyzed by Fisher’s exact test.
Fig 6.
Fusion-impairing LASV GPc mutation and fusion inhibitor impair viral membrane permeabilization.
(A) Wild-type and K33A mutant LASVpp-BlaM fusion with A549 cells. LASVpp was bound to A549 cells in the cold and viral fusion was initiated by shifting to 37°C and incubating for 2 hours. Data shown are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. **, p<0.01. (B) Mean YFP-Vpr intensity decay of LASV GPc WT and K33A mutant on the surface of A549 cells after applying membrane-impermeable pH 5.0 citrate buffer. K33A mutant abrogate LASVpp fusion. The exponential decay rates k are in 1/sec. Results were analyzed by Student’s t-test, ***, p<0.001. (C) Quantification of different types of single LASVpp YFP-Vpr quenching events for LASVpp WT or K33A GPc mutant on A549 cells after applying a pH 4.0, 4.5 or 5.0 citrate buffer at 37°C. (D) ST-193 inhibits LASVpp-BlaM fusion with A549 cells. LASVpp was bound to A549 cells in the cold and viral fusion was initiated by shifting to 37°C and incubating for 2 hours in the presence of 10 μM ST-193 or equal volume of solvent (DMSO). Data shown are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. ***, p<0.001. (E) Mean YFP-Vpr intensity decay of LASVpp on A549 cell surface after applying pH 5.0 citrate buffer in the presence or absence of 10 μM of ST-193. Exponential decay rates k are shown in 1/sec. Results were analyzed by Student’s t test, ***, p<0.001. Note that the different rates of YFP quenching for WT GPc in panels B and E are due to the presence of DMSO (vehicle) in experiments with ST-193. (F) Quantification of different types of single LASVpp YFP-Vpr quenching events on A549 cells in the presence or absence of 10 μM ST-193, after applying low pH. In panels (C) and (D), the total numbers of particles analyzed by Fisher’s exact test are shown above the bars. *, p<0.05; ***, p<0.001; NS, not significant.
Fig 7.
LASVpp membrane permeabilization is inhibited by fusion attenuating GPc mutations.
(A) Wild-type and mutant LASVpp fusion with A549 cells measured by a BlaM assay. LASVpp was bound to A549 cells in the cold, and the fusion was initiated by shifting to 37°C and incubating for 2 hours. Data shown are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001. (B, C) Mean YFP-Vpr intensity decay of LASVpp mutants on A549 cell surface after applying membrane-impermeable pH 5.0 citrate buffer. The exponential decay rates k are shown in 1/sec. Results were analyzed by Student’s t-test, **, p<0.01; ***, p<0.001; NS, not significant. (D) Quantification of different types of YFP-Vpr quenching of single LASVpp GPc mutants on A549 cells after applying low pH. Data was analyzed by Fisher’s exact test. **, p<0.01; ***, p<0.001; NS, not significant.