Fig 1.
Analysis of endogenous and ectopic expression and localization of hLAMP1 in A549 and DF-1 cells.
A549 or DF-1 cells were transduced with an empty pQCXIP vector (control) or vector expressing human LAMP1-WT or the LAMP1-d384 mutant. Cells were fixed and whether left untreated (bottom panels in A and B) or permeabilized with 125 μg/ml digitonin and immunostained for human LAMP1. (A, C) Images and quantification of hLAMP1 expression in A549 cells. Images acquired under same exposure conditions, but the brightness and contrast settings in panel A and B are different to ensure optimal display. (B, D) Images and quantification of hLAMP1 expression with DF-1 cells. Data shown are means ± SD of five fields of view for each condition. Blue asterisks show significance levels for the difference between LAMP1-WT and LAMP1-d384. Black asterisks on the top of bars represent significance relative to the vector control. Data were analyzed by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.
Fig 2.
LAMP1 expression enhances LASVpp fusion and infection in A549 and DF-1 cells.
(A) Efficiency of A549 and DF-1 cell infection through endosomal and forced fusion protocols, using luciferase-encoding LASVpp. Infection was measured at 48 hours post-infection (see Methods). (B) LASVpp-BlaM fusion with A549 and DF-1 cells. LASVpp entry through an endosomal pathway was initiated by pre-binding pseudoviruses in the cold, shifting to 37°C and incubating for 2 h. (C) Low pH-forced fusion of LASVpp with A549 and DF-1 cells. Cells were pretreated with 0.2 μM BafA1 for 1 h prior to binding pseudoviruses in the cold. Fusion was triggered by applying pH 5.0 citrate buffer at 37°C for 20 min followed by additional incubation in a neutral pH medium at 37°C for 30 min. (D) LASVpp infection of A549 and DF-1 cells. Luciferase-encoding LASVpp were bound to cells in cold. Cells were incubated in 37°C for 36 h to allow infection. (E) LASVpp infection through low pH bypass protocol in A549 and DF-1 cells described in panel C. After forced fusion, cells at 37°C for 36 h before reading the resulting luciferase signal. Data shown are means ± SD of three independent experiments. Data were analyzed by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001; NS, not significant. Blue asterisks show the significance levels for the difference between LAMP1-WT and LAMP1-d384, black asterisks on the top of bars represent significance relative to the vector control.
Fig 3.
LAMP1 expression enhances recombinant LCMV/LASV-GPC infection of A549 and DF-1 cells.
(A) Recombinant LCMV/LASV-GPC infection in A549 and DF-1 cells. Cells were allowed to bind LCMV/LASV-GPC in the cold and subsequently incubated at 37°C for 36 h. For infection in A549 and DF-1 cells achieved through forcing viral fusion by low pH exposure, cells were pretreated with 0.2 μM BafA1 for 1 h prior to binding LCMV/LASV-GPC in the cold. Fusion was triggered by applying pH 5.0 citrate buffer at 37°C for 20 min followed by additional incubation in a neutral pH medium at 37°C for 36 h. Infection was detected by immunostaining for LCMV nucleoprotein (NP). (B) Quantification of the fold change of the infection through an endosomal pathway. (C) Quantification of the fold change of the infection through forced fusion. Data shown are means ± SD of three independent experiments. Data were analyzed by Student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.
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).
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.
Fig 6.
Low pH-forced fusion of single LASVpp with the DF-1 cell plasma membrane.
(A) Illustration of single mCherry-2xCL-YFP-Vpr labeled LASVpp fusion with the plasma membrane of DF-1 cell induced by exposure to low pH. Viral membrane permeabilization in acidic buffer leads to virus interior acidification and quenching of YFP signal. Subsequent formation of a small fusion pore with the plasma membrane results in virus interior re-neutralization and YFP dequenching. Fusion pore enlargement leads to mCherry release into the cytoplasm. (B) Low pH-forced single LASVpp fusion with instant mCherry release. Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp forced fusion in DF-1-LAMP1-d384 cell showing YFP de-quenching and mCherry loss at 6.3 min. (see S4 Movie). (C) Low pH-forced single LASVpp fusion 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 forced fusion with DF-1-pQCXIP cell resulting in YFP dequenching at 34 min and a subsequent loss of mCherry at 37.6 min. (see S5 Movie). (D) Low pH-forced single LASVpp fusion 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-pQCXIP cell showing YFP dequenching at 42.9 min without mCherry loss. (see S6 Movie).
Fig 7.
Human LAMP1 enhances low pH-forced fusion of LASVpp with DF-1 cells and dramatically increases the fusion kinetics.
(A) Efficiencies of low pH-forced single LASVpp fusion events with instant mCherry release, delayed mCherry release and without mCherry release (fusion pore dilation) with DF-1 pQCXIP, LAMP1-WT and LAMP1-d384 cells. Data shown are means ± SD of 2 independent experiments. Black asterisks inside the bars represent significance relative to the vector control. The red, green and blue asterisks show significance levels for the difference between LAMP1-WT and LAMP1-d384 for fusion events with instant mCherry release, delayed mCherry release and without mCherry release respectively. At least 500 cell-bound particles were analyzed for each condition. 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) Same as in B but plotted as a dot-plot (lines represents means and whiskers are standard deviations). (D) The kinetics of small pore formation (YFP dequenching) between single LASVpp and DF-1 pQCXIP, LAMP1-WT and LAMP1-d384 cells using a forced fusion protocol. Data in (A) and (C) are means ± SD of two independent experiments. Blue asterisks show the significance levels for the difference between LAMP1-WT and LAMP1-d384, black asterisks on the top of bars represent significance relative to the vector control. 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.
Fig 8.
Low pH-forced single LASVpp fusion with DF-1 cells in the presence of soluble LAMP1.
LASVpp were bound to cells in the cold, in the presence of 200 μg/ml soluble LAMP1 (sLAMP1) or BSA (control). Single LASVpp fusion with the plasma membrane was initiated by addition of 2 ml of warm pH 5.0 citrate buffer supplemented with 200 μg/ml sLAMP1 or BSA. (A) Time lapse images (top) and fluorescence traces (bottom) for single LASVpp fusion with DF-1 cell in the presence of sLAMP1 showing YFP dequenching at 11.7 min and mCherry loss at 12.7 min. (B) Time lapse images (top) and fluorescence traces (bottom) of single LASVpp fusion with DF-1 cell in the presence of sLAMP1 showing YFP dequenching at 11.7 min without mCherry loss. (C) Time lapse images (top) and fluorescence traces (bottom) of single LASVpp fusion with DF-1 cell in the presence of BSA (control) showing YFP dequenching at 36.4 min and mCherry loss at 46 min. (D) Same conditions as in (A), but small fusion pore formation (YFP dequenching) at 40.1 min does not culminate in mCherry release.
Fig 9.
Soluble LAMP1 accelerates low pH-forced single LASVpp fusion with DF-1 cells.
(A) Efficiencies of low pH-forced single LASVpp fusion events with instant mCherry release, delayed mCherry release and without mCherry release with DF-1 cells in the absence or presence of sLAMP1. Data shown are means ± SD of two independent experiments. Inset: normalized fractions of each category of fusion. Asterisk shows significant change relative to the BSA control. (B) The distribution of lag times between small fusion pore formation (YFP dequenching) and fusion (loss of mCherry) for forced LASVpp fusion with DF-1 cells in the absence or presence of sLAMP1. (C) Kinetics of forced single LASVpp fusion pore formation (YFP dequenching) with DF-1 cells in the absence or presence of sLAMP1. (D) Low pH-forced fusion of LASVpp with DF-1 cells in the presence of sLAMP1 measured by the BlaM assay. Cells were pretreated with 0.2 μM BafA1 for 1 h prior to binding pseudoviruses in the cold. Fusion was triggered by applying pH 5.0 citrate buffer at 37°C for 20 min followed by additional incubation at a neutral pH, 37°C for 30 min. sLAMP1 (200 μg/ml) or BSA were included throughout virus spinoculation onto cells and low pH triggering of fusion. Data shown are means ± SD of two independent experiments. 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 were analyzed by non-parametric Mann-Whitney test using GraphPad. Other results were analyzed by Student’s t-test. *, p<0.05; ***, p<0.001; NS, not significant.