HIV-1 Virus-Like Particles Production Figure 3. Gag min proteins show a diffuse distribution pattern. 293T cells were transfected either with Gag WT (upper panels), Gag min (middle panels) or pcDNA3.1 (+) vector (bottom panels). At 48 h post-transfection, cells were fixed and permeabilized for immunofluorescence assays as described in Materials and Methods. Nuclei were stained with DAPI. White arrows indicate a punctiform distribution of Gag proteins. The samples were visualized with a Nikon eclipse TE 300 inverted microscope (magnification: x 1,000). The experiment shown is representative of 3 independent experiments. doi:10.1371/journal.pone.0028314.g003 Subsequently, to visually confirm the results described above and to examine the site of intracellular particle formation of the two MA mutants, we performed TEM on HEK293T cells expressing Gag WT, MAD8–132 and MAD44–132. An empty control vector was also tested as well (data not shown). First, morphological analysis of the two MA mutants confirmed their typical VLP shape with spherical-like morphology and electron- dense ENV (Fig. 6). In addition, for the MAD8–132 VLPs we observed many points of intracellular assembly inside the cytoplasm (bottom right). We however could not see this phenomenon with the MAD44–132 VLPs, but rather more numerous particles in the extracellular space (bottom left). These observations confirm the discrepancy in particle release between these two MA mutants. Globally, the results above show that the MA domain can be dispensable for VLP formation, although the basic region seems to promote particle production efficiency. NC and p6 regions are required for VLP release, although they can be replaced with heterologous sequences hCREB and p2b We next investigated the possible contribution of NC and p6 domains in VLP formation. According to the literature, the p6 domain is important in the final release process from infected cells, whereas NC participates in Gag-Gag interactions [46,56]. We thus designed the deletion mutants Dp6 and DNCp6 as illustrated in Fig. 1. As with VLPs produced from molecular clones [42,43], the NC and p6 deletion constructs were unable to produce VLPs. Indeed, cell-free supernatant analysis of trans- fected HEK293T cells did not permit to detect any protein signal, be it with SDS-PAGE (Fig. 7A) or following an IP/ western blot assay (Fig. 7B). However, the two deletion mutants were clearly expressed because we were able to detect protein signals in cell lysate fractions. Respective molecular weights of 41 and 50 kDa were detected for DNCp6 (Mwth = 41.91 kDa) and Dp6 (Mwth = 50.14 kDa). Results from TEM analyses allowed us to confirm a default in VLP release for those two different constructs (Fig. 7C) (data not shown for the Gag WT control). The Dp6 mutant shows a pinching-off default, as VLPs remain tethered to the cell plasma membrane. On the other hand, DNCp6 showed an intriguing morphology. Indeed, VLP formation and assembly occur close to the inner leaflet of the plasma membrane, but the particles seem to accumulate at the membrane in large quantities, while showing no sign of pinching-off (Fig. 7C, bottom left). PLoS ONE | www.plosone.org 5 Month 2011 | Volume 6 | Issue 11 | e28314