1. Physics of Living Systems
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Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

  1. Kaushik Inamdar
  2. Feng-Ching Tsai
  3. Rayane Dibsy
  4. Aurore de Poret
  5. John Manzi
  6. Peggy Merida
  7. Remi Muller
  8. Pekka Lappalainen
  9. Philippe Roingeard
  10. Johnson Mak
  11. Patricia Bassereau
  12. Cyril Favard
  13. Delphine Muriaux  Is a corresponding author
  1. Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, France
  2. Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, France
  3. CEMIPAI, CNRS UAR3725, University of Montpellier, France
  4. Institute of Biotechnology, University of Helsinki, Finland
  5. MAVIVH UMR Inserm U1259, University of Tours, France
  6. Institute for Glycomics, Griffith University, Australia
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Cite this article as: eLife 2021;10:e67321 doi: 10.7554/eLife.67321

Abstract

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

Introduction

The cell plasma membrane is a dynamic structure, where crucial processes such as endocytosis and exocytosis take place through local membrane deformations. Several pathogens, such as bacteria and enveloped viruses, interplay with the plasma membrane in the course of their replication cycle. Pathogens often enter the cells by endocytosis (Grove and Marsh, 2011; Gruenberg and van der Goot, 2006) and exit by membrane vesiculation (Rheinemann and Sundquist, 2020; Welsch et al., 2007), which are processes linked to the generation of plasma membrane curvature; either inward or outward. HIV-1 is an enveloped positive-strand RNA virus belonging to the family Retroviridae, and it is known to assemble and bud outward from the host cell plasma membrane (Coffin et al., 1997). The structural Gag polyprotein of HIV-1, by itself, is responsible for particle assembly (Gheysen et al., 1989), and it can oligomerize at the inner leaflet of the plasma membrane forming virus-like particles (VLPs). The force required to bend the membrane to achieve VLP formation has been proposed to be provided by Gag self-assembly (Hurley et al., 2010). The self-assembly of Gag has also been recently shown to segregate specific lipids (Favard et al., 2019; Yandrapalli et al., 2016) and proteins (Sengupta et al., 2019), generating plasma membrane domains that could favor budding (Foret, 2014; Lipowsky, 1993). However, only a small proportion of Gag-initiated clusters reach the fully assembled state leading to VLP release in living CD4+ T cells (Floderer et al., 2018), while a majority of these clusters lead to aborted events. Therefore, the mechanism by which the virus overcomes the energy barrier associated with the formation of the full viral bud remains an open question. Recently, a coarse-grained model of HIV assembly has shown that the self-assembly of Gag might not be sufficient to overcome this energy barrier (Pak et al., 2017), leaving the assembly in intermediate states. This supports the fact that other factors may be necessary to assist Gag self-assembly during the generation of new VLPs.

Plasma membrane curvature can also be generated by diverse host cell proteins. For example, I-BAR domain proteins sense and induce negative membrane curvature at the nanometer scale (a few tens to one hundred nanometers), that is, in the HIV-1 particle diameter size range, while generating outward micrometer-scale membrane protrusions such as membrane ruffles, lamellipodia, and filopodia. IRSp53 was first discovered as a substrate phosphorylated downstream of the insulin receptor (Yeh et al., 1996). It is also the founding member of the membrane curving I-BAR domain protein family, whose other mammalian members are MIM (missing-in-metastasis), ABBA (actin-bundling protein with BAIAP2 homology), PinkBAR (planar intestinal and kidney-specific BAR domain protein), and IRTKS (insulin receptor tyrosine kinase substrate) (Zhao et al., 2011). The latter, IRTKS, displays functional redundancy with IRSp53 (Chou et al., 2017; Millard et al., 2007) in being able to curve membranes. In addition to interactions with the plasma membrane, IRSp53 binds both Rac1 through its N-terminal I-BAR domain (Miki et al., 2000) and Cdc42 directly through its unconventional CRIB domain (Krugmann et al., 2001), and also downstream effectors of these GTPases such as WAVE2, Mena, Eps8, and mDia can bind IRSp53 through the SH3 domain. Thus, IRSp53 functions as a scaffold protein for the Rac1/Cdc42 cascade (Scita et al., 2008). IRSp53 was reported to exhibit a closed inactive conformation that opens synergistically upon binding to Rac1/Cdc42 and effector proteins (Disanza et al., 2013Kast et al., 2014; Miki and Takenawa, 2002; Suetsugu et al., 2006). Regulation of IRSp53 activity was recently shown to occur through its phosphorylation and interaction with 14-3-3 (Kast and Dominguez, 2019). Structurally, the I-BAR domain of IRSp53 is composed of a rigid six alpha-helix bundle dimer that is crescent-shaped. Due to its concave membrane-binding surface and lipid interactions, IRSp53 is able to generate negative membrane curvature (Zhao et al., 2011). While capable of forming homo-dimers, IRSp53 is also able to recruit and form hetero-dimers with other proteins to form clusters for the initiation of membrane curvature (Disanza et al., 2013).

Since the Rac1/IRSp53/Wave2/Arp2/3 signaling pathway is involved in the release of HIV-1 particles (Thomas et al., 2015), we hypothesized that IRSp53 may be a prime candidate for membrane remodeling required during viral bud formation. Hence, we investigated the possible role of IRSp53 and its membrane curvature generating activity in HIV-1 Gag assembly and particle budding. Importantly, we discovered that IRSp53 is present in an intracellular complex with HIV-1 Gag at the cell membrane, incorporated in Gag-VLPs and it is associated with purified HIV-1 particles, supporting IRSp53’s function in HIV-1 assembly as a facilitator of optimal HIV-1 particle formation through its membrane-bending activity. Thus, we identified IRSp53 as an essential non-redundant novel factor in HIV-1 replication, and demonstrated that it is critical for efficient HIV-1 membrane curvature and full assembly at the cell plasma membrane.

Results

IRSp53 knockdown decreases HIV-1 Gag particle release by arresting its assembly at the cell plasma membrane

We report here that the partial knockdown of IRSp53 expression reduces HIV-1 particle release in host Jurkat T cells and in the model cell line HEK293T (Figure 1a,b), similar to our previously reported results in primary T lymphocytes (Thomas et al., 2015). Cells were treated with siRNA targeting IRSp53 or IRTKS (validated by extinction of the transfected ectopic IRSp53-GFP or IRTKS-GFP proteins – Figure 1—figure supplement 1b and c, respectively). In Jurkat T cells, we expressed the viral Gag proteins in the context of HIV-1(ΔEnv) in order to only monitor the late steps of the viral life cycle. Partial IRSp53 gene expression knockdown (resulting in a maximum of 50% protein depletion) reduced particle release threefold as compared to the control siRNA (Figure 1a, bottom), and a sixfold reduction was determined when taking into account the percentage of protein depletion. This reduction in HIV-1 particle release is highly significant (n = 3 independent experiments, p value = 0.00265, Student’s t-test, and ANOVA statistical test for multiple comparisons indicate p value = 0.0089) since the gene editing of IRSp53 cannot be complete, nor edited by CRISPR/Cas9 knockout, without being toxic for the cells. To compare the role of different I-BAR domain proteins from the same family, we also measured the effect of siRNA targeting IRSp53 and IRTKS (Figure 1b) on HIV-1 Gag VLP production in HEK293T cells (Figure 1—figure supplement 1a, see graph Figure 1b, and immunoblots Figure 1—figure supplement 1d,e). IRTKS shares similar protein domain organization and high sequence homology with IRSp53 (40% amino acid sequence identity and 59% sequence similarity, Figure 1—figure supplement2), and displays some functional redundancy with IRSp53 (Chou et al., 2017). IRTKS can also induce plasma membrane curvature (Saarikangas et al., 2009). Partial knockdown of IRSp53 (~50% protein depletion) resulted in a two- to threefold decrease in HIV-1 Gag particle production (n = 3 independent experiments, p value = 0.000487, Student’s t-test) (Figure 1b, bottom, Figure 1—figure supplement 1d). In contrast, knockdown of IRTKS (Figure 1b, Figure 1—figure supplement 1e) did not have any significant effect on HIV Gag particle release (n = 3 independent experiments, p value = 0.0924, Student’s t-test), thus precluding the possibility of redundant functions between IRSp53 and IRTKS in the context of HIV-1 Gag particle formation.

Figure 1 with 3 supplements see all
Partial knockdown of IRSp53 decreases HIV-1 Gag particle release by arresting assembly at the cell plasma membrane.

(a) siRNA knockdown of IRSp53 expression in Jurkat T lymphocytes leads to a significant decrease in pNL4-3ΔEnv Gag particle release (see graph and immunoblots for IRSp53, p = 0.00265, Student’s t-test, and loading controls beneath the graph). (b) Similarly, knockdown of IRSp53 expression in Gag expressing HEK293T cells led to a significant decrease in HIV-1 Gag particle release (p = 0.00487, Student’s t-test), as compared to siRNA IRTKS (p = 0.0116, Student’s t-test). On the other hand, knockdown of IRTKS expression (a closely related I-BAR protein) did not have a significant effect on particle release (p = 0.0924, Student’s t-test, upper graph, immunoblots for IRSp53, IRTKS, and loading controls beneath the graph) (n = 3 independent experiments). Another multiple comparisons statistical test ANOVA was applied to compare the three siRNA conditions showing a significative difference with a p value = 0.0089. (c) Transmission electron microscopy images of HEK293T cells expressing HIV-1 Gag with siRNA control (upper panel) and siRNA IRSp53 (lower panel). Scale bar is 0.5 µm (upper image) and is 1 µm (lower image). (d) Transmission electron microscopy zoomed images of viral buds from HIV-1 Gag expressing cells treated with siRNA-mediated knocked down of IRSp53 (lower panel) showing arrested buds at the plasma membrane as compared to the siRNA control cells (upper panel) which display a normal range of buds in different stages of assembly and budding (scale bar = 100 nm). (e) Measurement of the bud dimensions (height and width median with interquartile) in the control siRNA and siRNA IRSp53 conditions (n = 145 buds from 14 different cells for each condition, n = 2 independent experiments). The knocked down cells exhibit a narrow range of heights corresponding to the arrested buds visible in the images, while the control cells display a wider range of heights corresponding to assembly progression (left graph, ‘Height of bud’). Distribution of the height values in the two conditions is significantly different (p = 1.05 × 10−28, Kolmogorov-Smirnov test). On the opposite, the widths of the buds in both conditions did not display significant differences in distributions (p = 0.0609, Kolmogorov-Smirnov test).

Figure 1—source data 1

Immunoblot Quantification using Fiji for Figure 1B graph.

https://cdn.elifesciences.org/articles/67321/elife-67321-fig1-data1-v2.xlsx
Figure 1—source data 2

Heights and Widths of HIV-1 Gag bud measurements for Figure 1E graph.

https://cdn.elifesciences.org/articles/67321/elife-67321-fig1-data2-v2.xlsx

Electron microscopy imaging of siRNA IRSp53 treated HEK293T cells expressing HIV-1 Gag revealed arrested particle budding at the cell plasma membrane (Figure 1c, lower panel), as compared to the siRNA-control cells (Figure 1c, upper panel). While the control cells exhibited the normal phenotype of Gag-VLP budding from the cell plasma membrane, the IRSp53 knockdown cells displayed a series of viral buds arrested in assembly decorating the cell plasma membrane (Figure 1c, Figure 1—figure supplement 3). These results revealed an arrest in Gag assembly at the membrane and thus the involvement of IRSp53 in the assembly process. Since IRSp53 is an I-BAR protein involved in cell membrane curvature, we measured the curvature exhibited by HIV-1 buds in IRSp53 knockdown cells. While control cells displayed a range of HIV-1 Gag particles at different stages of assembly and budding, the cells knocked down for IRSp53 instead displayed arrested buds at an early assembly stage (Figure 1d). By measuring the dimensions of these arrested buds, we found that buds from cells knocked down for IRSp53 displayed a narrower range of curvature height (48 ± 22 nm), as compared to the control (85 ± 53 nm) (n = 145 buds from 14 different cells, p value = 1.053 × 10−28, Kolmogorov-Smirnov test), while the bud widths presented no difference between siIRSp53 (135 ± 64 nm) and the control (140 ± 87 nm) (n = 145 buds from 14 different cells, p value = 0.0609, Kolmogorov-Smirnov test) (Figure 1e). The control cells thus exhibited a range of heights and widths consistent with the range of buds seen at the membrane of these cells. The result indicates that in the absence of IRSp53, the viral buds were unable to progress beyond a certain curvature.

HIV-1 Gag expression in cells increases IRSp53 membrane binding and allows their complexation

Since both Gag and IRSp53 target the cell plasma membrane upon interaction with PI(4,5)P2 (Favard et al., 2019; Mattila et al., 2007; Prévost et al., 2015; Saarikangas et al., 2009; Sengupta et al., 2019; Takemura et al., 2017; Yandrapalli et al., 2016), we then tested if Gag and IRSp53 could associate directly or indirectly using immuno-precipitation (IP) assays (Figure 2a,b). Our results showed that IP of endogenous IRSp53 resulted in co-precipitation of Gag (Figure 2a, lane 1), as compared to the controls (lanes 2–4). Unfortunately, we could not assess the amount of IRSp53 pulled down by the antibody between conditions because the IgG signal masked the endogenous IRSp53 signal. To overcome this issue, we performed an IP/co-IP experiment of ectopic IRSp53-GFP/HIV-1 Gag proteins with an anti-GFP antibody, and confirmed the pull-down of IRSp53-GFP (Figure 2b, lanes 2 and 3) and the co-precipitation of Gag (lane 3) while nothing was detected in the controls (lanes 1 and 4). Input and flowthrough were in accordance with the results, showing less IRSp53-GFP in the flowthrough (lane 3). We then concluded that HIV-1 Gag and endogenous IRSp53, or ectopic IRSp53-GFP, were components of the same intracellular complex, interacting directly or indirectly through other factors or membrane domains. IRSp53 is a cellular protein that switches from the cytosol to the cell plasma membrane for inducing membrane ruffles upon activation by Rac1 and its effectors (Miki and Takenawa, 2002; Suetsugu et al., 2006). We have previously shown that Gag cellular expression triggers Rac1 activation (Thomas et al., 2015), on which IRSp53 membrane localization and function are dependent. Thus, here, we compared the relative membrane binding of IRSp53 upon cellular expression of HIV-1 Gag using membrane flotation assays (Figure 2c,d). In the absence of Gag (‘HEK293T control' cells), one could observe the presence of IRSp53 both in the cytosol (Figure 2c, fractions 6–8, labeled with the ribosomal S6 biomarker) and in the cell membranes fractions (Figure 2c, fractions 1–3, labeled with the membrane Lamp2 biomarker). The lysosomal membrane protein Lamp2 and the cytosolic ribosomal protein S6 were used as controls to validate the correct separation of the membrane and cytosolic fractions. Thus, at equilibrium, 19 ± 8% of IRSp53 was bound to the cell membranes (Graph, Figure 2d). The same experiment was repeated with cells expressing HIV-1 Gag, where 66 ± 9% of Gag was bound to the cell membranes (Graph, Figure 2d). Notably, we observed a twofold increase with 44 ± 5% of IRSp53 bound to the cell membranes upon HIV-1 Gag expression (Graph Figure 2d) (n = 5 independent experiments, p value = 0.000129, Student’s t-test), while none with IRTKS. This effect was comparable with the one of Tsg101, a protein of the ESCRT-I complex known to interact mainly with the p6 domain of Gag (Garrus et al., 2001; Pornillos et al., 2003; von Schwedler et al., 2003) and partially with the NC domain (El Meshri et al., 2018). The cellular endosomal sorting complex required for transport (ESCRT) machinery has been involved in the mechanism of vesicular budding of intracellular multi-vesicular bodies and also hijacked by the HIV-1 Gag protein for viral particle budding. Here, we observe a twofold increase in cell membrane binding of Tsg101 upon Gag expression, passing from 36 ± 10% without Gag to 79 ± 8% in the presence of Gag (Figure 2c) (n = 5 independent experiments, p value = 0.00517, Student’s t-test).

Figure 2 with 1 supplement see all
Intracellular HIV-1 Gag and IRSp53 complexation and cell membrane binding.

(a) Co-immunoprecipitation of HIV-1 Gag/IRSp53 with an anti-IRSp53 antibody. HIV-1 Gag is enriched in the anti-IRSp53 pulldown (lane 1), as compared to the controls (lane 2: IP with an anti-rabbit serum; lane 3: no antibody; lane 4: mock, i.e., without Gag). (b) Co-Immunoprecipitation of HIV-1 Gag/ectopic IRSp53-GFP with an anti-GFP antibody upon overexpression of HIV-1 Gag and ectopic IRSp53-GFP in transfected HEK293 T cells. HIV-1 Gag is enriched in the anti-IRSp53-GFP pulldown (lane 3: transfected HEK293T cell lysate containing IRSp53-GFP and HIV-1 Gag), as compared to the controls (lane 1: mock without Gag or IRSp53-GFP; lane 2: IRSp53 alone; lane 4: Gag alone). Input, IP anti-GFP and flowthrough after IP are shown. (c) Membrane flotation assay protocol: (1) 293T cells were dounced, (2) the post-nuclear supernatant was loaded on a discontinuous sucrose gradient, and (3) following ultracentrifugation, cell membranes (lysosomal associated membrane protein, Lamp2 biomarker, fractions 1–3) were separated from the cytosolic fraction (ribosomal S6 protein biomarker, fractions 6–8). (d) Immunoblots of the indicated proteins (on the left) and quantification of the % of protein membrane binding in the graph below show that upon HIV-1 Gag expression in cells, IRSp53 is significantly enriched by twofold in the cell membrane fraction (p value = 0.000129; ***Student’s t-test) (n = 5 independent experiments). A similar increase is observed for Tsg-101, a known interactor of the p6 domain of Gag (p value = 0.00517; **, Student’s t-test).

Figure 2—source data 1

Membrane flotation Quantification using Fiji for Figure 2D graph.

https://cdn.elifesciences.org/articles/67321/elife-67321-fig2-data1-v2.xlsx

Furthermore, we examined if Gag/IRSp53 complexation was dependent on the p6 domain of Gag to reveal if this could be independent of ESCRT recruitment by Gag. We, thus, used a C-terminal mutant of Gag, GagΔp6, which is deficient in ESCRT-Tsg101 recruitment (von Schwedler et al., 2003), but is still capable of binding the plasma membrane and assembling particles that poorly bud. GagΔp6 viral particles are tethered and remain attached to the plasma membrane (see Floderer et al., 2018 for the characterization of Gag(i)mEos2Δp6). Our experiments revealed that Gag, Gag(i)mEos2, and GagΔp6(i)mEos2 were all pulled down with IRSp53 (Figure 2—figure supplement 1), showing that the addition of the internal mEos2 protein - a necessary tag for super resolution microscopy (SRM) imaging of Gag (see the following section) - did not affect the complexation of Gag with IRSp53. This was to show that the Gag used in the SRM imaging studies behaved similarly to wild-type Gag. Moreover, we showed that the p6 domain was not required for Gag/IRSp53 molecular interplay suggesting that it occurs before ESCRT recruitment.

Taken together, these results suggest that there is a complexation between HIV-1 Gag and IRSp53, reinforcing the idea of a strong molecular interplay between these two proteins directly or indirectly but in the same membrane domain. We observed that cellular Gag expression, possibly by triggering Rac1 activation (Thomas et al., 2015), favors cell membrane binding of IRSp53.

Single-molecule localization microscopy reveals IRSp53 surrounding HIV-1 Gag assembly sites

Our finding that IRSp53 and HIV-1 Gag are present in the same molecular complex at the cell membrane motivated us to assess whether IRSp53 was present specifically at the Gag assembly sites. Because HIV-1 assembly is ~100 nm in diameter (Floderer et al., 2018; Manley et al., 2008), we used PALM (Photo-Activated Localization Microscopy) coupled to dSTORM (direct Stochastic Optical Reconstruction Microscopy) with TIRF illumination, to investigate with high precision the localization of I-BAR proteins in Gag(i)mEos2 assembly sites at the plasma membrane (Figure 3, Figure 3—figure supplement 1, Figure 3—figure supplement 2).

Figure 3 with 4 supplements see all
Super-resolution microscopy imaging reveals preferential IRSp53 localization at HIV-1 Gag budding sites.

(a, b) Super-resolved PALM/STORM dual-color images of HEK293T cells expressing Gag(i)mEos2 (green) and immunolabeled for IRSp53 (red, a) or IRTKS (red, b) (scale bar = 10 µm) with a magnified view (scale bar = 500 nm) and selected zoom-in images of single Gag(i)mEos2 clusters (scale bar = 100 nm). (c, d) Quantification of coordinate-based colocalization (as in Malkusch et al., 2012) at Gag assembly sites: CBC values for IRSp53 (c) and IRTKS (d) were plotted as relative frequencies. IRSp53 CBC distribution (c) shows that 27% of all IRSp53 localizations are highly correlated with Gag(i)mEos2 localizations (>0.5). On the other hand, only 14% of IRTKS localizations (d) are highly correlated, while IRTKS CBC distribution exhibits a peak of anti-correlated/non-correlated (−0.5 to 0) localization. (e, f) Super-resolved dual-color PALM/STORM images of Jurkat T cells expressing Gag(i)mEos2 (green) and immunolabeled for IRSp53 (red, e) or IRTKS (red, f) with a magnified view (scale bar = 500 nm) and single Gag(i)mEos2 cluster zoom (scale bar = 100 nm). (g, h) Relative frequency distribution plot of the CBC values for IRSp53 (g) and IRTKS (h). (g) shows that 26% of IRSp53 localizations are highly correlated with Gag(i)mEos2 localizations (>0.5) while (h) shows that IRTKS localisations are mainly anti-correlated to non-correlated (−0.5 to 0) with Gag(i)mEos2 localizations.

We first checked our ability to correctly identify ongoing assembly sites by quantifying the sizes of Gag clusters. For this, we used purified HIV-1 Gag(i)mEos2 VLPs (Figure 3—figure supplement 3b). When using the same size estimation method described in Floderer et al., 2018, we measured individual VLP sizes around 130–140 nm (Figure 3—figure supplement 3b). VLP size was correctly estimated (114 ± 37 nm, median ± 1st IQR) from the size distribution of 800 different clusters identified in our VLP images using a density-based spatial scan (DBSCAN) clustering method (Figure 3—figure supplement 3b). Finally, DBSCAN showed us that these HIV-1 Gag(i)mEos2 cluster size distributions had similar median values for each cell type in each condition (HEK 293T (96 ± 44 nm, Figure 3—figure supplement 3c) or Jurkat T cells (105 ± 66 nm, Figure 3—figure supplement 3e) when IRSp53 was immunolabelled, HEK 293T (116 ± 68 nm, Figure 3—figure supplement 3d) or Jurkat T cells (96 ± 50 nm, Figure 3—figure supplement 3f) when IRTKS was immunolabelled), allowing us to directly compare IRSp53 and IRTKS organization close to these assembly sites.

In HEK293T and Jurkat T cells, reconstructed dual-color PALM/STORM images exhibited Gag(i)mEos2 assembly sites close to or overlapping with IRSp53 (Figures 3a,eFigure 3—figure supplement 1aFigure 3—figure supplement 2a) whereas Gag clusters did not seem to overlap with IRTKS (Figures 3b,f, Figure 3—figure supplement 1b, Figure 3—figure supplement 2b). These results are consistent with the siRNA data presented in Figure 1b. In order to quantify these observations, we performed coordinate-based colocalization (Malkusch et al., 2012) (CBC) analysis of HIV-1 Gag and IRSp53 (or IRTKS) in close proximity of HIV-1 assembly sites. We first isolated the assembly sites by segmentation and retrieved their center positions. We then kept all HIV-1 Gag located within a distance of 80 nm from this center (70–80% of all cluster sizes measured by DBSCAN are found within this distance) and all the IRSp53 (or IRTKS) found in a distance of 150 nm from this center (~twofold the assembly site size, see Figure 3—figure supplement 4 for details on the process workflow). We chose this IRSp53 (or IRTKS) cutoff distance to minimize the contribution of cross-colocalization between different HIV-1 Gag clusters. In contrast to classical colocalization analysis, CBC takes into account the spatial distribution of biomolecules to avoid excessive colocalization due to local densities of single molecules and provides a colocalization value for each single-molecule localization. This CBC value ranges from −1 to +1, where −1 corresponds to anti-correlation, 0 indicating non-correlation, and +1 corresponds to perfect correlation between the two molecules. Since CBC values are calculated for each localization, we plotted the CBC values as frequency distributions for all localizations of IRSp53/Gag and IRTKS/Gag. As shown in Figure 3c (for n = 4 HEK293T cells) or in Figure 3g (for n = 5 Jurkat T cells), the CBC distribution for IRSp53/Gag has a higher proportion of values exhibiting high colocalization (27% in HEK293T cells or 26% in Jurkat T cells of CBC > 0.5) in comparison to IRTKS/Gag values (14% for both cell types, n = 5 for HEK293T and Jurkat T cells of CBC > 0.5) which instead show a very high proportion (close to 75%) of anti-correlation (CBC < 0) (Figure 3d,h). This comparison directly shows that IRSp53 displays stronger single-molecule colocalization with Gag in assembling clusters than IRTKS does.

Although CBC values give a quantitative value of the colocalization, it does not provide direct information on the average positions of IRTKS or IRSp53 molecules with respect to Gag molecules within the assembling clusters. Moreover, as for all the colocalization methods used to analyze molecule proximities, the CBC method is a strongly parameterize method, the results of which can depend on the parameter values (total distance of search for colocalization, number of searching circles…). Thus, to gain more insight into these colocalization quantification, we performed simulations to generate different patterns of PALM/STORM localizations (Figure 4), and analyzed them with the same set of parameters (total distance and distance steps) that the one we used for the experimental data. Since IRSp53 and IRTKS are membrane curvature sensors and since we observed that a lack of IRSp53 stops the assembling bud at half completion (Figure 1e), we hypothesis that they will be located around the ongoing assembly site. We therefore simulated an annulus of IRSp53 or IRTKS STORM localizations with different waists located at increasing distances from the center of Gag clusters (Figure 4a and Figure 4—figure supplement 1). Numerically generated images were then analyzed identically to our experimental images (see 'Materials and methods' for details) and CBC distributions were generated (Figure 4b). Experimental and simulated cumulative distribution functions of the CBC values were compared by performing a root mean square error (RMSE) quantification (Figure 4—figure supplement 1, g,h,i,j). The lowest RMSE value was considered as the best similarity between the two distributions. This comparison indicated that IRSp53 localization, on average, displays a restricted pattern around and in the assembly sites. This corresponds to a circular ring surrounding the assembly site at 80 nm from the center of the Gag budding sites with a width of 80 nm (Figure 4c,e). On the other hand, IRTKS was present as a large diffuse pattern located at 140 nm from the Gag assembly site center with a width of 200 nm, explaining why fewer IRTKS molecules were detected in the assembly sites (Figure 4d,f). Interestingly, the maps of the RMSE values show that, on average, the surrounding belt center (independently of its waist) is located nearer to the HIV-1 Gag assembly center in the case of IRSp53 as compared to that of IRTKS (Figure 4—figure supplement 1g,h,i,j). Our results thus show that IRSp53 indeed specifically localizes at HIV-1 Gag assembly sites at the cell plasma membrane, whereas IRTKS poorly does.

Figure 4 with 1 supplement see all
Numerical simulations confirm preferential IRSp53 localization around HIV-1 Gag budding sites.

(a) Schematic representation of the different configurations used in the numerical simulation mimicking belts of given waists (w) of IRSp53 or IRTKS localizations (red) surrounding an HIV-1Gag(i)mEos2 assembly site (green) at a given distance ( d). (b) Numerically simulated cumulative distribution function of CBC values obtained for a 20 nm waist belt of IRSp53 or IRTKS at different distances from 0 to 160 nm (top). Below are found the schematic representations (upper part, input) and the ThunderSTORM reconstructed images obtained from the simulated positions (lower part, output, see 'Materials and methods' for details) for the 20 nm waist belt at different positions. (c, d, e, f) Experimental CBC values for IRSp53 in HEK (c) or Jurkat T cells (e) and IRTKS in HEK (d) and Jurkat T cells (f) were plotted as cumulative frequency distributions and compared to simulated distributions obtained at different distances and structures (see Figure 4—figure supplement 1 for the whole data sets). For both cell type, IRSp53 shows a cumulative CBC distribution corresponding to a simulation with a waist of 40 nm (width 80 nm) at a distance of 80 nm (left graph, bold gray lines correspond to the experimental data for IRSp53, bold blue lines correspond to the simulated values closest to experimental data, see Figure 4—figure supplement 1g,h,i,j for statistics). IRSp53 thus corresponds to a restricted pattern in and around a Gag assembly site (panel 1 schematic of simulated data, panel 2 simulated data, and panel 3 experimental data). On the other hand, the IRTKS experimental CBC distribution (bold gray line in the graph) is similar to simulations with a waist of 100 nm (width 200 nm) at a distance of 140 nm (bold red line). IRTKS is more diffuse and spreads out (panel 1 schematic of simulated data, panel 2 simulated data, and panel 3 experimental data). Scale bar in the panels = 100 nm.

The involvement of IRSp53 around Gag assembly sites seems to be conserved regardless of the cell type, reinforcing the idea of a specific role for IRSp53 in HIV-1 Gag particle assembly.

IRSp53 is incorporated in HIV-1 particles

To assess IRSp53 incorporation into HIV-1 Gag particles, we purified Gag-VLPs from cells transfected with Gag-mCherry and several GFP-tagged I-BAR domain proteins (Figure 5a). The IRSp53-I-BAR-GFP construct only contains the membrane curving I-BAR domain of IRSp53. PH-PLCδ-GFP, a PI(4,5)P2 binding protein, was used as a control, because it binds PI(4,5)P2 but does not generate membrane curvature. Fluorescent VLPs were purified from these transfected cells, then visualized for two colors (green: GFP and red: mCherry), and then a Mander’s coefficient was calculated as an indicator of incorporation of the ectopic (green) GFP-tagged proteins within the (red) Gag-mCherry VLPs and vice-versa (see Materials and Methods) (Figure 5a,b). We found a high correlation (Mander’s coefficient = 0.95–1) between IRSp53-GFP and Gag-mCherry (Figure 5b, left graph, red column), indicating that almost all Gag-mCherry VLPs contained IRSp53-GFP, while the reverse (green column) was not true, indicating that all the IRSp53-GFP-labelled vesicles produced by the cells were not all positive for Gag. When using the IRSp53-I-BAR domain alone, we also obtained a high Mander’s coefficient, that is, ~0.8 (Figure 5b). In contrast, for IRTKS-GFP, the Mander’s coefficient was 0.4–0.5, indicating no significant correlation between IRTKS-GFP and Gag-mCherry. This indicates that even if a domain (PH-PLCδ) or another I-BAR protein (IRTKS) can recognize PI(4,5)P2 at the cell membrane, it is not sufficient for incorporation into Gag-VLPs. Finally, we found that the reverse correlation of GFP-tagged proteins with Gag-mCherry was under the random 0.5 coefficient (Figure 5b, right graph, green column), indicating that, on average, GFP-tagged vesicles produced by the cells did not contain Gag. Taken together, these results show the preferential incorporation of IRSp53-GFP into released HIV-1 Gag-mCherry VLPs.

IRSp53 is incorporated into HIV-1 particles in a Gag-dependent manner.

(a) Schematic for the protocol followed for imaging and analysis. HIV-1 Gag VLPs were purified from HEK293T cells expressing HIV-1 Gag/Gag-mCherry and IRSp53-GFP, or other GFP tagged proteins binding PI(4,5)P2 (IRSp53-IBAR-GFP, IRTKS-GFP, and PH-PLCδ-GFP). Purified Gag VLPs were then spotted over a poly-lysine treated glass slide and imaged by TIRF-Microscopy (particles were imaged in the red, and IRSp53 in the green channel). For each condition, 3000 particles (~300 particles/image, 10 images) were counted. Fluorescence correlation (Mander’s coefficient, see Materials and Methods for details) was determined for Gag-mCherry and for IRSp53-GFP, IRTKS-GFP, and PH-PLCδ-GFP and reported in the graphs. (b) The 0.5 value indicates a random incorporation level (indicated by black line across the graph). IRSp53-GFP and IRSp53-IBAR show high correlation values (0.95–1 and 0.8, respectively). The other I-BAR domain proteins were not significantly correlated with Gag-mCherry particles (0.4–0.5). PH-PLCδ-GFP, a known marker of the phospholipid PI(4,5)P2, shows a slightly higher correlation (0.6), since HIV-1 Gag is known to associated with this phospholipid. (c) Incorporation of IRSp53 into wild-type pNL4-3 HIV-1 or (d) Gag VLPs revealed by immunoblots against Gag(p24), IRSp53, IRTKS, Tsg101, or actin, as indicated. Following a 25% sucrose cushion purification, IRSp53 was found to be associated with released wild-type HIV-1 (left panel) and Gag VLPs (right panel). Tsg101, known to be incorporated into released particles, was also associated with viral particles. IRTKS, a closely related I-BAR protein to IRSp53, was not incorporated in purified HIV-1 viral particles or Gag-VLPs. (e) Protocol of VLPs purification using sucrose cushions and an iodixanol gradient. Briefly, pellets obtained after ultracentrifugation of cell culture medium of HEK293T transfected with pNL4.3HIV-1 or pGag were deposed on an iodixanol gradient (20%, 30%, and 60%). 20 fractions of 200 µL were collected from the top of the tube. Fractions collected following an iodixanol gradient purification of NL4-3ΔPolΔEnv Gag VLPs were analyzed using Western blots for IRSp53 and Gag, TSG101 and ALIX, CD81 and CD63 revealed respectively on the same membrane (blots 1 and 2) revealing IRSp53 association with Gag viral particles and known cofactors.

To study the incorporation of endogenous IRSp53 in HIV-1 particles, cells were transfected with plasmids expressing either wild-type infectious HIV-1 or codon-optimized immature HIV-1 Gag protein (without genomic RNA). The virus particles were purified through a 20%-sucrose cushion or further through a continuous iodixanol density gradient (as in Grigorov et al., 2009 and Grigorov et al., 2006). IRSp53 was found to be associated with the viral particles in both conditions, that is, in infectious HIV-1 and in Gag VLPs, indicating that Gag alone is sufficient to recruit IRSp53 in the viral particles (Figure 5c,d, ‘IRSp53’). Tsg101 also showed an association with viral particles in both conditions (Figure 5c,d, ‘Tsg101’), as reported previously (Garrus et al., 2001; Hammarstedt and Garoff, 2004; Pornillos et al., 2003). In contrast, IRTKS was not associated neither with Gag-VLP nor HIV-1 particles (Figure 5c,d, ‘IRTKS’). Upon further purification (Figure 5e), IRSp53, and the ESCRT proteins, Tsg101 and ALIX, were found to be associated within the same fractions containing the HIV-1 Gag viral particles, together with other well-known viral particle cofactors such as CD81, CD63 tetraspanins (Grigorov et al., 2009; Grigorov et al., 2006). Thus, endogenous IRSp53 is most probably incorporated in HIV-1 particles in a Gag-dependent manner.

HIV-1 Gag is enriched at membrane tube tips generated by IRSp53 I-BAR domain

The results above demonstrate that not only is IRSp53 incorporated in Gag-VLPs, but it is present at the budding sites and its deletion strongly reduces HIV-1 particle release in a Gag-dependent manner by arresting its bud assembly at the cell plasma membrane (Figure 1). In order to advance molecular mechanistic understanding of the role of IRSp53 locally at Gag assembly sites, we assessed IRSp53 I-BAR/Gag interplay on model membranes giant unilamellar vesicles (GUVs). Previous in vitro studies showed that when placing IRSp53 I-BAR domain outside PI(4,5)P2-containing GUVs, the I-BAR domain can deform GUV membranes, generating tubes toward the vesicle interior (Jarin et al., 2019; Prévost et al., 2015; Saarikangas et al., 2009Jarin et al., 2021). Consistent with the previous observations, we observed I-BAR driven tubulations on GUVs at low I-BAR concentrations (0.005–0.06 μM, see Figure 6, Figure 6—figure supplement 1c as an example); surprisingly, when increasing I-BAR concentrations to 0.1 μM and up to 1 μM, we observed a decrease in the number of GUVs having tubes. Future work is required to investigate this seemingly puzzling observation.

Figure 6 with 2 supplements see all
IRSp53 I-BAR domain enhances Gag recruitment to GUV-membranes and at the tip of I-BAR domain-induced tubes.

(a) (Left) AX488 Gag fluorescence intensity on membranes in the absence of I-BAR domain (named ‘Gag only’), in the presence of I-BAR domain where GUVs were first incubated with I-BAR domain and then Gag (named ‘I-BAR + Gag’) and GUVs were first incubated with Gag and then I-BAR domain (named ‘Gag + I-BAR’). Each circle presents one GUV analysis. N = 82 GUVs, n = 4 sample preparations for ‘Gag only,’, N = 67 GUVs, n = 4 sample preparations for ‘I-BAR + Gag,’ and N = 104 GUVs, n = 4 sample preparations for ‘Gag + I-BAR’. To pool all data points from the four sample preparations, in each preparation for all three conditions, Gag intensities were normalized by the mean Gag intensity in the ‘Gag only’ condition. Protein bulk concentrations: 0.3 µM for AX488 Gag and 0.5 µM for I-BAR domain (not fluorescently labeled). (Right) Representative confocal images of AX488 Gag on GUV membranes in ‘I-BAR + Gag’ condition. To visualize GUV membranes, 0.5 mol% of BODIPY-TR-C5-ceramide was incorporated in the membranes. (b) Representative confocal images of AX594 Gag in I-BAR domain-induced tubules. Inverted grayscale images are shown for I-BAR domain and Gag. Protein bulk concentrations: 0.3 μM for AX594 Gag and 0.05 µM for I-BAR domain (70% unlabeled and 30% AX488 labeled I-BAR domain). Cyan arrowheads point out I-BAR domain-induced tubules and white arrows indicate the colocalization of Gag and I-BAR domain at the tips of the tubules. (c and d) Representative confocal images of AX488 Gag (green) in I-BAR domain-induced tubules. Protein bulk concentrations: in (c) 0.1 µM for AX488 Gag and 0.05 µM for I-BAR domain (not fluorescently labeled); in (d) 0.3 µM for AX488 Gag and 0.05 µM for I-BAR domain (not fluorescently labeled). To visualize GUV membranes, 0.5 mol% of BODIPY-TR-C5-ceramide (magenta) was incorporated in the membranes. In (c), inverted grayscale images were shown for membranes and Gag. The cyan arrowhead points out an I-BAR domain-induced tubule and white arrow indicates Gag signals at the tip of the tubule. Sorting map was obtained by calculating the fluorescence intensity ratio of Gag and membranes (see Material and Methods for more details). In (d), dashed white lines indicate I-BAR domain-induced tubules. Scale bars, (a–c) 5 µm and (d) 2 μm. GUV, giant unilamellar vesicle.

For our Gag membrane binding assay, we first used a high concentration of IRSp53 I-BAR domain (0.5 µM), while keeping the PI(4,5)P2 concentration constant. This was done in order to prevent the generation of tubes by IRSp53 I-BAR domain and to focus on analyzing the membrane-binding efficiency of Gag in the presence of IRSp53 I-BAR domain on flat, non-deformed GUVs. We found that Gag binding to GUV membranes is increased ~7-fold when IRSp53-I-BAR domain was introduced first on GUVs before adding Gag (median value 6.7), compared to the condition of Gag only (median value 0.9) (<0.0001, Student’s t-test) (Figure 6a, ‘Gag only’ vs. ‘I-BAR + Gag’). However, in the condition where Gag was introduced before adding the I-BAR domain, Gag intensity on GUV membranes increased only ~3-fold as compared to the Gag only condition (p < 0.0001, Student’s t-test) (Figure 6a, ‘Gag only’ vs. ‘Gag + I-BAR’). Notably, by comparing I-BAR + Gag and Gag + I-BAR conditions, we observed a twofold higher Gag intensity on GUV membranes in the first condition (< 0.0001, Student’s t-test) (Figure 6a, ‘I-BAR + Gag’ vs. ‘Gag + I-BAR’). Taken together, these results show that IRSp53 I-BAR domain facilitates Gag membrane binding on GUV in favor of increasing Gag concentration locally. Furthermore, given that in our cell experiments, we observed a relative increase of IRSp53 bound to the cell membranes upon HIV-1 Gag expression (Figure 2d), we also assessed if HIV-1 Gag could facilitate the membrane binding of IRSp53 I-BAR domain. Consistent with our cell experiment results, we observed a similar increase of the membrane binding (~1.5-fold) of IRSp53 I-BAR domain in the presence of HIV-1 Gag on GUVs (Figure 6—figure supplement 1a,b) suggesting a strong interplay between Gag and IRSp53 I-BAR domain.

Given these results, and that IRSp53 is a membrane curving protein involved in the early stages of cell protrusion generation (Disanza et al., 2013; Sathe et al., 2018), we asked whether the local membrane deformation induced by IRSp53 could be a preferred location for HIV-1 Gag assembly. We incubated GUVs with IRSp53 I-BAR domain at a low concentration (0.05 µM), which allows for the generation of inward membrane tubes, followed by the addition of Gag (Figure 6b, see Materials and Methods). This experiment revealed that Gag was sorted preferentially to the tips of the tubes generated by the IRSp53 I-BAR domain (Figure 6b,c,d, Video 1 and Figure 6—figure supplement 1c,d). We note that the majority of the tubes in GUVs were moving too rapidly, preventing us from acquiring images with high spatial resolution (see Videos 2 and 3). However, we observed that the Gag signals appeared dotty inside GUVs (Figure 6—figure supplement 2 and Videos 2 and 3), which is very different from the IRSp53 I-BAR domain signal that is clearly all along the tubes (Figure 6—figure supplement 1c). Moreover, for tubes that were not moving too fast, we found that for all the tubes (17 tubes, protein concentrations: 0.05 μM unlabeled I-BAR domain and 0.3 μM AX488 Gag), the Gag signal was exclusively located at the tips of the tubes (Figure 6d and Video 1). Finally, we observed that addition of HIV-1 Gag resulted in the formation of shorter I-BAR tubules as compared to GUVs incubated with the IRSp53-I-BAR domain alone (Figure 6—figure supplement 1c,d), indicating an interference in I-BAR tubule elongation when Gag sorted to the tubule tips, suggesting that Gag usurps the IRSp53 tubulation function.

Video 1
Imaging of an I-BAR domain-induced tubule having Gag signal at its tip.

Time-lapse imaging of an I-BAR domain-induced tubule. GUVs were first incubated with IRSp53 I-BAR domain (0.05 μM, unlabeled), followed by addition of HIV-1 Gag (0.3 μM, AX488 labeled, green). GUV membranes contained 0.5 mol% of BODIPY-TR-C5-ceramide (magenta). Frame interval = 0.6 s. Time in mm:ss. Scale bar = 2 μm. GUV, giant unilamellar vesicle.

Video 2
Imaging of a GUV having I-BAR domain-induced tubules.

GUVs were first incubated with IRSp53 I-BAR domain (0.05 μM, unlabeled), followed by addition of HIV-1 Gag (0.3 μM, AX488 labeled, green). GUV membranes contained 0.5 mol% of BODIPY-TR-C5-ceramide (magenta). Frame interval = 0.6 s. Time in mm:ss. Scale bar = 5 μm. GUV, giant unilamellar vesicle.

Video 3
Imaging of a GUV having I-BAR domain-induced tubules.

GUVs were first incubated with IRSp53 I-BAR domain (0.05 μM, unlabeled), followed by addition of HIV-1 Gag (0.3 μM, AX488 labeled, green). GUV membranes contained 0.5 mol% of BODIPY-TR-C5-ceramide (magenta). Frame interval = 0.6 s. Time in mm:ss. Scale bar = 5 μm. GUV, giant unilamellar vesicle.

Taken together, these results demonstrate that HIV-1 Gag binding to membranes is enhanced locally by the presence of IRSp53 I-BAR domain, and that Gag preferentially binds to highly curved membranes generated by the I-BAR domain of IRSp53.

Discussion

The findings of this study uncovered the role of the host cellular I-BAR factor IRSp53 in HIV-1 Gag assembly and membrane curvature upon bud formation. In vitro, on GUVs, we showed that the IRSp53 I-BAR domain enhances Gag membrane binding locally (Figure 6), and vice versa (Figure 6—figure supplement 1a,b), in agreement with cell membrane flotation assays also showing an increase of IRSp53 membrane retention upon Gag expression (Figure 2). Indeed, IRSp53 was found at, or in close vicinity to Gag assembly platforms at the cell membrane (Figures 3 and 4), and is incorporated into Gag-VLPs and in HIV-1 virions (Figure 5). Importantly, we revealed that the partial knockdown of IRSp53 gene expression arrested Gag assembly at the mid-bud formation stage (Figure 1) and that Gag preferentially localizes at the tube tips induced by IRSp53 I-BAR domain, interfering with its long tubule formation in vitro (Figure 6). Altogether, IRSp53 appears instrumental in membrane curvature upon HIV-1 budding and is locally subverted as an essential factor needed for full HIV-1 Gag particle assembly.

Using GUVs, we observed that Gag not only colocalizes with IRSp53 I-BAR domain on vesicles, but that the IRSp53-I-BAR domain increases Gag binding to these model membranes, mimicking the possible local Gag/IRSp53 interplay at the assembly site (Figure 6a). Indeed, BAR domain proteins, in general, and IRSp53 in particular, are known to induce strong PI(4,5)P2 clusters (Saarikangas et al., 2009; Zhao et al., 2013), PI(4,5)P2 was shown to play a role in Gag binding to the cell plasma membrane (Ono et al., 2004), as well as PI(4,5)P2 is strongly clustered during virus assembly (Favard et al., 2019; Ono et al., 2004; Yandrapalli et al., 2016). Thus, these results suggest that the membrane binding of Gag on IRSp53-enriched membrane domains could promote the plasma membrane binding of both proteins (Figure 6). This is in agreement with super-resolution imaging in cells, where Gag/IRSp53 interactions may take place at the Gag assembly sites as IRSp53 was localized in close proximity to Gag assembly sites in both HEK293T cells and CD4 Jurkat T cells (Figure 3). Our experiments suggest that Gag and IRSp53 are associated in a common complex at the cell plasma membrane (Figure 2). Given that upon Gag expression, IRSp53 increases its binding to the cell membrane (Figure 2), this suggests that Gag could activate IRSp53 through Rac1 activation (Thomas et al., 2015) or perhaps by releasing its auto-inhibition (Kast et al., 2014). However, these explanations remain to be tested.

HIV-1 particles are known to incorporate a large number of cellular proteins, many of which are directly involved in virus budding (Hammarstedt and Garoff, 2004). Here, we showed that IRSp53 is incorporated in Gag-VLPs, as well as in purified HIV-1 virions (Figure 5), which most likely depends on the I-BAR domain of IRSp53 (Figure 5). Using the IRSp53-I-BAR domain on GUVs, we induced membrane protrusions that have a negative mean curvature similar to a viral bud; Gag was found particularly at the tube tips that have a half-sphere geometry similar to a viral bud (Figure 6, Videos 1, 2 and 3). This indicates that Gag binds preferentially to IRSp53 I-BAR-curved membranes in vitro in contrast to other almost-flat areas of the GUVs. Similarly, in cells, single-molecule localisation images reveal some Gag clusters enriched at IRSp53 labeled protrusions at the plasma membrane (Figure 3—figure supplement 1). IRSp53 clusters have already been reported prior to filopodia formation (Disanza et al., 2013) and in negatively curved area at the onset of endocytic buds (Sathe et al., 2018). Moreover, it was shown that inducing local membrane curvature helps to initiate Gag lattice formation (Pak et al., 2017). We thus propose that IRSp53 induces local membrane curvature, most likely upon activation through Rac1/Cdc42 and effectors, which in turn can promote local Gag recruitment and initiation of the viral assembly (knowing that expression of Gag can activate Rac1, Thomas et al., 2015). This appears to be independent of the cell types (Figure 3).

Although the presence of RNA can facilitate the growth of the Gag network (Chen et al., 2014a; Floderer et al., 2018), favoring membrane bending due to the intrinsic curvature of assembling Gag hexamers, coarse-grained simulations of HIV-1 Gag assembly showed that, above a certain threshold, this Gag self-assembly is unable to overcome the free energy penalty required to curve the membrane. Here, we observed that siRNA knockdown of IRSp53 gene expression induces a decrease in viral particle production and arrests the assembly at half completion (Figure 1). Since IRSp53 stabilizes curvature by scaffolding (Prévost et al., 2015), another role of IRSp53 could be to lower this free energy barrier involved in the progression of the budding process beyond the half-sphere geometry by stabilizing long enough the bud curvature. This stabilization could be achieved either directly by organizing linearly around the assembly site (Jarin et al., 2019) and mechanically constricting the nascent bud, or indirectly with the help of actin polymerization. Interestingly, Ku et al., 2013 also observed that 60% of assembling particles exhibit a pause around the midway mark of the assembly process. This pause can provide a temporal window for IRSp53 to intervene in the progression of HIV-1 particle assembly as we propose here.

Finally, ESCRT recruitment occurs at the end of virus assembly, after the membrane has been curved, forming a vesicle ready to bud (Bleck et al., 2014; Johnson et al., 2018). Overexpression of a mutant of the ESCRT protein Tsg101 was previously shown to block HIV-1 budding at a late stage, arresting the budding with a characteristic bulb-shaped phenotype indicative of a defect in the late stage of the bud scission (Goila-Gaur et al., 2003), in contrast with our observations with the IRSp53 siRNA phenotype (Figure 1, Figure 1—figure supplement 3). Consequently, this suggests that Gag-IRSp53 association is ESCRT independent (as shown in Figure 2—figure supplement 1) and occurs at an earlier stage of virus assembly.

Another study (Mercenne et al., 2015) showed that angiomotin, which acts as an adaptor protein for HIV-1 Gag and the ubiquitin ligase NEDD4L, functions in HIV-1 assembly prior to ESCRT-I recruitment. Interestingly, angiomotin also contains a BAR domain (Moleirinho et al., 2014), but it is canonically involved in inducing positive curvature, as opposed to the negative curvature induced by I-BAR IRSp53. Thus, it is possible that angiomotin functions in another way, for example, at the viral bud neck which has both positive and negative curvatures by facilitating ESCRT recruitment.

IRSp53 itself is a scaffold protein for cofactors of cortical actin signaling (Zhao et al., 2011) and we have previously shown that a Rac1 signaling pathway, including IRSp53, is involved in HIV-1 particle production (Thomas et al., 2015). Thus, it is possible that IRSp53 could also play a role in generating local cortical actin density in the vicinity of the viral bud in formation. The role of cortical actin associated with IRSp53 scaffolding in that context remains to be elucidated.

Our work illustrates a novel role for the host cellular I-BAR factor IRSp53, which is subverted by the retroviral Gag protein, in HIV-1-induced membrane curvature and in favoring the formation of the fully assembled viral particle.

Materials and methods

Antibodies

A rabbit polyclonal anti-IRSp53 antibody (Merck Millipore), a rabbit polyclonal anti-IRTKS (Bethyl), a mouse monoclonal anti-CA (NIH AIDS Reagent Program), a rabbit polyclonal anti-GFP (Invitrogen), a mouse monoclonal anti-human CD63 (Santa Cruz Biotechnology), a mouse monoclonal anti-human CD81 (Santa Cruz Biotechnology), a rabbit monoclonal anti-human TSG101 (Abcam), and a rabbit polyclonal anti-human ALIX (Covalab) and secondary anti-rabbit Atto647N antibody (Sigma) were used in this study.

Plasmids

The plasmid expressing HIV-1 codon-optimized Gag (pGag(myc), named pGag), the plasmid expressing Pol and Env-deleted HIV-1 (named pNL4.3ΔPolΔEnv was a gift of E.Freed, HIVDRP, NIH, USA) encoding Gag alone with its packageable viral RNA (Chen et al., 2014b) and the plasmid expressing full wild-type HIV-1 (named pNL4.3) were described previously (Favard et al., 2019). Plasmids IRSp53-GFP, IRTKS-GFP, PinkBAR-GFP, and IRSp53-I-BAR-GFP were obtained from the University of Helsinki (Finland) (Saarikangas et al., 2009). Plasmids expressing PH-PLCδ-GFP was a gift of B.Beaumelle (IRIM, France), Gag(i)mCherry (named Gag-mCherry), Gag tagged with internal photo-activable mEos2 (named Gag(i)mEos2), p6-deleted Gag tagged with mEos2 (named pGagΔp6-mEos2) were described in Floderer et al., 2018.

siRNA

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Stealth siRNA (Invitrogen) targeting IRSp53 (BAIAP2) and IRTKS (BAIAP2L2), and Smartpools (Dharmacon) targeting IRSp53 (BAIAP2) or random sequence for siRNA controls were used in this study.

Cell culture and transfection

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Human embryonic kidney cells (HEK 293T-ATCC-CRL-1575TM) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) and human Jurkat T lymphocytes (ATCC-CRL-2899TM) were maintained in RPMI (GIBCO). All cell lines were tested negative for mycoplasma thanks to a MycoAlert Mycoplasma Detection Kit (Lonza Bioscience) done every month. Media was supplemented with 10% fetal bovine serum (FBS, Dominique Dutscher) and complemented with sodium pyruvate and antibiotics (penicillin-streptomycin). Cells were grown at 37°C in a 5% CO2 atmosphere. Transfection was performed by using the calcium phosphate precipitate method on HEK293T cells (as described in Grigorov et al., 2006) and the AMAXA (Lonza) method according to the manufacturer's instructions for the Jurkat T cells (as in Thomas et al., 2015). Based on different plasmid conditions, the cells were transfected (2 × 106 cells/transfection) with a total of 8 µg of plasmids. The amount of transfected plasmid was normalized by adding pcDNA3.1 empty plasmid DNA. The cell medium was replaced by fresh medium 6 hr post-transfection and the experiments were performed 24–48 hr post-transfection. SiRNA transfections in cells were performed with either RNAiMax (Invitrogen) or calcium phosphate buffer in HEK293T cells or by electroporation for Jurkat T cells. One day prior to transfection, 2 × 105 cells/well were seeded in 2 mL of growth medium without antibiotics in a six-well plate. Transfection was performed using the manufacturer’s protocol. After 24 hr of siRNA transfection, the cells were again transfected using the phosphate calcium buffer method. These cells were incubated at 37°C in a 5% CO2 atmosphere for 24/48 hr.

Immunoprecipitation assay

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Based on different plasmid conditions, HEK293T cells (2 × 106 cells) were transfected with pGag alone or pGag/pIRSp53-GFP plasmids (8 µg total) and the amount of transfected plasmid was normalized by adding pcDNA3.1 ‘mock’ plasmid. The cell medium was replaced 6 hr post-transfection. After 24 hr post-transfection, the cells were washed with cold 1× phosphate buffer solution (PBS) prior to collection with 800 µL of chilled lysis buffer (50 mM TRIS-HCl [pH = 7.4]; 150 mM NaCl; 1 mM EDTA; 1 mM CaCl2; 1 mM MgCl2; 1% Triton, 0.5% sodium deoxycholate; protease inhibitor cocktail [Roche] one tablet/10 mL lysis buffer). The cells were incubated on ice for 30 min and then centrifuged at 13,000 rpm/15 min/4°C. The supernatant was collected in a new tube and the pellet was discarded. For each condition, 1000 µg of protein (the collected supernatant) was incubated with 1 µg of anti-IRSp53 or anti-GFP antibody on a tube rotator overnight at 4°C. About 25 µL of beads (Dynabeads Protein A, Life Technologies) was added to each tube of protein-antibody complex and incubated for 2 hr on the tube rotator at 4°C. The samples were then washed five times with the lysis buffer, followed by addition of 20 µL 2× Laemmli’s buffer to the beads. The samples were denatured at 95°C for 10 min and then processed for Western blot.

Western blot and analysis

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About 50 µg of each protein (intracellular in cell lysates) samples or 20 µL of purified VLP samples were mixed with SDS loading dye, deposited, and resolved on a 10% SDS-PAGE gel. The gels were then transferred on to polyvinylidene PVDF membranes (Amersham). Immunoblotting was performed by incubating the membranes overnight with primary antibody at 4°C, and 2 hr with horseradish-peroxidase (HRP)-conjugated secondary antibody at room temperature. The Western blot signals were detected using ECL Prime/ECL Select substrate (Amersham) and images were taken using ChemiDoc (Bio-Rad).

VLP purification and quantification

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After 24 or 48 hr post-transfection, cell culture supernatants containing Gag-VLPs were collected, filtered through a 0.45 µm filter, and clarified at 800×g for 5 min at 4°C. The supernatant was then purified by loading it on a cushion of 25% sucrose in TNE buffer (25 mM Tris-HCl, 4 mM EDTA, and 150 mM NaCl) and ultracentrifuged at 100,000×g for 1 hr 30 min at 4°C in an SW41Ti rotor (Beckman Coulter). Dry pellets were resuspended in TNE buffer at 4°C overnight. Gag-VLP release was estimated by performing anti-CAp24 immunoblot and by quantifying Gag signal in the blots using Fiji software as described in Thomas et al., 2015. The calculation for Gag-VLP release is: % of Gag in VLP = Gagreleased/(Gagreleased + Gagintracellular normalized to GAPDH).

Membrane flotation assay

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For each condition, 4 × 106 cells were transfected and viral supernatants were harvested 48 hr post-transfection, as described above. The cells were washed with ice-cold PBS and resuspended in Tris-HCl containing 4 mM EDTA and 1× complete protease inhibitor cocktail (Roche). Every step was then performed at 4°C. Cell suspensions were lysed using a Dounce homogenizer, then centrifuged at 600×g for 3 min to obtain Post-Nuclear Supernatants (PNS). A cushion of 820 μL of 75% (wt/vol) sucrose in TNE buffer was loaded at the bottom of an ultracentrifuge tube and mixed with 180 μL of PNS adjusted to 150 mM NaCl. About 2 mL and 300 μL of 50% (wt/ml) sucrose cushion followed by 0.9 mL of 10% (wt/ml) sucrose cushion were then layered to obtain the gradient that was then centrifuged in a Beckmann SW60Ti rotor at 35,000 rpm, 4°C, overnight. Eight fractions of 500 μL were collected from the top to the bottom and analyzed by Western blotting.

Transmission electron microscopy

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siRNA treated HEK293T cells were fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 48 hr, washed with PBS, post-fixed in 1% osmium tetroxide for 1 hr, and dehydrated in a graded series of ethanol solutions. Cell pellets were embedded in EPON resin (Sigma) that was allowed to polymerize at 60°C for 48 hr. Ultrathin sections were cut, stained with 5% uranyl acetate and 5% lead citrate, and deposited on collodion-coated EM grids for examination using a JEOL 1230 transmission electron microscope.

Sample preparation for super-resolution PALM/STORM microscopy

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HEK293T cells expressing HIV-1 Gag/Gag(i)mEos2 cultured on poly-l-lysine (Sigma) coated 25 mm round #1.5 coverslips (VWR) were fixed using 4% PFA + 4% sucrose in PBS for 15 min at room temperature. Samples were subsequently quenched in 50 mM NH4Cl for 5 min. Samples were then washed in PBS and then blocked for 15 min iat room temperature using 1% BSA in PBS and subsequently in 0.05% Saponin in 1% BSA in PBS. Samples were stained using a 1:100 dilution of the primary antibodies (rabbit polyclonal anti-human IRSp53, Sigma and rabbit polyclonal anti-human IRTKS antibody, Bethyl) for 60 min at room temperature. Samples were washed three times for 5 min using 1% BSA in PBS followed by 60 min staining using a 1:2000 dilution of the anti-rabbit Atto647N antibody (Sigma). Samples were washed three times for 5 min with PBS and stored in light protected container in +4°C until imaged. Samples were mounted on a StarFrost slide with a silicon joint with the STORM buffer (Abbelight). Cells were imaged within 60 min after application of the STORM buffer.

PALM/STORM Imaging

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Single-molecule localization microscopy was performed on a Nikon inverted microscope equipped with 405, 488, 561, and 642 nm lasers, an EMCCD Evolve 512 Photometrics camera (512 × 512, 16 µm pixel size) with an oil immersion objective 100× NA1.49 Plan Apochromat. PALM imaging of Gag mEos2, activation was performed with laser irradiance set to 0.3 kW/cm² for 405 nm conversation and ~2.2 kW/cm² for 561 nm excitation. Illumination was performed over a 25 × 25 μm2 area in the sample (1/e2 spatial irradiance distance) in TIRF-mode. About 20–50,000 images were acquired for each cell with 50 ms integration time. The mean precision localization in PALM measurements was found to be 20 ± 5 nm (Figure 3—figure supplement 3a). 2D-STORM imaging of Atto647N was performed using a ~5 kW/cm² irradiance with the 642 nm excitation. About 25,000 images were acquired for each condition. Tetraspeck 100 nm multicolor beads (Life Technologies) as fiducial markers to correct for drift and chromatic abberation.

Single-molecule localization microscopy image reconstruction and analysis

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SMLM acquisitions were analyzed using the ThunderSTORM plugin in Fiji (Ovesný et al., 2014). The mean precision localization in PALM measurements was found to be 20 ± 5 nm (mean ± sd) and 27 ± 9 nm for STORM (Figure 3—figure supplement 3). During post-processing, a density filter was applied first to eliminate the background noise by identifying and discarding ‘isolated’ localizations, with a threshold of a minimum of five neighbors in a 50 nm radius for a molecule to be considered ‘not isolated.’ In the next step, molecules that converged to the same position were identified as duplicates and removed, keeping the localization with the smallest uncertainty as the valid coordinate. Furthermore, molecules reappearing with one ‘off-frame’ within 20 nm were merged into one single localization, with the first one appearing considered as the valid localization. Following these steps of post-processing, the sample drift was corrected by the drift correction module using fiducial markers described above. Each acquisition had at least two fiducial markers in the field of illumination. The resulting list of localizations was then used to reconstructing the respective PALM and STORM images and for the CBC analyses using the ThunderSTORM module. The module DBSCAN of the super-resolution quantification software SR Tesseler (Levet et al., 2015) was used to analyse the PALM localizations for quantification of Gag cluster sizes. In order to monitor the localization of I-BAR proteins in the vicinity of Gag assembling particles, the Gag particles were segmented by thresholding using Fiji, to generate a binary mask of the PALM images. The centers of each Gag assembling cluster were then determined and a custom MATLAB (Mathworks) code was used to extract localizations in a radius of 80 nm around each Gag cluster center and to extract I-BAR proteins localizations belonging to a disk of 150 nm radius around the center of each Gag clusters (see Results section). These subsets of coordinates were then used to calculate the experimental CBC, with the algorithm developed by Malkusch et al., 2012 and implemented in the ThunderSTORM plugin of Fiji (see Figure 3—figure supplement 4). The CBC values are calculated from single-molecule localization data of two species (Gag and IBAR proteins [IRSp53 or IRTKS]). A CBC value is assigned to each single localization of each species. We analyzed the distributions of these CBC values by plotting and comparing the distribution histograms of the CBCs obtained in the two conditions (IRSp53 vs. IRTKS).

We also performed a set of numerical simulations of super resolution images in Fiji. For this, we generated images by randomly choosing a molecule position within a radius of 60 nm from the cluster center for Gag [a compromise between the values found in the EM images (Figure 1e) and those obtained by DBSCAN analysis in the SMLM experimental images (Figure 3—figure supplement 3)]. For I-BAR proteins, we randomly assign positions within belts of different waists (from 0 to 100 nm) located at different distances (from 0 to 160 nm) from the Gag cluster center (see Figure 4 and Figure 4—figure supplement 1). Each localization was then randomly assigned a brightness such that the final pointing precision obtained at the end of the process for the Gag localization was equivalent to the experimental one. We then convolved each of these positions by a Gaussian 2D PSF, to generate a diffraction-limited image in which random noise was introduced using the ‘add noise’ function of Fiji. Super-resolution images of these simulated localizations were then reconstructed using the ThunderSTORM plugin of Fiji with the same parameters as the one used in the experimental data reconstruction. Finally, once the data set of localizations for both numerically simulated Gag clusters and associated surrounding I-BAR proteins were obtained, they were analyzed with the CBC function of ThunderSTORM with the same parameters as in the experimental analysis (analyzing all the neighbor positions within 10 successive radii of 20 nm each to estimate the CBC). Each localization was attributed CBC values and we compared the experimental cumulative distribution functions of CBC values to the simulated cumulative distribution functions using RMSE measurements, in order to establish the most probable configuration of I-BAR belts around the ongoing Gag assembly site.

Preparation and imaging of fluorescent VLPs

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After 24 hr seeding, 2 × 106 HEK293T cells were transfected with 8 µg of I-BAR-GFP expressing plasmid with or without 8 µg of pGag/pGag(i)mCherry plasmids (2/3 and 1/3, respectively). After 24 hr transfection, cell media (9 mL) was filtered before performing VLPs purification by ultracentrifugation in an SW41Ti rotor (Beckman Coulter) at 29,000 rpm, for 1 hr 30 min, at 4°C, on a 20% sucrose cushion in TNE buffer. Dry pellets were resuspended in 110 µL of TNE and allowed to sediment on round 25 mm coverslips for 45 min in an AttoFluor Cell Chamber (Invitrogen). VLPs were imaged with a Nikon Ti Eclipse 2 TIRF microscope. Images were taken with an Evolve EMCCD camera – 512 photometrics, using a NA = 1.45, 100× objective and using 488 and 561 nm lasers.

Image analysis for colocalization

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Images were acquired with a Zeiss LSM780 (for fixed cells) or a Nikon Eclipse Ti-2 in TIRF mode (for fluorescent viral particles). Colocalization analysis based on Mander’s coefficients was performed using JaCOP (Just another Colocalization Plugin) (Bolte and Cordelières, 2006). Mander’s coefficients are defined as M1=iAi,colociAi and M2=iBi,colociBi, A and B being the two respective channels (mCherry and GFP). 0 < M < 1, with 1 full colocalization and 0.5 random colocalization. The M1 and M2 coefficients were calculated for several images and then represented as column graphs with red columns representing the degree of overlap of mCherry images with GFP images, and green columns representing the inverse.

Iodixanol gradient

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Cell culture medium of HEK293T (2.5 × 106 cells plated) transfected with 8 µg of pNL4.3ΔpolΔenv plasmid was collected 48 hr after transfection and filtered using a 0.45 µm filter. The medium was then ultracentrifuged on a 20% sucrose cushion in TNE using an SW41Ti rotor (Beckman) at 40,000 rpm for 1 hr 30 min. A solution with 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl pH 7.4 was used to diluted the 60% iodixanol stock solution (OptiPrep from Sigma) and to prepare a 40% and 20% iodixanol solution. About 1.5 mL of each dilution (60%, 40%, and 20% iodixanol) was successively layered in an SW55Ti tube (Beckman) and the pellet of VLPs obtained after ultracentrifugation on a 20% sucrose cushion in TNE was loaded from the top. Tubes were ultracentrifuged at 50,000 rpm in an SW55Ti rotor (Beckman) at 4°C for 3 hr. Then, 20 fractions of 200 µL were collected from the top of the tube to the bottom. About 20 µL of each fraction was loaded for Western blotting.

GUV reagents

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Brain total lipid extract (131101P) and brain L-α-phosphatidylinositol-4,5-bisphosphate (PIP2, 840046P) were purchased from Avanti Polar Lipids/Interchim. BODIPY-TR-C5-ceramide, (BODIPY TR ceramide, D7540) and Alexa Fluor 488 C5-Maleimide (AX 488) were purchased from Invitrogen. β-casein from bovine milk (>98% pure, C6905) and other reagents were purchased from Sigma-Aldrich. Culture-Inserts 2 Well for self-insertion were purchased from Ibidi (Silicon open chambers, 80209).

Protein purification and fluorescent labeling

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Recombinant mouse IRSp53 I-BAR domain was purified and labeled with AX488 dyes, as previously described (Prévost et al., 2015; Saarikangas et al., 2009). Recombinant HIV-1 immature Gag protein was purified by J. Mak as described in Yandrapalli et al., 2016 and labeled with Alexa488 maleimide dyes (Invitrogen). Briefly, a 200 µM solution of the maleimide dye was incubated overnight at 4°C with a 20 µM solution of the Gag purified protein in a buffer of pH 8.0 with 1M NaCl and 50 mM Tris-HCl. Post incubation, the labeled mixture was subjected to dialysis with the Slide-A-Lyzer Mini Dialysis Device (Thermo Fisher Scientific), following the manufacturer’s instructions to remove the excess unbound dye from the solution.

GUV preparation and observation

Lipid and buffer compositions

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Lipid compositions for GUVs were brain total lipid extract (Seong-hyun et al., 2006) supplemented with 5 mol% brain PI(4,5)P2. If needed, 0.5 mol% BODIPY TR ceramide was present in the lipid mixture as a membrane reporter. The salt buffer inside the GUVs, named I-buffer, was 50 mM NaCl, 20 mM sucrose, and 20 mM Tris-HCl pH 7.5. The salt buffer outside the GUVs, named O-buffer, was 60 mM NaCl and 20 mM Tris-HCl pH 7.5.

GUV preparation

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GUVs were prepared by using the polyvinyl alcohol (PVA) gel-assisted method (Weinberger et al., 2013), except GUVs shown in Figure 6, Figure 6—figure supplement 1c in which the electroformation method was used (Méléard et al., 2009). For the PVA method, a PVA solution (5% (w/w) of PVA in a 280 mM sucrose solution) was warmed up to 50°C before spreading on a coverslip that was cleaned in advance by being bath sonicated with 2% Hellmanex for at least 30 min, rinsed with MilliQ water, sonicated with 1 M KOH, and finally sonicated with MilliQ water for 20 min. The PVA-coated coverslip was dried in an oven at 50°C for 30 min. About 5–10 µL of the lipid mixture (1 mg/mL in chloroform) was spread on the PVA-coated coverslip, followed by drying under vacuum for 30 min at room temperature. The PVA-lipid-coated coverslip was then placed in a 10 cm cell culture dish and 0.5 mL of the I-buffer was added on the coverslip, followed by keeping it stable for 45 min at room temperature to allow the GUVs to grow. For the electroformation method, a few μl of lipid mixture at 3 mg/mL were deposited onto platinum electrodes (Goodfellow). The lipid film was dried for at least 30 min under vacuum at room temperature, and then rehydrated in I-buffer under a voltage of 0.25 V and a frequency of 500 Hz overnight at 4°C (Méléard et al., 2009).

Sample preparation and observation

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GUVs were first incubated with either Gag or I-BAR domain at bulk concentrations depending on the designed experiments for at least 15 min at room temperature before adding either I-BAR domain or Gag, respectively, into the GUV-protein mixture. In experiments where there was only Gag but no I-BAR domain, the stock solution of I-BAR domain was used in order to obtain a comparable salt strength outside GUVs as those where I-BAR domain was present. The GUV-protein mixture was then incubated for at least 15 min at room temperature before observation. For the Gag/I-BAR membrane recruitment assay, samples were observed on a Nikon C1 confocal microscope equipped with a 60× water immersion objective (Nikon, CFI Plan Apo IR 60× WI ON 1.27 DT 0.17). For the Gag/I-BAR tubulation assay, samples were observed with an inverted spinning disk confocal microscope Nikon eclipse Ti-E, equipped with Yokogawa CSU-X1 confocal head, 100× CFI Plan Apo VC objective (Nikon) and a CMOS camera, Prime 95B (Photometrics).

For all experiments, coverslips were passivated with a β-casein solution at a concentration of 5 g.L−1 for at least 5 min at room temperature. Experimental chambers were assembled by placing a silicon open chamber on a coverslip.

GUV image analysis

Image analysis was performed by using Fiji (Schindelin et al., 2012).

Quantification of AX488 Gag binding on GUV membranes

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Fluorescence images were taken at the equatorial planes of GUVs using identical confocal microscopy settings. The background intensity of the AX488 channel was obtained by manually drawing a line with a width of 10 pixels perpendicularly across the membrane of a GUV. We then obtained the background intensity profile of the line where the x-axis of the profile is the length of the line and the y-axis is the averaged pixel intensity along the width of the line. The background intensity was obtained by calculating the mean value of the sum of the first 10 intensity values and the last 10 intensity values of the background intensity profile. To obtain Gag fluorescence intensity on the membrane of the GUV, we used membrane fluorescence signals to find the contour of the GUV (using the ‘Fit Circle’ function in Fiji). Then, a 10 pixel wide band centered on the contour of the GUV was used to obtain the Gag intensity profile of the band where the x-axis of the profile is the length of the band and the y-axis is the averaged pixel intensity along the width of the band. Gag fluorescence intensity was then obtained by calculating the mean value of the intensity values of the Gag intensity profile, following by subtracting the background intensity.

Gag sorting map

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Fluorescence images of GUVs were taken using identical confocal microscopy settings. For every GUV, we first calculated the fluorescence intensity ratio for every pixel of the Gag and membrane images of a GUV using (IGag-IbackgroundGag)/Imembrane, where IGag is the Gag intensity, IbackgroundGag is the background intensity in the Gag channel, and Imembrane is the membrane intensity. The sorting map was then obtained by converting the resulting image from the previous step to a pseudo-colored image via the ‘Look Up Table, Phase’ in Fiji. The background intensity value in the Gag channel was the mean intensity value of a 50 pixel wide square in the background outside GUVs. The sorting map of I-BAR domain was obtained by using the same procedure as those for Gag.

Statistics

All notched boxes show the median (central line), the 25th and 75th percentiles (the bottom and top edges of the box), the most extreme data points the algorithm considers to be not outliers (the whiskers), and the outliers (crosses).

Data availability

All data have been provided in the manuscript and supporting files in our submission that allows research reproductibility (see source data, reagents table and supplemental informations).

References

  1. Book
    1. Coffin JM
    2. Hughes SH
    3. Varmus HE
    (1997)
    Retroviruses
    Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Decision letter

  1. Felix Campelo
    Reviewing Editor; The Barcelona Institute of Science and Technology, Spain
  2. Vivek Malhotra
    Senior Editor; The Barcelona Institute of Science and Technology, Spain
  3. Felix Campelo
    Reviewer; The Barcelona Institute of Science and Technology, Spain
  4. Ricardo Henriques
    Reviewer; University College London, United Kingdom
  5. Zandrea Ambrose
    Reviewer

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This manuscript combines cell biology, biochemistry, and quantitative biophysics to understand how a new host cell factor, the human I-BAR domain protein IRSp53, promotes HIV type 1 (HIV-1) assembly and release. Since this new factor is a protein involved in the generation and sensing of negative membrane curvature, this manuscript will be of interest not only for retrovirologists and virologists in general but also for membrane biologists and biophysicists.

Decision letter after peer review:

Thank you for submitting your article "Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Felix Campelo as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ricardo Henriques (Reviewer #2); Zandrea Ambrose (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

We all agreed that this paper provides novel and important new data that will merit, after revision, publication in eLife. We believe that for the revision, we are not asking the authors for extensive extra work, just some key experimental controls or tests that could be duly due in a few months. We'd also appreciate that the authors go through a thorough revision of the text, in terms of clarity and quality (also on the figures and method description, see details below).

To summarize, the main points that the reviewers considered necessary for this article to be accepted at eLife are:

1) Improve quality of the IP/co-IP data as well as the biochemical measurements of membrane fraction pools.

2) Assess specificity of siRNA-mediated knockdowns.

3) It is unclear what the role of IRSp53 is in the membrane curvature of CD4+ T cells and whether expression levels and localization are consistent with Jurkat T cells.

4) Better description and quantitative comparison of simulated and experimental SMLM data.

5) Make sure that method descriptions are complete.

6) Discuss the very high similarity between histograms in Figure 3c,d (HEK293T) and Figure S10c,d (Jurkats).

Since the reviews are quite detailed and include very valuable comments that will for sure improve the quality of the manuscript, we include them in detail in this decision letter. Please, consider them as much as possible when submitting a revised version.

Reviewer #1 (Recommendations for the authors):

– Figure 1C: How specific is this? Can the authors express siRNA-resistant mutants of IRSp53 and see if this reverts the phenotype? Or at least test the effect with other siRNA oligoes?

– Line 152: To overcome the limitation caused by the IgGs masking the IRSp53 signal in the WBs, there are nowadays different ways of probing the membranes with secondary antibodies that do not detected denatured IgGs (available commercially from different companies). Along these lines, is this Ig G band the band seen in Figure S4a between the 55 and 70 kDa marker bands? What is the mature MW of IRSp53? Is it 53 kDa or higher? If 53 kDa, then IRSp53-GFP should be around 80 kDa (53+27) and not >100 kDa as in the IP in S4A. Could the authors discuss about this?

– Overexpression of IRSp53-GFP does not lead to more Gag being pulled down (lanes 3 and 4 in S4a). Is the GFP-tagged form of IRSp53 functional (at least in respect to Gag binding)? Could the authors elaborate a bit more on this?

– Figure 2c: To asses if the increased binding of IRSp53 to membranes in the presence of Gag is specific for this IBAR protein, a control where the membrane bound pool of another IBAR protein (e.g. IRTKS) should be included.

– This is possible obvious for people in the field, but I missed at understanding why relatively low concentration (0.05 uM) of IRSp53 IBAR induces invagination into GUVs but 10x larger concentration does not seem to deform those GUVs (Figure 5). Could the authors clarify this please?

Reviewer #2 (Recommendations for the authors):

1. Gag and IRSp53 complexing is shown in an immunoprecipitation assay (Figure 2a). However, the quantification of membrane fraction enrichment of IRSp53 upon Gag expression in the membrane floatation assay the presented data is not convincing. Inamdar and colleagues claim a 2-fold increase of IRSp53 in the membrane fraction. Still, based on the dataset shown in Figure 2c, the findings don't seem to match the minor differences observed in the respective bands in the gel image. In the presented data set, the membrane fractions of Gag and IRSp53 do not appear in the same lane (Lane 1). I, therefore, suggest that the authors provide a more detailed description of their analysis method and the full data set to convincingly confirm the difference in membrane-bound IRSp53 upon Gag-expression compared to control cells, and the amount of membrane-bound Gag relative to its cytosolic counterpart. If the previously reported results hold, cross-validate them using a different methodology, for instance, by measuring the colocalisation of IRSp53 and Gag with the plasma membrane using microscopy. If the new calculations do not match the results previously reported, the authors should adjust their conclusions accordingly.

2. Dual-color SMLM is used to elucidate the IPSp53/IRTKS organisation patterns at Gag clusters sites by PALM/dSTORM imaging. Inamdar and colleagues quantify the Gag cluster size distribution and show that IPSp53 shows a considerable colocalized fraction, while IRTKS tends toward an anticorrelated distribution. In the next step, the authors try to deduce the spatial organisation of IPSp53 and IRTKS around Gag clusters based on simulated datasets of Gag and IPSp53/IRTKS CBC distribution geometries.

While dual-color SMLM is the right approach, the study presented here will benefit from a consistent and comprehensive description of the experimental details (see also Mn7) and the simulation. I propose adding a schematic of the geometric simulation parameters shown in Figure S8a in Figure 3. Also, it should be highlighted in Figure 3e and 3f that the simulated distributions presented are based on different waist diameters.

A second point that should be addressed is the lack of a quantitative measure of how well the experimental results are described by the simulated data. Also, the authors should comment on the effects of the choice of the radius in the data filtering (as shown in Figure S7) and over parameterization.

3. In the controlled in-vitro environment of GUVs, the authors show that Gag membrane recruitment is enhanced in the presence of the IRSp53 I-Bar domain and preferentially locates to the areas of high curvature in I-Bar domain induced membrane tubes. While I fully support the conclusions drawn from this elegant experiment, a complete description of the GUV preparation and imaging methods is missing. Also, from the methods section and the captions in Figure 5 and S11, it is unclear which membrane label was used in the experiment.

Reviewer #3 (Recommendations for the authors):

It would have been better to incorporate Jurkat CD4+ T cell data into the main figures to highlight the biological relevance of the findings.

Additional recommendations to strengthen the manuscript:

1) The role and expression levels of IRSp53 in CD4+ T cells should be described and/or shown, as these are the relevant target cells for HIV-1 infection.

2) It is recommended that "gene extinction" in lines 105 and 107 be revised to "protein depletion" or "reduced gene expression" or something similar, as siRNA do not affect the gene but rather gene expression.

3) Figure 1e (both schematic and graph data points) is nearly impossible to see as it is small and very faint. It is recommended that darker colors and thicker lines and symbols be used, similar to other figures in the manuscript.

4) The order of the supplemental figures should be in chronological order from when they are referenced in the text, which is not the case in this manuscript. And all parts of each figure should be described in the text (e.g. Figure S1a).

5) It is suggested that the reference of GUVs on page 7 be removed. Otherwise, a description of what they are and the significance of the finding described here should be included.

6) Likewise, description of Gag(i)mEos2 is referred on page 8 but it is not clear why this construct was used. Presumably this was to show that the Gag used in the imaging studies behaved similarly to wild-type Gag, but this is not clear.

7) On lines 192-4, it is stated, "We evidenced that cellular Gag expression, most probably by triggering Rac1 activation (Thomas et al., 2015), favors cell membrane binding of IRSp53." As Rac1 was not evaluated here, it is suggested that the sentence be modified to, "We observed that cellular Gag expression, possibly by triggering Rac1 activation (Thomas et al., 2015), favors cell membrane binding of IRSp53."

8) The manuscript overall would benefit from strong editing for grammar, punctuation, and better word usage.

https://doi.org/10.7554/eLife.67321.sa1

Author response

Essential revisions:

We all agreed that this paper provides novel and important new data that will merit, after revision, publication in eLife. We believe that for the revision, we are not asking the authors for extensive extra work, just some key experimental controls or tests that could be duly due in a few months. We'd also appreciate that the authors go through a thorough revision of the text, in terms of clarity and quality (also on the figures and method description, see details below).

To summarize, the main points that the reviewers considered necessary for this article to be accepted at eLife are:

1) Improve quality of the IP/co-IP data as well as the biochemical measurements of membrane fraction pools.

The IP/co-IP datas have been update with new experiments that summarize all the points of the reviewers, including new controls (Input, IP/coIP, and FlowThrough), better quality gels, and better explanation in Materials and methods section for sake of clarity. See new figure 2b.

Biochemical measurements of membrane fraction pools were made several times (and with 3 independent people in the lab – KI, RD and PM), so we have now included better western blot images of membrane flotation assays that fit with the graph (see figures 2c,d); we also added the ribosomal S6 marker as a cytosolic control fraction, and IRTKS western blotting of the fractions.

2) Assess specificity of siRNA-mediated knockdowns.

We have tried several different commercial and home-designed siRNA targeting IRSp53 from different companies (providing single siRNA and multiple siRNA mix): we have summarizing all in Author response image 1. One can see that indeed only 2 siRNA were effective in extinguishing IRSp53 gene: one from Invitrogen on endogenous IRSp53 and ectopic IRSp53-GFP and one from Dharmacon that was only effective on ectopic IRSp53-GFP, as revealed by Western Blot (Author response image 1A). Furthermore, the specificity of the siRNA was challenge by testing siRNA IRSp53 on human IRSp53-GFP and on mouse I-BAR-GFP in HEK293T transfected cells and visualized by fluorescence microscopy. Results show in Author response image 1B that only siIRSp53 is able to extinguished human IRSp53-GFP and not mouse I-BAR-GFP. SiIRTKS and siCtrl are not extinguishing any of these genes. Overall these results confirm the specificity of IRSp53 siRNA-mediated knockdowns.

Author response image 1
Specificity of siRNA-mediated knockdowns.

(A) Western blots of HEK293T cells lysates probed with anti-IRSp53 antibody (and house-keeping gene GAPDH) showing a series of different siRNA IRSp53 (and siRNA Control, CTRL from Invitrogen, Dharmacon or Σ) on endogenous and ectopic IRp53 genes in human HEK293T cells and their efficacy in specifically down regulating IRSp53. (B) siRNA IRSp53 from Invitrogen was tested for its specificity in extinguishing human IRSp53-GFP protein expressed in transfected HEK293T cells, but not mouse I-BAR-GFP, and as compare to siRNA control and IRTKS, revealed by fluorescence imaging (GFP).

To further answer the reviewers’ comments, we also perform one rescue experiment of the phenotype as shown Author response image 2. We observed that, upon co-transfection of pGag+pIRSp53-GFP+siRNA IRSp53 (lane 2), about 50% of the ectopic IRSp53-GFP was extinguished (since this construct is not siRNA resistant), leaving 50% of this ectopic protein expressed in the cells. In this context, one can observe that Gag-VLP release is ~50% (lane 2), similar to the condition pGag+siCTRL (lane 3). When we compare this to pGag+siIRSp53 (lane 4) which is reduced by 2-3 fold (data from Figure 1b of the manuscript), we can say that the remaining IRSp53-GFP in the Lane 2 seems to rescue the defect caused by extinction of the endogenous IRSp53. In the condition pGag+pIRSp53-GFP +siCTRL, VLP-Gag release was slightly reduced. This is an atypical rescue experiment since we do not have an IRSp53-GFP that is resistant to the siRNA IRSp53 used in this study (Author response image 1B), but it suggests that if IRSp53-GFP is overexpressed in the presence of Gag and the siRNA IRSp53, VLP-Gag release is at a normal 50% level in contrast to the absence of IRSp53-GFP (compare lane 2 with lane 4). Unfortunately, due to limited time and by the siRNA IRSp53 out of stock, and the delay in supply, we could only provide one experiment. We thus decided to show it for answering the reviewers but not as part of a figure in the final manuscript.

Author response image 2
Rescue of siRNA IRSp53 knock-down with overexpression of IRSp53-GFP.

293T cell were transfected with pGag, pIRSp53 and siRNA control (siCTRL, lane 1) or siRNA IRSp53 (lane 2); cell lysat and VLP wre loaded on SDS-PAGE gels and immunoblots were revealed with anti-GFP (for IRSp53-GFP) and anti-CAp24 (for HIV-1 Gag). One graph on the left shows the percentage of IRSp53-GFP expression upon siRNA IRSp53 cell treatment (lane 2) as compare to the siRNA CTRL (lane 1). The graph on the right shows the resulting gel quantification for the % of Gag-VLP release upon siRNA IRSp53 cell treatment (lane 2) as compare to the siRNA CTRL (lane 1) in the presence of IRSp53-GFP over-expression, or without (lane 3 and 4, as in Figure 1b). N=1 rescue experiment.

3) It is unclear what the role of IRSp53 is in the membrane curvature of CD4+ T cells and whether expression levels and localization are consistent with Jurkat T cells.

We have published that IRSp53 (using siRNA) is involved in HIV-1 particle release on primary T cells (PBMC derived T cells) in Thomas et al., JVI 2015, so high probability is that it would be the same in different cell type, transfected HEK293T cells, transfected or infected Jurkat T cells and infected primary T cells. But we have not done the extensive super-resolution microscopy on infected primary T cells because this would require time overconsuming study. We are currently proceeding in setting up condition with an infectious HIV-1 virus carrying mEOS2 photoactivable protein for being able to infect primary T cells and go on for further research using infectious relevant system and super-resolution microscopy, but it is not ready for this current manuscript as it would require months of extra work and experiments.

Although, we agree with the reviewer #3 that the localization of Gag in Jurkat T cells and in primary CD4 T cells is different at the cellular level, but at the nanoscopic level of the budding sites, chances are that it would be similar (to be checked in further studies).

4) Better description and quantitative comparison of simulated and experimental SMLM data.

We have addressed this point in the manuscript by

i) describing in detail the method used to record, extract and analyse the experimental data, for this we have added extended description of methods used in quantifying SMLM data, we have introduced a section in the methods explaining how we performed the simulations. We have also described more in details the analysis of SMLM data in the Results section.

ii) performing root mean square error quantification in order to compare the experimental to the simulated cumulative distribution functions. We have added maps of values of the RMSE to show similarities between the different numerical schemes (waists and distance from the center of the surrounding belt) and the experimental data in the supplemental figure 9 (FigS9). We have changed the text of the manuscript in the Results section accordingly. We want to point out here that the idea of these simulations is not to find a perfect similarity between experiments and simulation distribution of the CBC value. This numerical simulation has been performed more in a view of discriminating among different possible scenarii of I-BAR proteins (IRSp53 or IRTKS) localisation around a Gag on going bud. We show here the scenario that fits the best to the experimental CBC distribution in order to illustrate that there are clear differences in between IRSp53 and IRTKS in terms of organisation around the on-going bud.

5) Make sure that method descriptions are complete.

We improved the method descriptions, by extending it and going more into details, in particular for the SMLM and simulation analysis but also for the transfection methods and the GUV data analysis.

6) Discuss the very high similarity between histograms in Figure 3c,d (HEK293T) and Figure S10c,d (Jurkats).

The very high similarity between histograms of cbc values in the case of HEK 293T and Jurkats T cells suggest that the organisation of these two I-Bar proteins around the budding site is independent of the cell type.

We did not discuss it in the previous version of the manuscript as we found this result to be not so surprising. In fact, we and others have previously shown that, on average, the bud assembly kinetics was independent of the cell type (Floderer et al., Sci Reports 2018; Ivachenko et al., Plos Pathogens 2009). Interestingly, we also observe (as others) that the final diameters of the released particles are equivalent independently of the cell type where they are produced, Jurkat T cells (Floderer et al., Sci Reports 2018); HEK 293T (this study), HeLa cells (Manley et al., 2009). These two mechanics and kinetics features are therefore very similar independently of the cell type, suggesting that the time evolution of the curvatures generated during the assembly of the virus is certainly equivalent in both cell type. Supposing that these processes rely on the same molecular interplay with the same kinetics, with Gag as an orchestrator and knowing that IRSp53 as well as IRTKS proteins are express in both cell types (Jurkat and HEK 293T) where they recognise equivalent curvatures and have the same functions at the plasma membrane, we think that these observations can explain why this colocalization based CBC histograms are very similar.

Since the reviews are quite detailed and include very valuable comments that will for sure improve the quality of the manuscript, we include them in detail in this decision letter. Please, consider them as much as possible when submitting a revised version.

Reviewer #1 (Recommendations for the authors):

– Figure 1C: How specific is this? Can the authors express siRNA-resistant mutants of IRSp53 and see if this reverts the phenotype? Or at least test the effect with other siRNA oligoes?

See the above general response to the reviewers. All the answers to these questions are presented in Author response images 1 and 2 to confirm siRNA IRSp53 specificity.

– Line 152: To overcome the limitation caused by the IgGs masking the IRSp53 signal in the WBs, there are nowadays different ways of probing the membranes with secondary antibodies that do not detected denatured IgGs (available commercially from different companies). Along these lines, is this Ig G band the band seen in Figure S4a between the 55 and 70 kDa marker bands? What is the mature MW of IRSp53? Is it 53 kDa or higher? If 53 kDa, then IRSp53-GFP should be around 80 kDa (53+27) and not >100 kDa as in the IP in S4A. Could the authors discuss about this?

– Overexpression of IRSp53-GFP does not lead to more Gag being pulled down (lanes 3 and 4 in S4a). Is the GFP-tagged form of IRSp53 functional (at least in respect to Gag binding)? Could the authors elaborate a bit more on this?

In order to simplify and overcome the difficulty to read IP/co-IP with IRSp53 (53 KDa) and Gag (55KDa) due to IgG similar sizes, we perform new IP/co-IP experiment with ectopic IRSp53-GFP (80KDa); the results are shown in the new figure 2b that present very clear results on IRSp53-GFP/Gag complexing. Thus, the figure S4a has been removed.

As for the “real” migration size of IRSp53 (below 55KDa) and IRSp53-GFP (below 100KDa, between 70 and 100 KDa) on SDS-PAGE gels, we cannot explain better, but we observe using the siRNA IRSp53 (see Figure 1 – Supplemental 1, Author response images 1 and 2) that we are indeed knocking down IRSp53 or ectopic IRSp53-GFP. The other band above MW 55KDa is indeed IRSp58 another isoform of IRSp53/58 that has been reported [for example, see DOI: 10.1523/JNEUROSCI.19-17-07300. 1999], and that can be seen in the IRSp53 immunoblot in figure S1d.

– Figure 2c: To asses if the increased binding of IRSp53 to membranes in the presence of Gag is specific for this IBAR protein, a control where the membrane bound pool of another IBAR protein (e.g. IRTKS) should be included.

As suggested by the reviewer, IRTKS has been included in the new figure 2d. The gel band quantification reveals that IRTKS membrane binding is not changing upon Gag expression, contrary to IRSp53 or Tsg101.

– This is possible obvious for people in the field, but I missed at understanding why relatively low concentration (0.05 uM) of IRSp53 IBAR induces invagination into GUVs but 10x larger concentration does not seem to deform those GUVs (Figure 5). Could the authors clarify this please?

The reviewer is right about this seemingly nontrivial observation that puzzles us as well. We verified this point by measuring the percentages of GUVs having inward membrane tubes generated by the I-BAR domain at different bulk concentrations outside the GUVs. We observed an increase of GUVs having tubes at concentrations ranging from 0.005 μM to 0.06 μM; however, when we increased I-BAR concentrations from 0.1 μM up to 1 μM, we observed a decrease in the amount of GUVs having tubes. See the following table for the quantification.

Author response table 1
I-BAR concentrationPercentage of GUVs with tubes (%)Total number of GUVs
0.005 uM2478
0.02 uM90260
0.06 uM87189
0.1 uM22159
0.2 uM22120
0.5 uM19123
1 uM877

Given that the observed concentration-dependent tubulation of I-BAR domain is not the focus of this paper, we decided not to include the abovementioned results. However, to clarify the point mentioned by the reviewer, we have added the following sentences in the manuscript:

“Previous in vitro studies showed that when placing IRSp53 I-BAR domain outside PIP2-containing GUVs, I-BAR domain can deform GUV membranes, generating tubes towards the interior of the vesicles (Jarin et al., 2019; Prévost et al., 2015; Saarikangas et al., 2009). […] Future work is required to investigate on this seemingly puzzled observation.”

If the reviewer thinks that these results would be useful for the readers, we could include the abovementioned quantification in SI.

Reviewer #2 (Recommendations for the authors):

1. Gag and IRSp53 complexing is shown in an immunoprecipitation assay (Figure 2a). However, the quantification of membrane fraction enrichment of IRSp53 upon Gag expression in the membrane floatation assay the presented data is not convincing. Inamdar and colleagues claim a 2-fold increase of IRSp53 in the membrane fraction. Still, based on the dataset shown in Figure 2c, the findings don't seem to match the minor differences observed in the respective bands in the gel image. In the presented data set, the membrane fractions of Gag and IRSp53 do not appear in the same lane (Lane 1). I, therefore, suggest that the authors provide a more detailed description of their analysis method and the full data set to convincingly confirm the difference in membrane-bound IRSp53 upon Gag-expression compared to control cells, and the amount of membrane-bound Gag relative to its cytosolic counterpart. If the previously reported results hold, cross-validate them using a different methodology, for instance, by measuring the colocalisation of IRSp53 and Gag with the plasma membrane using microscopy. If the new calculations do not match the results previously reported, the authors should adjust their conclusions accordingly.

2. Dual-color SMLM is used to elucidate the IPSp53/IRTKS organisation patterns at Gag clusters sites by PALM/dSTORM imaging. Inamdar and colleagues quantify the Gag cluster size distribution and show that IPSp53 shows a considerable colocalized fraction, while IRTKS tends toward an anticorrelated distribution. In the next step, the authors try to deduce the spatial organisation of IPSp53 and IRTKS around Gag clusters based on simulated datasets of Gag and IPSp53/IRTKS CBC distribution geometries.

While dual-color SMLM is the right approach, the study presented here will benefit from a consistent and comprehensive description of the experimental details (see also Mn7) and the simulation. I propose adding a schematic of the geometric simulation parameters shown in Figure S8a in Figure 3. Also, it should be highlighted in Figure 3e and 3f that the simulated distributions presented are based on different waist diameters.

We thank the reviewer for his suggested improvements leading to a better comprehension of our work by the future reader. We now have changed the figures and split the experimental part and the simulated part of the dual-colour SMLM into 2 different figures, namely figure 3 and figure 4. We have added the geometric simulation parameters in figure 4 and highlighted that the simulated distributions are based on different waist of the assembly surrounding belt.

A second point that should be addressed is the lack of a quantitative measure of how well the experimental results are described by the simulated data.

In order to quantify the similarity between experimental and simulated CBC, we performed a RMSE quantification of the difference between experimental and simulated CBC cumulative distribution functions as these functions are the one represented in figure 4. A map of the different RMSE values obtained for all the different parameters used in the simulation is depicted in the supplementary figure (Figure 4 – supplemental 1). The minimal RMSE in the maps is found for a distance of 80nm and a belt waist of 40 nm in the case of IRSp53, whereas this minimum is displaced towards higher distance (140 nm) and larger waist (100 nm) in the case of IRTKS. However, the idea of performing simulation with this simple configurations (belts of IRSp53/IRTKS surrounding Gag assembly) was not to retrieve the experimental configuration observed here but to (i) show that differences observed here in CBC can be interpreted as different geometries of I-BAR proteins (Irsp53/IRTKS) surrounding HIV-Gag on going assemblies and (ii) relate that on average, surrounding IRSp53 belt is smaller and located closer to the centre of the assembly than IRTKS is.

Also, the authors should comment on the effects of the choice of the radius in the data filtering (as shown in Figure S7) and over parameterization.

We have added different sentences to precise these choices (data filtering and parametrization) in the main manuscript, as well as in the supplementary figures.

Regarding the choice of the radius for the data filtering, we have justified it in the results (line 215-221):

“We then kept all HIV-1 Gag located within a distance of 80 nm from this center (70 to 80% of the all clusters size described above are found within this distance) and all the IRSp53 (or IRTKS) found in a distance of 150 nm from this center (~2x the assembly site size, see Figure 3 – supplemental 4 for details on the process workflow). We choose this IRSp53 (or IRTKS) cut-off distance to avoid cross-colocalization between different HIV-1 Gag clusters in dense areas.”

Regarding the parameterization, we have introduced the following text in the Results section (line 240-248):

“Although CBC values gives a quantitative value of the colocalization, it does not provide direct information on the average positions of IRTKS or IRSp53 molecules with respect to Gag molecules within the assembling clusters were unclear. […] Thus, to gain more insight into these colocalisation quantification, we performed simulations to generate different patterns of PALM/STORM localizations (Figure 4), and analyzed them with the same set of parameter (total distance and number of circles) that the one we used for the experimental data.”

We hope these comments and justification answer to the referee’s suggestion and somehow maybe make our final statements more precisely related to the method we used.

3. In the controlled in-vitro environment of GUVs, the authors show that Gag membrane recruitment is enhanced in the presence of the IRSp53 I-Bar domain and preferentially locates to the areas of high curvature in I-Bar domain induced membrane tubes. While I fully support the conclusions drawn from this elegant experiment, a complete description of the GUV preparation and imaging methods is missing. Also, from the methods section and the captions in Figure 5 and S11, it is unclear which membrane label was used in the experiment.

We thank the reviewer for pointing this out. In Figure 5 (now Figure 6) and Figure 6—figure supplement 2 (now Figure 6—figure supplement 1), 0.5mole% of BODIPY-TR-C5-ceramide is incorporated in GUV membranes for visualizing the membranes. We have clarified this in the figure legends. We have also gone through the GUV preparation and imaging method descriptions to make sure they are completed.

Reviewer #3 (Recommendations for the authors):

It would have been better to incorporate Jurkat CD4+ T cell data into the main figures to highlight the biological relevance of the findings.

Additional recommendations to strengthen the manuscript:

1) The role and expression levels of IRSp53 in CD4+ T cells should be described and/or shown, as these are the relevant target cells for HIV-1 infection.

Currently, the very few results that we have on IRSp53 in HIV infected primary CD4 T cells are too preliminary and cannot be reasonably included in this study.

2) It is recommended that "gene extinction" in lines 105 and 107 be revised to "protein depletion" or "reduced gene expression" or something similar, as siRNA do not affect the gene but rather gene expression.

Thank you for pointing that out. We have correcting it all over the main text by saying “siRNA knock-down gene expression”.

3) Figure 1e (both schematic and graph data points) is nearly impossible to see as it is small and very faint. It is recommended that darker colors and thicker lines and symbols be used, similar to other figures in the manuscript.

The reviewer is right. Thus, all the letters/writing in the figure 1 have been increased in size in agreement with the reviewer’s comment, which makes the new figure 1 more readable.

4) The order of the supplemental figures should be in chronological order from when they are referenced in the text, which is not the case in this manuscript. And all parts of each figure should be described in the text (e.g. Figure S1a).

We apologize for this error that is now corrected in the new version of the manuscript.

5) It is suggested that the reference of GUVs on page 7 be removed. Otherwise, a description of what they are and the significance of the finding described here should be included.

This sentence was removed from the part 2 of the results (page 7). In the current manuscript, we described this GUV experiment and results in section 5 together with all the GUV experiments. Then the description of Figure 6—figure supplement 1a, b results was inserted in the section 5 (Page 14, lanes 334-339) and the significance of the finding is now included in the results and in the Discussion section (page 15 lanes 365-66).

6) Likewise, description of Gag(i)mEos2 is referred on page 8 but it is not clear why this construct was used. Presumably this was to show that the Gag used in the imaging studies behaved similarly to wild-type Gag, but this is not clear.

As it is written page 8, Gag and Gag(i)mEOS2 were both pull down with IRSp53 during IP/co-IP. We had to check that Gag(i)mEOS2 that we used for PALM/STORM microscopy imaging was immunoprecipitated the same as Gag. So, yes, it was to show that the labelled Gag(i)mEOS2 protein used in the imaging studies behaved similarly to wild-type Gag. For a sake of clarity, we added one sentence lane 188 page 8.

7) On lines 192-4, it is stated, "We evidenced that cellular Gag expression, most probably by triggering Rac1 activation (Thomas et al., 2015), favors cell membrane binding of IRSp53." As Rac1 was not evaluated here, it is suggested that the sentence be modified to, "We observed that cellular Gag expression, possibly by triggering Rac1 activation (Thomas et al., 2015), favors cell membrane binding of IRSp53."

Thank you; this has been modified accordingly to the reviewer’s advice (page 8, lanes 193-194).

8) The manuscript overall would benefit from strong editing for grammar, punctuation, and better word usage.

The manuscript in its final new version has been given to a native English for editing.

https://doi.org/10.7554/eLife.67321.sa2

Article and author information

Author details

  1. Kaushik Inamdar

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5959-6409
  2. Feng-Ching Tsai

    Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6869-5254
  3. Rayane Dibsy

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Data curation, Formal analysis, Validation, Methodology
    Competing interests
    No competing interests declared
  4. Aurore de Poret

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Data curation, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  5. John Manzi

    Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  6. Peggy Merida

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Data curation, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  7. Remi Muller

    CEMIPAI, CNRS UAR3725, University of Montpellier, Montpellier, France
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  8. Pekka Lappalainen

    Institute of Biotechnology, University of Helsinki, Helsinki, Finland
    Contribution
    Resources, Writing - review and editing
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6227-0354
  9. Philippe Roingeard

    MAVIVH UMR Inserm U1259, University of Tours, Tours, France
    Contribution
    Data curation, Formal analysis, Supervision, Validation, Visualization, Methodology
    Competing interests
    No competing interests declared
  10. Johnson Mak

    Institute for Glycomics, Griffith University, Brisbane, Australia
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
  11. Patricia Bassereau

    Institut Curie, Université PSL, Sorbonne Université, CNRS UMR168, Laboratoire Physico Chimie Curie, Paris, France
    Contribution
    Resources, Supervision, Funding acquisition, Validation, Investigation, Writing - review and editing
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8544-6778
  12. Cyril Favard

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    Additional information
    ORCID: 0000-0002-8304-2980
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8304-2980
  13. Delphine Muriaux

    Infectious disease Research Institute of Montpellier (IRIM), CNRS UMR 9004, University of Montpellier, Montpellier, France
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    delphine.muriaux@irim.cnrs.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8517-9342

Funding

Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ECTZ88374)

  • Delphine M Muriaux

Agence Nationale de la Recherche (ANR10-INBS-04)

  • Patricia Bassereau

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors greatly acknowledge the Montpellier MRI-CNRS and CEMIPAI microscopy facility for access to the PALM/STORM microscopes. The authors thank Eric Freed (NIH, Frederick, MD, USA) for providing the pNL43GagΔPolΔEnv plasmid and A. Cimarelli (CIRI, Lyon, France) for providing the pGag(myc) plasmid. The authors greatly acknowledge the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infractucture France-BioImaging (ANR10-INBS-04). DM and CF are members of the ImaBio Consortium of the CNRS (GDR ImaBio). This work was supported by the ANRS Grant ECTZ35754. KI was the recipient of an ANRS fellowship for 3 years (2017–2020). RD is a recipient of a SIDACTION fellowship. The authors thank Michael Henderson for careful English reading of the manuscript.

Senior Editor

  1. Vivek Malhotra, The Barcelona Institute of Science and Technology, Spain

Reviewing Editor

  1. Felix Campelo, The Barcelona Institute of Science and Technology, Spain

Reviewers

  1. Felix Campelo, The Barcelona Institute of Science and Technology, Spain
  2. Ricardo Henriques, University College London, United Kingdom
  3. Zandrea Ambrose

Publication history

  1. Received: February 7, 2021
  2. Accepted: June 10, 2021
  3. Accepted Manuscript published: June 11, 2021 (version 1)
  4. Version of Record published: July 6, 2021 (version 2)

Copyright

© 2021, Inamdar et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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