EPI TIR-FM min

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a 15 b EPI 45 75 min TIR-FM c min Supplementary figure 1. Fluorescence microscopy of Gag- GFP.

a, Images of a single cell observed for 75 min under epifluorescence illumination illustrating the transition from a diffuse to a punctate Gag-GFP distribution.

1 a 15 b EPI min TIR-FM c min Supplementary figure 1. Fluorescence microscopy of Gag- GFP. HeLa cells were transfected with Gag and Gag-GFP and imaged at 5-6 hpt. a, Images of a single cell observed for 75 min under epifluorescence illumination illustrating the transition from a diffuse to a punctate Gag-GFP distribution. b, (top) Epifluorescence illumination or (bottom) TIR-FM were used to image the same cell, using identical focal planes on the adherent surface. c, Images of a cell observed over a -minute period using TIR-FM illustrating the transition from a diffuse to a punctate Gag-GFP distribution (see movie 1). 1

2 a Slowly appearing Gag puncta Rapidly appearing Gag puncta b Rapidly appearing Gag puncta Slowly appearing Gag puncta Lateral velocity (µm/sec) % of appearing Gag puncta hpt, Vpu hpt, Vpu hpt, Vpu hpt, Vpu + Supplementary figure 2. Two distinct behaviors of HIV-1 Gag puncta appearing at the plasma membrane. a, Quantification of the mean lateral velocity of (left) 25 slowly appearing puncta and (right) 25 rapidly appearing puncta. b, Enumeration of slowly and rapidly appearing puncta in the presence or absence of Vpu-mCherry at 5-6 or hpt. For each group, 3 cells imaged for minutes were analyzed and all the appearing events were classified as slow or rapid. NEXT PAGE: Supplementary figure 3. Endocytic reporters mark rapidly but not slowly appearing Gag puncta. Images illustrating rapidly (a) and slowly (b) appearing Gag puncta with corresponding clathrin-lightchain or CD63 fluorescence, as indicated. For each sequence, corresponding fluorescent intensity plots are shown on the right. Fields are 5.5x5.5μm. 2

3 a : :5 : :15 : :25 min Gag mix Clathrin Overlay Gag fluorescence intensity (a.u) 2 1 Gag Clathrin Clathrin fluorescense intensity (a.u) : :5 : : :35 :45 min Gag mix Gag CD63 CD63 Overlay Fluorescence intensity (a.u) b : :5 1: 1:55 2: 4: min Gag mix Clathrin Gag 8 Clathrin Gag fluorescence intensity (a.u) 1 7 Clathrin fluorescence intensity (a.u) Overlay : :5 2: 3:35 8:25 11: min Gag mix 1 Gag CD63 CD63 Overlay Maximun fluorescence intensity (a.u) Supplementary figure 3 3

a GFP BT b 543 nm 488 nm VLPs Cells VLPs Cells c GFP Raw mcherry

mcherry pre bleach mcherry post bleach GFP variation - -8 - - - -

FRET between Gag-GFP and Gag-mCherry in fluorescent puncta in

excited with the 488-nm laser and fluorescence emissions were

laser in cells expressing Gag-mCherry or VLPs containing

The intensity of the mcherry signal when excited by the 488 nm

c, Comparison of raw and corrected mcherry signal.

mcherry and GFP emission, upon subsequent illumination with the

(Left panel) A field of VLPs pre- and post- mcherry bleach (Right

4 a GFP BT b 543 nm 488 nm VLPs Cells VLPs Cells c GFP Raw mcherry mcherry corrected VLPs Cells d GFP pre bleach GFP post bleach mcherry pre bleach mcherry post bleach GFP variation Cherry variation - Supplementary figure 4. FRET between Gag-GFP and Gag-mCherry in fluorescent puncta in live HeLa cells and unfixed single VLPs collected from cell supernatants. a, Measurement of the bleed through of the donor into the acceptor channel. Cells expressing Gag-GFP or isolated VLPs containing Gag-GFP were excited with the 488-nm laser and fluorescence emissions were collected simultaneously through both the GFP-emission and the mcherry-emission channels. The bleed through of GFP emission into the mcherry channel was 5% of the GFP signal in the GFP channel. b, Measurement of direct excitation of mcherry by the 488 nm laser in cells expressing Gag-mCherry or VLPs containing Gag-mCherry. The intensity of the mcherry signal when excited by the 488 nm laser was 25% of its value when excited by the 543 nm laser. c, Comparison of raw and corrected mcherry signal. d, Effect of bleaching mcherry with the 543 nm laser on the mcherry and GFP emission, upon subsequent illumination with the 488 nm laser. (Left panel) A field of VLPs pre- and post- mcherry bleach (Right panel). The effect of mcherry bleaching on mcherry and GFP signals (in arbitrary units) in VLPs was analyzed. Note that when mcherry was bleached by using a 543nm laser in cell-free VLPs, the mcherry signal in individual VLPs decreased, but the GFP signal increased, confirming that the corrected emission from mcherry was the consequence of FRET from the Gag-GFP within individual particles. 4

5 GFP mcherry Ratio GFP mcherry Ratio GFP mcherry Rat Normalized fluorescence intensity (a.u) Normalized fluorescence intensity (a.u) Normalized fluorescence intensity (a.u) Supplementary figure 5. FRET analysis of three slowly appearing Gag puncta. Plots of the normalized fluorescence intensity over-time for GFP, corrected mcherry and the mcherry:gfp ratio (the FRET coefficient) during appearance of Gag puncta. 5

6 Pre-bleach Post-bleach : 3:25 5: 7: 11:35 16: 21:15 min Supplementary figure 6. Images showing a Gag-GFP punctum that fully recovered (black arrow) and a punctum that did not recover (red arrow) after photobleaching. Fields are 2.25x2.25μm. The corresponding plots of fluorescence intensity over-time are given in figure 3a. The image on the right is an overlay of a pre-bleach image in which Gag puncta have been colored in red with a post-bleach image in which puncta have been colored in green. Yellow puncta are, therefore, those that recovered. 6

7 a Gag-GFP/ Gag, ratio 1:5 : : 1: 7: 9: :5 min Gag-GFP/ Gag, ratio 1: : :15 1:35 3:15 5:45 6:55 min Gag-mCherry/Gag, ratio 1:1 : : :35 1:45 3:25 4:5 min b Fluorescence intensity (a.u) Fluorescence intensity (a.u) Gag-GFP/Gag, ratio 1: Gag-mCherry/Gag, ratio 1: Fluorescence intensity (a.u) c Kinetic of assembly (min) Gag-GFP/Gag, ratio 1: GagGFP/Gag 1:, n= GagGFP/Gag 1:5, n= GagCherry/Gag 1:1, n= GagGFP/Gag 1:1, n=37 NL4.3-MAYFP-BL2/NL4.3-BL2 1:1, n= Supplementary figure 7. The observed kinetics of assembly were not affected by the proportion of GFP-tagged molecules in VLPs, nor by the maturation time of the fluorophore linked to Gag. HeLa cells were transfected with variable ratios of plasmids encoding Gag and Gag-GFP or Gag and Gag-mCherry, and observed at 5-6 hpt for to minutes. a, Images of assembly events. Each field is 5.5x5.5μm. b, Plots of the fluorescence intensity over time for the events shown in (a). c, The average time to complete assembly (mean ± sd) for VLPs composed of Gag/Gag-fluorescent proteins at the various indicated ratios and for HIV-1 virions assembled using a proviral plasmid carrying YFP embedded in the stalk region of matrix (NL4.3-MAYFP-BL2, see fig 4 and supplementary fig 8). 7

min Supplementary figure 8. Assembly of HIV-1 particles from full length proviral plasmids. Hela cells were transfected with a 1:1 mix of NL4.3-BL2 and NL4.

8 min Supplementary figure 8. Assembly of HIV-1 particles from full length proviral plasmids. Hela cells were transfected with a 1:1 mix of NL4.3-BL2 and NL4.3- MAYFP-BL2. Cells were observed at hpt using TIR-FM. Images of a cell observed over a -minute period illustrating the appearance of Gag-YFP puncta. 8

9 doi:.38/nature6998 SUPPLEMENTARY INFORMATION Methods Expression vectors Plasmids expressing HIV-1 Gag and Gag-GFP proteins, namely pcr3.1/syn-gag and pcr3.1/syn-gag-egfp, were previously described 4. pcr3.1/syn-gag-egfp was used to generate pcr3.1/syn-gag-mcherry and pcr3.1/syn-gag-phluorin. A derivative of pcr3.1/syn-gag-egfp lacking the functional bipartite late budding domain 9 was used to generate pcr3.1/syn-gag-ld - -phluorin. pcr/vpu-mcherry was derived from pcr/vpu-yfp 9. pcr/cd63-mcherry plasmid 4 and a plasmid expressing dsred-clathrin-light-chain (a gift from T. Kirchhausen) were used to generate a LNCX (Invitrogen, Carlsbad, CA) -based retroviral vectors expressing CD63-mCherry and dsred-clathrin-light-chain. A HIV-1 NL4.3 proviral plasmid derivative, termed NL4.3-BL2 was generated by deleting the Env gene and introducing inactivating mutations in both integrase (D116A) and reverse transcriptase (from YMDD to YMAA). Similarly, a BL-2 version of the previously described NL4.3-MAYFP 4 was generated. Cells and transfection HeLa and 293T cells were cultured as before 4. To generate HeLa-derived cell lines stably expressing CD63-mCherry or dsred-clathrin-light-chain, 7% confluent 293T cells in six-well plates were transfected using polyethylenimine (PolySciences, Warrington, Pennsylvania, United States) with ng pcmv/vsv-g (expressing the glycoprotein of vesicular stomatitis virus), 7ng of pmlv-gagpol (expressing the murine leukemia virus Gag-Pol) and ng of the LNCX retroviral vectors encoding CD63-mCherry or dsred-clathrin-light-chain cdnas. The 293T supernatants were collected 48hrs later, filtered (.22μm) and used to transduce HeLa cells. Selection with G418 (.5mg/ml) was applied 24hrs later. Resistant cells were trypsinized a few days later and single cell clones derived by limiting dilution. Clones expressing low level of proteins were selected for the experiments. For imaging, HeLa cells were plated onto glass-bottom dishes (MatTek, Ashland, MA) and transfected with a total of 2μg of DNA. Cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA), as described previously 4. Plasmids expressing Gag-GFP were mixed with plasmids expressing untagged Gag at a 1:1 ratio, or, where indicated, at a 1:5 or 1: ratio. For experiments in which Vpu was co-expressed, cells were transfected at a Gag/Vpu ratio of 1:2. Cells were imaged 5- hours after transfection in Cell Imaging Media (CIM; HBSS + mm Hepes + 5% FBS [ph 7.4]). Cells were observed over a to minute period with one image acquired every 5 seconds. Isolation of cell-free VLPs from culture supernatants 293T cells in six-well plates were transfected with 2μg of plasmids expressing unlabelled Gag and Gag-fluorescent protein fusions, as above. Culture supernatants were collected 48hrs later and filtered (.22μm). VLPs were pelleted by ultracentrifugation through a % sucrose- PBS cushion for 2h at 27, rpm, re-suspended in 1μl PBS, filtered again and deposited on 9

10 doi:.38/nature6998 SUPPLEMENTARY INFORMATION polylysine coated glass-bottom dishes (MatTek, Ashland, MA) for 1 h before being imaged in CIM at 37 C. Image acquisition Through-the-objective TIR-FM and epifluorescence microscopy were performed using an inverted Olympus IX-7 microscope equipped with an APO x, N.A TIR objective (Olympus Scientific, Center Valley, PA) and a 12-bit cooled CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan), as previously described 13,15. The microscope was enclosed in a home-built chamber and all imaging was performed at 37 C. For TIR-FM, GFP was excited with the 488-nm line of an argon laser (Omnichrome; Melles Griot, Carlsbad, CA) reflected off a dichroic beamsplitter (z488rdc). The evanescent field decayed to 1/e in 7 nm. Simultaneous dual-color TIR-FM imaging of GFP and mcherry or GFP and dsred was achieved by exciting GFP with the 488-nm laser line of the argon laser and red fluorescent proteins with the 543-nm HeNe laser (model 5-LGR-193, Melles Griot) reflected off a 488/543 polychroic mirror. The emission was spectrally separated by means of an emission splitter (Dual-View; Optical Insights, Santa Fe, NM) equipped with a 515/ bandpass filter and a 58lp filter. For epifluorescence, the light of a xenon short-arc lamp [model UXL-1M (Ushio, Tokyo, Japan) in an OPTI QUIP (Highland Mills, NY) model 77 housing with power supply model ] was passed through a 515/ bandpass excitation filter. All mirrors and filters were obtained from Chroma Technologies Corp. (Brattleboro, VT, USA). Image processing and quantitative analysis All data analyses used MetaMorph software (Molecular Devices). For dual-color movie sequences, the images acquired through the emission splitter were separated, aligned with an accuracy of a single pixel, and analyzed. For measuring fluorescence intensity, a region was drawn around an area of interest of by pixels and the maximum intensity within this region recorded. For tracking VLPs over time, the tracking object option of Metamorph was used. This option allowed the calculation of velocities. All quantitative data (fluorescent measurements, tracking coordinates, background, etc) were transferred to Excel for analysis. FRET analysis Analyses were performed using cells expressing different combinations of untagged Gag, Gag-GFP and Gag-mCherry, as well as on cell-free VLPs. We chose the GFP/mCherry pair for FRET studies for three reasons. First, they show significantly greater photostability than CFP/YFP. Second, the emission of GFP (the donor) in the emission region of the mcherry (the acceptor) is significantly lower (5% under our imaging conditions, supplementary fig 4a) than for the more commonly used CFP/YFP pair (63% under our imaging conditions). Thus, while the overall FRET efficiency is reduced, there is an even greater reduction of the background, offering an improvement in signal to noise 31. Finally, since the spectral overlap is less, FRET between the GFP/mCherry pair may be more dependent on the higher order oligomeric interactions that could be expected during VLP assembly. Fluorophore emission bleed-through from the green channel into the red was measured by collecting GFP emission in both the donor and acceptor channel in the absence of mcherry

11 doi:.38/nature6998 SUPPLEMENTARY INFORMATION (Supplementary fig. 4A). The average intensities were measured from fields and the bleedthrough of GFP into the red channel was approximately 5%, both in cells and cell free VLPs. Direct excitation of mcherry by the 488-nm laser was measured by comparing average intensities of mcherry signal excited with the 543-nm or the 488-nm laser in the absence of GFP (supplementary fig. 4B). This source of contamination of mcherry into the FRET channel by direct excitation at 488 nm was 25% of the mcherry emission when excited at 543 nm. The corrected mcherry signal (i.e. the FRET signal) was obtained by subtracting these values from the raw mcherry signal using the arithmetic option (supplementary fig. 4C). During image acquisition the two fluorophores were excited exclusively with the 488-nm laser. The emission was separated as described above. To facilitate comparison, the fluorescence intensities have been normalized to their maximum. The FRET coefficient was calculated by dividing the corrected mcherry emission by the intensity of the GFP emission in the area of interest. Fluorescence recovery analysis Cells were imaged at 5hpt for 5, photobleached for 3 and observed for an additional. During the bleach, the opening of an iris placed in front of the TIR-FM laser was reduced to bleach only a part of the cell. The intensity of the 488nm-laser was unchanged throughout the experiment. Recovery is given as the percentage of the intensity regained after the bleach compared to the pre-bleach intensity. CO 2 mediated acidification Cells were imaged at 5hpt for 5. The gas that was perfusing the solution above the cells was switched to % pco 2 saturated with H 2 O for seconds and then returned to air for 15-. The increased pco 2 acidifies both the solution and the cytosol. However, the acidification in the cytosol is significantly faster as a consequence of the cytosolic carbonic anhydrase. We have previously demonstrated that a 15 second replacement of the air above the solution is sufficient to start acidifying the cytosol 23. To facilitate comparison, the fluorescence intensities given in Fig 3d have been normalized to their pre-acidification average. The data shown in Fig 3e,f have been collected from 5 cells expressing Gag-LD - -phluorin, from 4 cells expressing Gag-pHluorin and from cell-free VLPs. Statistical analysis All data were analysed with Excel software (Microsoft). The Vpu effects at early and late time points were analysed by a Chi-square test while all other analysis were performed using Student s t-test. 11

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION sirna pool: Control Tetherin -HA-GFP HA-Tetherin -Tubulin Supplementary Figure S1. Knockdown of HA-tagged tetherin expression by tetherin specific sirnas. HeLa cells were cotransfected with plasmids expressing