Fig 2: HIV Gag curvature mutants can impact Arp2/3 dependent cortical F-Actin (A–E) HIV Gag mutants used. (A) Wild type HIV Gag. (B) HIV Gag late domain mutant with p6 deleted. (C) NC deletion mutant with the Leucine Zipper (LZ) derived from Saccharomyces cerevisiae GCN4 to rescue Gag oligomerisation. (D,E) HIV Gag curvature mutants (D) P99A and (E) EE75,76AA. (F) Enumeration of filopodia per cell in Diaph2-ve cell clones. *** p < 0.0001. (G,H) Representative FIB-SEM 3D rendered images of (G) WT vs. curvature mutants (H) P99A and (I) EE75,76AA. Data is derived from three independent HIV infections using each mutant.

Fig 3: Depletion of F-actin regulators reveals long filopodial networks to be driven by Arp2/3 and Diaph2. (A) F-Actin staining by phalloidin Alexa-647 reveals extensive and long filopodia in uninfected scramble shRNA control cells (Scr-CTL), short abundant filopodia upon deletion of the formin Diaph2, and extensive lamellipodia when both Diaph2 and Arp2/3 are co-depleted. (B) Schematic representation of FIB-SEM imaging data. (C–E) FIB-SEM 2D images (upper) and 3D reconstructions (lower) of (C) non-depleted control cells (Scr-CTL), (D) Diaph2-depleted cells and (E) Diaph2 and Arp2/3 co-depleted cells. All scale bars are 5 µm. (F–H) Schematic representation of phenotypes induced by loss of F-Actin regulators. (F) Wildtype scenario, (G) Diaph2 deficiency, (H) Diaph2 & Arp2/3 deficiency. Note in (H) we represent reduced levels of Arp2/3, given its high cellular abundance and residual levels of Arp2/3 in our knockdown cells (Figure S3). From here on, in all figure legends, all cells used to observed the dynamics of F-Actin during HIV infection are based on the U937 cell line or related genetically manipulated clones.

Fig 4: HIV budding enriched to positively curved cortical F-Actin. (A) Representative FIB-SEM images of HIV virions associated with (B) Filopodia and (C,D) plasma membrane of a HIV infected cell depleted of Diaph2. (E,F) 3D rendering FIB-SEM images of HIV infected (E) Diaph2-ve and (F) Diaph2-veArp2/3-ve cell clones. HIV buds are shaded in red to highlight their location. (G,J) enumeration of HIV-buds in association with positively curved cortical F-Actin structures. (G) filopodia and (J) lamellipodia are pseudocolored using a colour spectrum from blue (positive curvature), green (neutral curvature) to red (negative curvature). (H,I) Enumeration of total HIV buds in association with the filopodia in Diaph2-ve cells. (H) Absolute viral bud counts and I. HIV bud density per µm2. (K,L) Enumeration of total HIV buds in association with the lamellipodia in Diaph2-ve Arp2/3-ve cells. (K) Absolute viral bud counts and (L) HIV bud density per µm2. All scale bars are 0.5 µm, with the exception of (B–D), which is 0.1 µm. Bar graphs in (H,I) represent mean and standard deviations of virion counts from n = 15 filopodial structures. (K,L) bar graphs represent mean and standard deviations of virion counts from n = 15 lamellipodial structures. Statistics for panels (G–L) are summarised in Table S1.

Fig 5: Cortical F-Actin regulators enriched at the final stages of HIV egress are revealed through HIV proteomics. (A,B) Viral proteome analysis of proteins associated with cortical F-Actin regulation. Proteins with increased abundance relative to untreated cells are shown in green, whereas those with relative decreased abundance are shown in red. (A) Virions produced in Diaph2-ve cells. 1. Highlights the Arp2/3 complex node where the amounts of ACTR2 and Cdc42 are increased in virion proteomes. 2. Indicates a GTPase node in association with IQGAP1. 3. Highlights a node of integrin and related proteins. 4. Highlights a node involved in cadherin adhesion that is downregulated upon Diaph2 depletion. (B) Virions produced in Diaph2-veArp2/3-ve cells. Note that in 1. Arp2/3 components are predictably depleted compared to untreated cells, whilst in 2. the GTPase node and IQGAP1 remain unchanged.