Distribution of ESCRT Machinery at HIV Assembly Sites Reveals Virus Scaffolding of ESCRT Subunits Schuyler B. Van Engelenburg et al. Science 343, 653 (2014); DOI: 10.1126/science.1247786
This copy is for your personal, non-commercial use only.
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of June 21, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6171/653.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2014/01/15/science.1247786.DC1.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6171/653.full.html#related This article cites 33 articles, 15 of which can be accessed free: http://www.sciencemag.org/content/343/6171/653.full.html#ref-list-1 This article appears in the following subject collections: Virology http://www.sciencemag.org/cgi/collection/virology
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
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REPORTS
Distribution of ESCRT Machinery at HIV Assembly Sites Reveals Virus Scaffolding of ESCRT Subunits Schuyler B. Van Engelenburg,1 Gleb Shtengel,2 Prabuddha Sengupta,1 Kayoko Waki,3 Michal Jarnik,1 Sherimay D. Ablan,3 Eric O. Freed,3 Harald F. Hess,2 Jennifer Lippincott-Schwartz1* The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.
Fig. 1. Interferometric superresolution microscopy resolves the 3D organization of cell-associated HIV particles. (A) Diffraction-limited (left) and iPALM-based superresolution (right) images of COS7 cells expressing HIV Gag-FLAG labeled with AlexaFluor 647 (AF647)– conjugated antibody (Life Technologies, Grand Island, NY). Gag clusters from different surfaces of the membrane are clearly resolved (pseudocolored for height; white arrowheads). Scale bars, 10 mm (full image); 100 nm (right). (B) Correlative iPALM (left) and SEM (middle) demonstrate HIV Gag-FLAG clusters to be membrane-enveloped particles (overlay, right). Scale bars, 250 nm. (C) Probability density of aligned clusters from HIV Gag-internal-FLAG (top, n = 1181 clusters) and HIV Gag-FLAG (bottom, n = 1398 clusters) highlight the shell-like lattice (isosurface threshold = 0.5 localizations × nm−3). (D) Probability slices (10 nm) display a ring-like distribution for Gag-internal-FLAG clusters (top middle) compared with the more compact Gag-FLAG clusters (bottom middle). Distance between crosshatch is 100 nm (right). Radial averages highlight the density differences owing to probe position within the HIV Gag molecule (inset model).
1 Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. 2Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. 3Virus-Cell Interaction Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
*Corresponding author. E-mail: [email protected]
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the membrane. Otherwise, virus egress is severely inhibited (1–5). Despite great progress in demonstrating the ESCRT machinery’s role in mediating HIV abscission, the nanoscale organization, and thus function, of ESCRT subcomplexes at native HIV assembly sites remains poorly defined.
Z Position (nm)
ne of the key host factors human immunodeficiency virus (HIV) relies on to complete its infection cycle is the endosomal sorting complexes required for transport (ESCRT). By recruiting this machinery, HIV is able to mediate the final step of virus particle fission from
ESCRT machinery participates in important cellular membrane remodeling events, such as cytokinesis and multivesicular body biogenesis (MVB), organizing into three general sets of subcomplexes. One set includes ESCRT-0, -I, and -II, which interact directly with cargo or scaffold proteins to direct assembly of downstream ESCRT complexes. Another set includes ESCRT-III subunits, which are thought to polymerize on membranes into a helical architecture to constrict and abscise membrane buds formed by early ESCRT/ cargo complexes (6, 7). The final set consists of the AAA+ adenosine triphosphatases (ATPases) Vps4A and Vps4B, which are thought to be necessary for HIV abscission through ESCRT-III filament remodeling and/or recycling (7–9). Together, the three sets of ESCRT subcomplexes cooperate to drive cellular membrane remodeling. Structural
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REPORTS information obtained by means of in vitro assembly (7, 10) and cellular overexpression studies (11) suggest a model of bud formation and abscission in which ESCRT-III filaments encircle and constrict the aperture of a membrane protrusion, acting on the base of the neck, in trans from the protrusion head. On the other hand, when directed by the midbody scaffold during cytokinesis (12, 13) ESCRT-III filaments appear to polymerize from the scaffold and constrict the membrane, acting in cis with respect to the scaffolding structure. Whether ESCRT-III filaments polymerize at HIV bud sites in cis or trans to the scaffolding structure is not known because of the small dimensions of a budding HIV virion (120 to 140 nm diameter). Consequently, it remains unclear how ESCRT machinery acts to mediate viral membrane abscission and propagate HIV infection. We used interferometric photoactivatedlocalization microscopy (iPALM) (14) to decipher the three-dimensional (3D) nanoscale organiza-
A
tion of ESCRT components at HIVassembly sites and thereby gained insight into the mechanism for viral membrane abscission (supplementary text). ESCRT subcomplexes were selected for our analysis on the basis of their direct interaction with the structural HIV protein Gag [ESCRT-I subunit tumor susceptibility gene 101 (Tsg101)] (15) and their role in HIV membrane abscission (ESCRTIII and Vps4; CHMP2A, CHMP4B, and Vps4A) (16). The ESCRT proteins were modified with either green fluorescent protein (GFP) or photoswitchable cyan fluorescent protein 2 (PSCFP2) and stably expressed in COS7 cells. Stable cell lines expressing fluorescent ESCRT probes were validated for proper function (supplementary text). Superresolution image analysis of HIV Gag demonstrated the ability of the iPALM method to sensitively locate and resolve subviral details of membrane-enveloped particles emanating from the plasma membrane of expressing cells (Fig. 1 and supplementary text). With the capacity to differ-
B
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entiate subviral protein organization within HIV bud structures, we next performed two-channel iPALM imaging of both HIV Gag and ESCRT subcomplexes. Imaging of HIV Gag and ESCRT-I (PSCFP2-Tsg101) revealed a small cytosolic pool of Tsg101 protein punctuated by clustering at Gag assembly sites (Fig. 2A and fig. S6A). PSCFP2Tsg101 was localized within the interior of the Gag lattice of assembling particles (Fig. 2D), which is consistent with the direct interaction between the p6 domain of HIV Gag and the UEV domain of Tsg101 (3). PSCFP2-CHMP2A and -CHMP4B (ESCRT-III) cell lines expressing HIV Gag showed high cytosolic pools of PSCFP2 followed by membrane-localized co-clustering with Gag clusters (Fig. 2, A to D, and fig. S6B). When coclustering was observed, the 3D distribution of ESCRT-III and Gag at assembly sites was similar and distinctly separable from the cytosolic pool of PSCFP2-CHMP2A or -CHMP4B (Fig. 2, A, B, and D). To correlate the apparent observation
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PSCFP2-CHMP2A Plasma membrane (tdEOS-CAAX)
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Gag-FLAG CHMP4B
Gag-FLAG CHMP2A
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Fig. 2. Two-color 3D superresolution imaging E localizes ESCRT-I and ESCRT-III subunits within Gag-FLAG PSCFP2-CHMP2A the HIV Gag lattice of budding particles. (A and B) HIV Gag fused internally to mEOS2 protein (Gag-imEOS2) detects assembly sites containing ESCRT-I subunits Tsg101 [(A), top] and ESCRT-III subunits CHMP4B [(A), bottom] and CHMP2A (B) fused to PSCFP2 protein. (B) (Top) iPALM measurements resolve the cytoplasmic pool of PSCFP2Total Probability per nm2 CHMP2A (top arrowhead), peripheral membrane-associated 0.00085 0.00733 0.02057 Gag-imEOS2 (middle arrowhead), and densely coclustered 0.00011 0.00497 0.01953 Gag and CHMP2A structures extending from the membrane HIV Gag lattice. Scale bars, 50 nm. (E) Correlative two-color iPALM and SEM of (bottom arrowhead). Scale bars, 100 nm (A); 500 nm (B). (C) PSCFP2-CHMP2A two virus-like particles (white arrowheads) emanating from a cell expressing coexpressed with unlabeled HIV Gag and membrane marker tdEOS-farnesyl Gag-FLAG (red) and PSCFP2-CHMP2A (green). The left membrane bud shows (red) show displacement of a CHMP2A cluster (green) extending beyond the confinement of PSCFP2-CHMP2A to the head of the particle. The particle on the plane of the plasma membrane. Scale bar, 500 nm. (D) Single clusters repreright is at an earlier stage of budding, apparent from the extended lateral sentative of AF647-labeled Gag-FLAG and PSCFP2-ESCRT probes. Axial (top) fluorescence distribution (right arrowhead). Scale bar, 250 nm. and lateral (bottom) views show localization of PSCFP2-ESCRT probes within the
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Fig. 4. Released VLPs contain ESCRT-I and ESCRTIII subunits, but reduced levels of CHMP2A relative to CHMP4B. (A) Quantitative fluorescence microscopy analysis of GFP-ESCRT and Gag-GFP containing single particles. The fluorescence intensity of Gag-GFP is predicted to arise from ~600 to 1000 molecules (assuming 3000 to 5000 Gag molecules per particle), suggesting that signal from GFP-ESCRT particles corresponds to hundreds of molecules per particle (data represent ≥25,000 particles from ≥2 independent samples for each probe). (B) Correlative two-color iPALM and scanning electron micrographs of a single released (top, red) Gag- and (top, green) CHMP4B-positive VLP. Image registration residual uncertainty of 11 nm. Scale bar, 50 nm. (C) Western blot analysis of purified VLP fraction from GFP-ESCRT stable cell lines. GFP-Tsg101 and GFP-CHMP4B are detected in released particles (top); however, GFP-CHMP2A levels are dramatically reduced from VLPs upon particle release (bottom; asterisk indicates proteolysis product). Reduction in the levels of GFP-CHMP2A in released particles correlates between the biochemical and fluorescence data. (D) Proposed virus-scaffolding model for ESCRT-mediated HIV particle release from host cells. A pre-scission pool of ESCRT-I subunit Tsg101 (gray) interacts with HIV Gag (red), leading to assembly of ESCRT-III subunits CHMP4B and CHMP2A (green). Hypothetical ESCRT-III polymerization beyond the Gag lattice and Vps4 (orange) remodeling of CHMP2A subunits leads to membrane aperture constriction and particle abscission (middle and right).
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Fig. 3. Single-cluster averaging demonstrates ESCRT subunits within the interior of the HIV Gag lattice. (A to D) Cell-associated HIV Gag-FLAG clusters from PSCFP2-ESCRT–expressing cells were subjected to 3D single-cluster averaging [(A) PSCFP2-Tsg101, n = 313 clusters; (B) PSCFP2-CHMP2A, n = 460 clusters; (C) PSCFP2-CHMP4B, n = 509 clusters; and (D) PSCFP2CHMP4B/HIV Gag DPTAP, n = 278 clusters]. Two-color isosurfaced probability densities show a high-probability core of PSCFP2-Tsg101 (A) and ESCRT-III subunits PSCFP2-CHMP2A/CHMP4B [(B) and (C)] (green) with respect to the HIV Gag lattice (red) (isosurface threshold, HIV Gag-FLAG = 2.5 × 10−4 nm−3 and PSCFP2-ESCRTs = 2.5 × 10−5 nm−3). Right insets [(A) to (D)] depict 40-nm sections along the z axis [top HIV Gag (red), bottom PSCFP2-ESCRT (green)]. (D) A marked reduction in the probability density of PSCFP2-CHMP4B signal was observed upon expression of release-defective HIV Gag-FLAG DPTAP. (E) Radial average plots of sections from (A) to (D) define the highest-probability densities of ESCRT (green) subunits residing within the HIV Gag probability shell (red). Approximately 90% of PSCFP2-Tsg101 (top left), 80% of PSCFP2-CHMP2A (top right), and 61% of PSCFP2-CHMP4B (bottom right) integrated probability reside within the halfmaximum Gag probability shell, whereas 10% of the integrated probability for PSCFP2-CHMP4B remains when ESCRT-I recruitment is inhibited (Gag-FLAG DPTAP; bottom right). Line thickness represents SEM and is
This copy is for your personal, non-commercial use only.
Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of June 21, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6171/653.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2014/01/15/science.1247786.DC1.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6171/653.full.html#related This article cites 33 articles, 15 of which can be accessed free: http://www.sciencemag.org/content/343/6171/653.full.html#ref-list-1 This article appears in the following subject collections: Virology http://www.sciencemag.org/cgi/collection/virology
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on June 21, 2014
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here.
REPORTS
Distribution of ESCRT Machinery at HIV Assembly Sites Reveals Virus Scaffolding of ESCRT Subunits Schuyler B. Van Engelenburg,1 Gleb Shtengel,2 Prabuddha Sengupta,1 Kayoko Waki,3 Michal Jarnik,1 Sherimay D. Ablan,3 Eric O. Freed,3 Harald F. Hess,2 Jennifer Lippincott-Schwartz1* The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.
Fig. 1. Interferometric superresolution microscopy resolves the 3D organization of cell-associated HIV particles. (A) Diffraction-limited (left) and iPALM-based superresolution (right) images of COS7 cells expressing HIV Gag-FLAG labeled with AlexaFluor 647 (AF647)– conjugated antibody (Life Technologies, Grand Island, NY). Gag clusters from different surfaces of the membrane are clearly resolved (pseudocolored for height; white arrowheads). Scale bars, 10 mm (full image); 100 nm (right). (B) Correlative iPALM (left) and SEM (middle) demonstrate HIV Gag-FLAG clusters to be membrane-enveloped particles (overlay, right). Scale bars, 250 nm. (C) Probability density of aligned clusters from HIV Gag-internal-FLAG (top, n = 1181 clusters) and HIV Gag-FLAG (bottom, n = 1398 clusters) highlight the shell-like lattice (isosurface threshold = 0.5 localizations × nm−3). (D) Probability slices (10 nm) display a ring-like distribution for Gag-internal-FLAG clusters (top middle) compared with the more compact Gag-FLAG clusters (bottom middle). Distance between crosshatch is 100 nm (right). Radial averages highlight the density differences owing to probe position within the HIV Gag molecule (inset model).
1 Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. 2Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. 3Virus-Cell Interaction Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
*Corresponding author. E-mail: [email protected]
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the membrane. Otherwise, virus egress is severely inhibited (1–5). Despite great progress in demonstrating the ESCRT machinery’s role in mediating HIV abscission, the nanoscale organization, and thus function, of ESCRT subcomplexes at native HIV assembly sites remains poorly defined.
Z Position (nm)
ne of the key host factors human immunodeficiency virus (HIV) relies on to complete its infection cycle is the endosomal sorting complexes required for transport (ESCRT). By recruiting this machinery, HIV is able to mediate the final step of virus particle fission from
ESCRT machinery participates in important cellular membrane remodeling events, such as cytokinesis and multivesicular body biogenesis (MVB), organizing into three general sets of subcomplexes. One set includes ESCRT-0, -I, and -II, which interact directly with cargo or scaffold proteins to direct assembly of downstream ESCRT complexes. Another set includes ESCRT-III subunits, which are thought to polymerize on membranes into a helical architecture to constrict and abscise membrane buds formed by early ESCRT/ cargo complexes (6, 7). The final set consists of the AAA+ adenosine triphosphatases (ATPases) Vps4A and Vps4B, which are thought to be necessary for HIV abscission through ESCRT-III filament remodeling and/or recycling (7–9). Together, the three sets of ESCRT subcomplexes cooperate to drive cellular membrane remodeling. Structural
Gag-FLAG MA CA
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REPORTS information obtained by means of in vitro assembly (7, 10) and cellular overexpression studies (11) suggest a model of bud formation and abscission in which ESCRT-III filaments encircle and constrict the aperture of a membrane protrusion, acting on the base of the neck, in trans from the protrusion head. On the other hand, when directed by the midbody scaffold during cytokinesis (12, 13) ESCRT-III filaments appear to polymerize from the scaffold and constrict the membrane, acting in cis with respect to the scaffolding structure. Whether ESCRT-III filaments polymerize at HIV bud sites in cis or trans to the scaffolding structure is not known because of the small dimensions of a budding HIV virion (120 to 140 nm diameter). Consequently, it remains unclear how ESCRT machinery acts to mediate viral membrane abscission and propagate HIV infection. We used interferometric photoactivatedlocalization microscopy (iPALM) (14) to decipher the three-dimensional (3D) nanoscale organiza-
A
tion of ESCRT components at HIVassembly sites and thereby gained insight into the mechanism for viral membrane abscission (supplementary text). ESCRT subcomplexes were selected for our analysis on the basis of their direct interaction with the structural HIV protein Gag [ESCRT-I subunit tumor susceptibility gene 101 (Tsg101)] (15) and their role in HIV membrane abscission (ESCRTIII and Vps4; CHMP2A, CHMP4B, and Vps4A) (16). The ESCRT proteins were modified with either green fluorescent protein (GFP) or photoswitchable cyan fluorescent protein 2 (PSCFP2) and stably expressed in COS7 cells. Stable cell lines expressing fluorescent ESCRT probes were validated for proper function (supplementary text). Superresolution image analysis of HIV Gag demonstrated the ability of the iPALM method to sensitively locate and resolve subviral details of membrane-enveloped particles emanating from the plasma membrane of expressing cells (Fig. 1 and supplementary text). With the capacity to differ-
B
Total Probability per nm2 Z
0.00000
0.00662
0.01324
Y
X
Z X
X
0.00000
0.00903
entiate subviral protein organization within HIV bud structures, we next performed two-channel iPALM imaging of both HIV Gag and ESCRT subcomplexes. Imaging of HIV Gag and ESCRT-I (PSCFP2-Tsg101) revealed a small cytosolic pool of Tsg101 protein punctuated by clustering at Gag assembly sites (Fig. 2A and fig. S6A). PSCFP2Tsg101 was localized within the interior of the Gag lattice of assembling particles (Fig. 2D), which is consistent with the direct interaction between the p6 domain of HIV Gag and the UEV domain of Tsg101 (3). PSCFP2-CHMP2A and -CHMP4B (ESCRT-III) cell lines expressing HIV Gag showed high cytosolic pools of PSCFP2 followed by membrane-localized co-clustering with Gag clusters (Fig. 2, A to D, and fig. S6B). When coclustering was observed, the 3D distribution of ESCRT-III and Gag at assembly sites was similar and distinctly separable from the cytosolic pool of PSCFP2-CHMP2A or -CHMP4B (Fig. 2, A, B, and D). To correlate the apparent observation
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0.00280
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0.00659
0.10881
0.25122
0.00039
0.02700
0.08710
0.00014
0.02707
0.11940
PSCFP2-CHMP2A Plasma membrane (tdEOS-CAAX)
Z X
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Gag-FLAG Tsg101
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Fig. 2. Two-color 3D superresolution imaging E localizes ESCRT-I and ESCRT-III subunits within Gag-FLAG PSCFP2-CHMP2A the HIV Gag lattice of budding particles. (A and B) HIV Gag fused internally to mEOS2 protein (Gag-imEOS2) detects assembly sites containing ESCRT-I subunits Tsg101 [(A), top] and ESCRT-III subunits CHMP4B [(A), bottom] and CHMP2A (B) fused to PSCFP2 protein. (B) (Top) iPALM measurements resolve the cytoplasmic pool of PSCFP2Total Probability per nm2 CHMP2A (top arrowhead), peripheral membrane-associated 0.00085 0.00733 0.02057 Gag-imEOS2 (middle arrowhead), and densely coclustered 0.00011 0.00497 0.01953 Gag and CHMP2A structures extending from the membrane HIV Gag lattice. Scale bars, 50 nm. (E) Correlative two-color iPALM and SEM of (bottom arrowhead). Scale bars, 100 nm (A); 500 nm (B). (C) PSCFP2-CHMP2A two virus-like particles (white arrowheads) emanating from a cell expressing coexpressed with unlabeled HIV Gag and membrane marker tdEOS-farnesyl Gag-FLAG (red) and PSCFP2-CHMP2A (green). The left membrane bud shows (red) show displacement of a CHMP2A cluster (green) extending beyond the confinement of PSCFP2-CHMP2A to the head of the particle. The particle on the plane of the plasma membrane. Scale bar, 500 nm. (D) Single clusters repreright is at an earlier stage of budding, apparent from the extended lateral sentative of AF647-labeled Gag-FLAG and PSCFP2-ESCRT probes. Axial (top) fluorescence distribution (right arrowhead). Scale bar, 250 nm. and lateral (bottom) views show localization of PSCFP2-ESCRT probes within the
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7 FEBRUARY 2014
VOL 343
SCIENCE
www.sciencemag.org
REPORTS
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Fig. 4. Released VLPs contain ESCRT-I and ESCRTIII subunits, but reduced levels of CHMP2A relative to CHMP4B. (A) Quantitative fluorescence microscopy analysis of GFP-ESCRT and Gag-GFP containing single particles. The fluorescence intensity of Gag-GFP is predicted to arise from ~600 to 1000 molecules (assuming 3000 to 5000 Gag molecules per particle), suggesting that signal from GFP-ESCRT particles corresponds to hundreds of molecules per particle (data represent ≥25,000 particles from ≥2 independent samples for each probe). (B) Correlative two-color iPALM and scanning electron micrographs of a single released (top, red) Gag- and (top, green) CHMP4B-positive VLP. Image registration residual uncertainty of 11 nm. Scale bar, 50 nm. (C) Western blot analysis of purified VLP fraction from GFP-ESCRT stable cell lines. GFP-Tsg101 and GFP-CHMP4B are detected in released particles (top); however, GFP-CHMP2A levels are dramatically reduced from VLPs upon particle release (bottom; asterisk indicates proteolysis product). Reduction in the levels of GFP-CHMP2A in released particles correlates between the biochemical and fluorescence data. (D) Proposed virus-scaffolding model for ESCRT-mediated HIV particle release from host cells. A pre-scission pool of ESCRT-I subunit Tsg101 (gray) interacts with HIV Gag (red), leading to assembly of ESCRT-III subunits CHMP4B and CHMP2A (green). Hypothetical ESCRT-III polymerization beyond the Gag lattice and Vps4 (orange) remodeling of CHMP2A subunits leads to membrane aperture constriction and particle abscission (middle and right).
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Fig. 3. Single-cluster averaging demonstrates ESCRT subunits within the interior of the HIV Gag lattice. (A to D) Cell-associated HIV Gag-FLAG clusters from PSCFP2-ESCRT–expressing cells were subjected to 3D single-cluster averaging [(A) PSCFP2-Tsg101, n = 313 clusters; (B) PSCFP2-CHMP2A, n = 460 clusters; (C) PSCFP2-CHMP4B, n = 509 clusters; and (D) PSCFP2CHMP4B/HIV Gag DPTAP, n = 278 clusters]. Two-color isosurfaced probability densities show a high-probability core of PSCFP2-Tsg101 (A) and ESCRT-III subunits PSCFP2-CHMP2A/CHMP4B [(B) and (C)] (green) with respect to the HIV Gag lattice (red) (isosurface threshold, HIV Gag-FLAG = 2.5 × 10−4 nm−3 and PSCFP2-ESCRTs = 2.5 × 10−5 nm−3). Right insets [(A) to (D)] depict 40-nm sections along the z axis [top HIV Gag (red), bottom PSCFP2-ESCRT (green)]. (D) A marked reduction in the probability density of PSCFP2-CHMP4B signal was observed upon expression of release-defective HIV Gag-FLAG DPTAP. (E) Radial average plots of sections from (A) to (D) define the highest-probability densities of ESCRT (green) subunits residing within the HIV Gag probability shell (red). Approximately 90% of PSCFP2-Tsg101 (top left), 80% of PSCFP2-CHMP2A (top right), and 61% of PSCFP2-CHMP4B (bottom right) integrated probability reside within the halfmaximum Gag probability shell, whereas 10% of the integrated probability for PSCFP2-CHMP4B remains when ESCRT-I recruitment is inhibited (Gag-FLAG DPTAP; bottom right). Line thickness represents SEM and is
