Journal: bioRxiv

Article Title: HIV-1 Assembly is Assisted by IRSp53 Through an Interaction that Extends Gag Conformation

doi: 10.64898/2026.10.05.756744

Figure Lengend Snippet: (A) Immunoblots showing Gag VLP production in the absence and in the presence of IIRSp53-GFP. IRSp53-GFP in cell lysates and in VLPs is revealed using anti-GFP and HIV-1 Gag using anti-CAp24 antibodies, as indicated. Results show that IRSp53-GFP is released with Gag VLPs. (B) STED confocal images showing the top view of an HeLa Cells producing HIV-1 Gag(i)GFP and immune-stained for IRSp53 (revealed using anti-IRSp53 and anti-StarRed antibodies). Cells are also labelled with phalloidin-StarOrange for labelling F-actin. A zoomed image shows that IRSp53 is located at close proximity of HIV-1 budding sites, as it was reported in transfected 293THEK cells and Jurkat T cells (Inamdar et al., eLife 2021). STED confocal imaging of HeLa cells (top view) expressing HIV-1 Gag showing HIV-1 Gag buds at the cell surface (green dots) with endogenous IRSp53 (red, anti-IRSp53 labelled with StarRed secondary antibody) and F-actin (Magenta, labelled with Phalloidin). (C) Scheme of the IRSp53-GFP constructs used in this study with the corresponding IRSp53 mutants: Superactive IRSp53-234, W413G, only the N-terminal membrane binding domain of IRSp53, IMD or the C-ter domain dIMD, binding cellular and actin-related factors. (D) Scheme of the GFP-trap system allowing to immunoprecipitated the IRSp53-GFP proteins, WT and mutants, and to reveal by Western blots for their interactions, directly or indirectly, with HIV-1 Gag. (E) Western blots (anti-CAp24) showing that only IRSp53-GFP WT is able to interact well with Gag. Input is the whole Gag present in the cell lysates. FT is the flow-through after application of the cell lysates on the beads. IP is the Gag protein remaining in interaction with the beads carrying IRSp53-GFP or mutants. Lanes 1: WT IRSp53-GFP + Gag; 2: 234+Gag; 3: IMD+Gag; dIMD+Gag; 5:W413G+Gag; 6: GFP+Gag; 7: mock+Gag; 8:none (showing that the upper band is not

Article Snippet: Human embryonic kidney cells (HEK 293T-ATCC-CRL-1575TM) were maintained in Dulbecco’s Modi fied Eagle’s Medium (DMEM, GIBCO) and human HeLa cells (ATCC-CRL-CCL-2).

Techniques: Western Blot, Confocal, Staining, Transfection, Imaging, Expressing, Construct, Membrane, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: HIV-1 Assembly is Assisted by IRSp53 Through an Interaction that Extends Gag Conformation

doi: 10.64898/2026.10.05.756744

Figure Lengend Snippet: Interactions between the IRSp53 SH3 and the Gag CANTD domains. (A) The initial state for molecular dynamics was the Gag CANTD from previous work and PDB 2M8L for the SH3 domain fit to the AlphaFold-predicted structure of the interface. (B) We ran 6 replicates and tracked the W59-1222 distance to measure stability. The colors correspond to the colors outlining the final snapshots in (C). There are two main modes of interactions corresponding to interactions with different prolines in the PIAP and some dissociation. The DNS domain is heavily involved in the stable interfaces. (D) The initial state for molecular dynamics was the gag CANTD from previous work and PDB 2KXC for the SH3 domain fit to the AlphaFold-predicted structure of the interface. (E) We ran replicates and tracked the W59-1222 distance to measure stability. The colors correspond to the colors outlining the final snapshots in (F). There are two main modes of interactions corresponding to interactions with different prolines in the PIAP and some dissociation. The SNK domain is heavily involved in the stable interfaces. (G) Immunoprecipitation results revealed by western blots (anti-CAp24) confirm that DNS mutation in the IRSp53 SH3 domain results in a much weaker interaction between Gag and IRSp53 than the WT. IP is Gag immunoprecipitated with IRSp53-GFP thanks to the GFP-Trap kit; Input is the total Gag proteins; the Flow-through is the Gag proteins that are not bound to IRSp53-GFP-beads complex during the experiment. These are representative western blots of 3 independent IP experiments. (H) Western blots showing Gag-VLP release from transfected HeLa cells expressin Gag and either exogenous GFP, IRTKS-GFP, IRSp53-GFP WT or GNG mutants, showing that Gag-VLP release is impaired by the IRSp53-GFP GNG mutant. WM is the weight marker in kDa.

Article Snippet: Human embryonic kidney cells (HEK 293T-ATCC-CRL-1575TM) were maintained in Dulbecco’s Modi fied Eagle’s Medium (DMEM, GIBCO) and human HeLa cells (ATCC-CRL-CCL-2).

Techniques: Stability, Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Marker