*3.3. Cryogenic Transmission Electron Microscopy (cryo-TEM)*

Ultrastructural analysis of VLP, EV and BV populations was conducted in harvested supernatants by cryo-TEM (Figure 3B–F). Gag-eGFP VLPs were observed as electrodense nanoparticles surrounded by a lipid envelope with a granular-like heterogeneous internal structure (Figure 3B,C, white arrows). Gag-eGFP VLPs produced in both platforms displayed an average size of 202 ± 68 nm (*n* = 59) for HEK 293 and 146 ± 42 nm (*n* = 188) for Sf9 cells (Figure 3G,H, respectively). In parallel, different EV subpopulations could be detected, including exosomes (30–100 nm), microvesicles (50–2000 nm) and multivesicular bodies (MVB) (Figure 3D–F) [6]. Similar nanoparticle populations were detected in conditioned medium samples from both cell lines (Supplementary materials S1), indicating a basal expression of EVs in these cell lines. As for infected Sf9 supernatants, a large concentration of BVs was detected (black arrows), encompassing different BV phenotypes: the occlusion-derived BV (ODV), a relaxed form of the BV (rBV) (Figure 3E) and the typical infective BV (budded virus) (Figure 3F). ODVs presented several rod-shaped nucleocapsids arranged in parallel disposition inside vesicular bodies (light blue arrow), while a spiral-like nucleocapsid organization could be clearly distinguished in rBV (mid blue arrow) [7]. Budded BVs displayed ovoid-like structures containing one nucleocapsid, with the DNA highly compacted in supercoiled structures (black arrow). Moreover, the apical spikes and the BV lateral pocket, which is the space between the lipid bilayer and the nucleocapsid, were resolved using this technique (Figure 3G) [7]. Other BV forms could be identified in infected Sf9 samples and consisted of BV containing vesicles or protein structures besides the nucleocapsid (cBV, dark blue arrow, Figure 3E). Differences in Gag-eGFP VLPs (green) and Gag VLPs (brown) were also evaluated by cryo-TEM (Figure 3I,J). Gag VLPs evidenced a higher internal degree of ordered Gag arrangement in comparison to Gag-eGFP VLPs, similar to that of immature HIV-1 virions [38], while Gag-eGFP VLPs did not attain such level of structural organization.

**Figure 3.** Cryo-TEM analysis of HIV-1 Gag-eGFP VLPs produced in HEK 293 and Sf9 cells harvested at 72 hpt and 72 hpi, respectively. (**A**) Sample preparation protocol. Supernatant from HEK 293 (**B**) and Sf9 cells (**C**). Both samples were prepared and visualized in Holey carbon grids. (**D**–**F**) Morphological

characterization of contaminant particles including multivesicular bodies (MVB) (**D**), occlusion-derived BV (ODV, light blue), a relaxed-form of the BV (rBV, mid blue) or BV-containing vesicles or protein structures (cBV, dark blue) (**E**), and analysis of the infective BV (budded virus) structure: nucleocapsid (NC), lateral pocket side (P) and apical spikes (SP) (**F**). (**G**–**H**) PSD analysis of HEK 293 and Sf9 supernatants, respectively. White arrows indicate the presence of VLPs, dashed grey arrows point EVs and infective BVs are shown in black arrows. (**I**,**J**) Ultrastructural organization of an HIV-1 Gag-eGFP VLP (**I**) and an HIV-1 Gag VLP (**J**) produced in Sf9 cells by BV infection. LM: lipid membrane, MA: matrix; CA: capsid, NC: nucleocapsid. Negative controls were assessed using the same conditions as in VLP samples (Supplementary materials S1).
