Methods
Plasmid derivation
Plasmids expressing codon-optimized HIV-1 Gag and Gag-mCherry proteins, namely pCR3.1/HIV-Gag and pCR3.1/HIV-Gag-mCherry, were previously described1. A derivative of pCR/HIV-1Gag and pCR3.1/HIV-Gag-mCherry lacking the bipartite L-domain in p6 (PTAP and LRSLF were mutated to LTAL and LRSPS, respectively) was generated using overlapping PCR methods2. Plasmids expressing codon-optimized equine infectious anemia virus (EIAV) Gag/Gag-mCherry proteins (pCR3.1/EIAVGag and pCR3.1/EIAVGag-mCherry) were generated by inserting Gag gene into pCR3.1 and pCR3.1/mCherry. A derivative of pCR/EIAV-1Gag and pCR3.1/EIAV-Gag-mCherry in which the Alix binding site was mutated (YPDL to AADA) was generated using overlapping PCR methods. Plasmids expressing HIV-1 Vpu, namely, pCR3.1/Vpu, were previously described2.
pCR3.1-GFP-Alix, pCR3.1-GFP-Tsg101, pCR3.1-Chmp1b-GFP, pCR3.1-GFP-Chmp4b, pCR3.1-GFP-Chmp4c and pCR3.1-GFP-Vps4A were previously described 3, 4 and were used to used to generate LNCX (Clontech, Mountain View, CA )(Alix, Tsg101, Chmp4b, Chmp4c and Vps4A) or LMNI (Chmp1b) -derived retroviral vectors. LMNI is a MLV-based retroviral vector in which expression of the inserted gene cDNA is driven by the murine leukemia virus LTR and is linked to sequences encoding an internal ribosome entry site and a blasticidin resistance gene. The plasmid encoding the VPS4 mutant E228Q, namely pCR3.1-VPS4-DN was previously described 5 and was used to generate a pCR3.1-VPS4-DN-mCherry. pCR3.1-GFP-Vps4-K173Q was derived from pCR3.1-GFP-Vps4A by using PCR-based methods.
Cells and transfection
Hela cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. HeLa cell lines stably expressing GFP tagged versions of the ESCRT proteins were generated by retroviral transduction as described previously1. Single cell clones expressing low levels of each GFP-fusion proteins were selected for the experiments. HeLa cells were transfected with untagged Gag and Gag-mCherry in a 10:1 ratio using Lipofectamine 2000 (InVitrogen, Carlsbad, CA).
Multinucleation assays
Approximately 105 Hela cells stably expressing GFP-tagged ESCRT proteins were plated on glass-bottomed dishes (MatTek, Ashland, MA). As a control, cells were transfected for 24 hours with 1 µg of plasmid expressing either GFP alone of GFP-tagged Vps4 K173Q, which over-expression is known to inhibits cytokinesis 6. Cells were fixed and stained with anti-tubulin antibodies and with DAPI. 300 cells from 3 independent experiments were analyzed for the presence of more than one nucleus per cell. Cells connected by midbodies were considered multinucleated.
Virus release assays and Western blot analysis
Virus-like particle (VLP) release assays were carried as previously described7 by transfecting approximately 3 x 106 cells in 10-cm dishes with 2 µg of Gag and 1 µg of Vpu expression plasmids. Cells and extracellular virus particles were harvested at 24 hours as described previously2. Virion and cell lysates were separated on 4 to 12% acrylamide gels, and proteins were probed with various antibodies: anti-Vps4A (UT2889, 1:1000 dilution)8, anti-Alix9 (1:1000 dilution) (gifts from W. Sundquist), anti-HIV-1 p24CA (183-H12-5C, 1:100 dilution), anti-EIAV equine serum (1:200 dilution) (VMRD Inc, Pullman, WA) and anti-GFP (1:5000 dilution) (Roche). Blots were subsequently probed with anti-horse and anti-mouse antibodies (1:10000 dilution) conjugated to IRDye680 (LI-COR Biotechnology, Lincoln, NE). Fluorescent signals were detected using a LICOR Odyssey scanner (LI-COR Biotechnology).
Image acquisition
For microscopic analysis, HeLa cells were plated on glass-bottomed dishes (MatTek, Ashland, MA) and transfected with a total of 1mg of plasmids expressing Gag/Gag-mCherry, as described above. For epifluorescence microscopy, cells were fixed 18-20 hours after transfection and nuclei were stained with DAPI, as previously described2, and/or with a mouse antibody against a-tubulin (clone B-512, 1:10000 dilution) (Sigma) followed with an anti-mouse Alexa fluor 594 (Molecular probes). Fluorescent imaging of fixed cells was done using a Deltavision microscopy suite, as previously described2.
Live cell TIR-FM was performed as previously described1, 10, with an inverted Olympus IX-70 microscope with a 60x, 1.45 N.A. TIR objective (Olympus Scientific, Center Valley, PA) and a 12-bit cooled CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan). Cells were imaged 5-8 hours after transfection. The microscope was enclosed in a home-built chamber and all imaging was performed at 37°C. The evanescent field decayed to 1/e in 70 nm. Simultaneous dual-color TIR-FM imaging of GFP and mCherry was achieved by exciting GFP with the 488-nm laser line of the argon laser of an argon laser (Omnichrome; Melles Griot, Carlsbad, CA) and red fluorescent proteins with the 543-nm HeNe laser (model 05-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 515/30 bandpass filter and a 580lp filter. All mirrors and filters were obtained from Chroma Technologies Corp. (Brattleboro, VT, USA). Time-lapse movies were acquired over a 30 to 60 minute period with one image acquired every 5 seconds, as previously described1,3. The camera and shutters were controlled using MetaMorph software (Molecular Devices, Sunnyvale, CA).
Data analysis
All data analyses used MetaMorph software. 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 the intensity of fluorescent puncta, a region of 8 x 8 pixels was drawn around an area of interest and the maximum fluorescent intensity within this region recorded. The time to complete assembly was defined as the elapsed time from the image at which a fluorescent Gag signal is first detectable to the point when Gag intensity reaches a plateau.
For analysis of co-localization, regions of 8 x 8 pixels were drawn around VLPs, which were selected in the Gag-mCherry channel (red), and the fluorescent intensity in the corresponding region, in the GFP-ESCRT protein channel (green) was recorded. Alternatively, GFP-positive puncta were selected and the fluorescent intensity in the corresponding region, in the Gag-mCherry protein channel (red) was recorded. The fluorescent intensities in both channels were quantified and transferred to Excel for analysis.
FACS analysis
The distribution of GFP expression of Hela and Hela expressing GFP-tagged ESCRT proteins was analyzed with a BD LSRII FACS. Approximately 100000 cells were analyzed per cell line.
Statistical analysis
- Error bars in Figure 2a and Figure 2b indicate standard deviation calculated with Excel using .
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3. Martin-Serrano, J., Yarovoy, A., Perez-Caballero, D. & Bieniasz, P.D. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc Natl Acad Sci U S A 100, 12414-12419 (2003).
4. Martin-Serrano, J., Zang, T. & Bieniasz, P.D. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat Med 7, 1313-1319 (2001).
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6. Morita, E. et al. Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. Embo J 26, 4215-4227 (2007).
7. Jouvenet, N. et al. Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin. J Virol 83, 1837-1844 (2009).
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9. Fisher, R.D. et al. Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding. Cell 128, 841-852 (2007).
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