Release of RNPs from endosomes and viral protein synthesis in the presence of nocodazole.

<p>Confocal images of cells inoculated with rVSV-MLG at t-0 (A) and t-120 (B and C) post-NH₄Cl washout in the presence of cycloheximide and nocodazole (B), or just nocodazole (C), and stained for VSV N protein using N mAb conjugated to Alexa Fluor-568. Quantification of the amount of colocalization was determined for 50 individual cells using the colocalization function in the LSM software version 3.2. Percent of N protein that colocalized with MLG is indicated for t-0 and t-120 (n  =  50 cells). Bars  =  5 µm.</p

Localization of MLG at the nuclear envelope.

<p>(A) BHK cells were pre-treated with bafilomycin A1 (Baf) and then rVSV-MLG and Tfn-TR were bound to cells in the cold in media containing Baf for 90 minutes. The inoculum was removed and the cells were incubated for 150 minutes at 37°C in media containing Baf. Cells were imaged by LSCM as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000994#ppat-1000994-g003" target="_blank">Fig. 3</a>. Bars  =  10 µm. (B) Cells were infected with rVSV-MLG in the presence of NH₄Cl for 90 minutes to allow accumulation of virus in endosomes and then entry was initiated by incubation in media without NH₄Cl, but containing cycloheximide to prevent new viral protein synthesis. Cells were fixed at t-0 (top panels) and t-60 (bottom panels), stained for M protein using a rhodamine conjugated anti-M mAb (red) and a Nup62 antibody labeled with Alexa Fluor-647 (blue), and examined by LSCM. White arrows indicate the localization of MLG at the nuclear envelope. Note the M mAb does not detect M protein localized to the nuclear envelope (middle panels, yellow arrows). Bars  =  2 µm.</p

Cell fractionation and analysis of the distribution of RNPs and M protein during virus entry.

<p>rVSV-wt was bound to cells in the cold for 90 minutes in the presence of cycloheximide to prevent new protein synthesis, the inoculum was removed and the cells were washed and then either removed from the dish immediately (t-0) or warmed to 37°C and then harvested for fractionation at the times indicated. Fractions were subjected to immunoblot analysis using polyclonal anti-VSV sera. Relevant regions of the immunoblot are shown. NV (no virus) indicates cells that were mock infected and harvested at t-0. Graphs below the blots show quantification of N or M protein in each fraction. (A) N protein detected in the P16 (plasma membrane-associated virions) and NDG pellet (detergent-resistant nucleocapsids) fractions. Times post-entry are shown above the immunoblot. VSV N is a lane containing purified virus. (B) M protein detected in the S16 (plasma membrane and mitochondrial membrane) and NDG supernatant (endosomal and nuclear membrane) fraction. NV (no virus) mock infected cells. VSV M is the lane containing purified virus. (C) The four fractions probed with antibodies to the indicated cellular markers.</p

Spatio-temporal distribution of MLG during virus entry and uncoating.

<p>A synchronized entry assay was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000994#ppat-1000994-g004" target="_blank">Fig. 4B</a> using either rVSV-MLG (A) or “bald” ΔG-MLG (B) and the cells were imaged 15 minutes after addition of 37°C media. A region having a high concentration of endosomes was magnified to 500× using the “crop” function of the LSM software to show intracellular vesicles containing MLG and Tfn-TR. Arrows indicate an asymmetric distribution of MLG within Tfn-TR positive endosomes in rVSV-MLG infected cells. Bars  =  5 µm. (C) MLG distribution 150 minutes post-entry. (D) The sizes of the MLG “patch” found on Tfn-TR endosomes (n  =  30 endosomes) from a live cell experiment similar to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000994#ppat-1000994-g003" target="_blank">Fig. 3</a> were measured using the Profile function of the Zeiss LSM510 software, version 3.2 and plotted as a function of time post-entry.</p

Kinetics of RNP separation from MLG-endosomes.

<p>(A) rVSV-MLG was bound to cells in the cold as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000994#ppat-1000994-g003" target="_blank">Fig. 3</a> except Tfn-TR was omitted. Cells were fixed at the times indicated, permeabilized and RNPs were detected by staining for N protein using an N mAb conjugated to Alexa Fluor-568. Images were collected by LSCM using identical detector, offset, and gain settings. Bars  =  10 µm. (B) Colocalization of RNPs with MLG from the images in (A) as a function of time post-entry. Significance was determined using Student's t-test (n  =  9 cells). (C) The red and green channel levels for the t-5 and t-60 images from (A) were adjusted in Adobe Photoshop to give similar red and green intensities, which shows better the colocalization of RNPs with MLG detected by the LSM software for the t-5 time point.</p

Endocytosis of surface bound Tfn-TR and rVSV-MLG.

<p>rVSV-MLG and Tfn-TR were bound to cells at 4°C for 90 minutes and the cells were either (top panels) fixed immediately with ice-cold 3% paraformaldehyde (t-0), or (middle panels) treated with acidic desferrioxamine to strip Tfn-TR from the cell surface (t-0, AW) and then fixed, or (bottom panels) warmed by replacing the inoculum with 37°C media for 2 minutes followed by quickly cooling with the addition of ice-cold PBS, and then acid-desferrioxamine washed on ice. After the acid wash the cells were warmed to 37°C, the cells were incubated for an additional 3 minutes (t-5, AW) and then examined without fixation by LSCM on a heated stage. Bars  =  5 µm. DIC  =  differential interference contrast.</p

Time-line of VSV entry and uncoating events.

<p>A model for VSV entry and uncoating based on the spatio-temporal imaging analysis of MLG and RNP from rVSV-MLG. Relevant figures from the analyses are shown in parentheses. Observed times of the events are shown in red-colored font. In the model, virions attach to the cell surface, are endocytosed and trafficked to early endosomes in a Rab5-dependent manner where low pH induces conformational changes G resulting in acidification of the virion interior (A) and subsequent fusion of the viral and endosomal membrane (B). Acidification of the virion interior has an effect on the M protein to destabilize interactions between membrane-associated M (green circles with orange borders) and the condensed RNP, which results in release of the RNP into the cytoplasm after membrane fusion (C). The bulk of M remains associated with endosomes, but begins to diffuse across the endosomal membrane after RNP release (D). A smaller fraction of M is released into the cytosol and reaches the NPCs in a microtubule and actin independent manner (E).</p

Effect of DN-Rab expression on VSV infection.

<p>Cells were transfected with plasmids expressing DN- or wt-Rab-eGFP proteins and infected with rVSV-wt or influenza virus at 18 hours post-transfection. Cells were fixed, permeabilized and stained for VSV N protein or influenza NP at 8 hr post-infection. (A) Fluorescence and brightfield micrographs showing the effect of DN-Rab5 (top panels), DN-Rab7 (middle panels), and DN-Rab11 (bottom panels) on VSV infection. Arrows mark cells expressing Rab-eGFP proteins. (B) Inhibition of virus infection was quantified by determining the number of Rab-positive cells that were also infected by VSV or influenza virus. Statistical significance for effect on VSV infection was determined using Student's t-test (* =  p < 0.002) from 152 cells (Rab5), 153 cells (Rab7), or 156 cells (Rab11).</p

Uncoating and distribution of M protein in live cells.

<p>Cells were inoculated with rVSV-MLG and Tfn-TR at 4°C for 90 minutes and either fixed immediately with ice-cold 3% paraformaldehyde (t-0) or were quickly warmed by the addition of 37°C media and examined without fixation by LSCM on a heated stage at the indicated times using separate 35mm glass bottomed dishes for each time point to reduce signal loss by photobleaching. Bars  =  10 µm. Arrowheads indicate MLG localization to NPCs.</p

Colocalization of MLG with markers for recycling endosomes.

<p>A synchronized fusion assay was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000994#ppat-1000994-g004" target="_blank">Fig. 4B</a>, the cells were fixed at t-60, stained for (A) LAMP-1, (B) mannose-6-phosphate receptor, (C) Tfn-TR, or (D) Rab11 and then examined by LSCM (Bars  =  10 µm). Percent colocalization of MLG with the indicated marker from approximately 20 individual cells from two independent experiments is shown in the upper right-hand corner of the merged images.</p