Figure 7. Domains NC and p6 are required for VLP release, although they can be replaced with heterologous hCREB and p2b sequences. (A) 293T cells were transfected either with Gag WT or expression vectors encoding for NC or P6 mutants (i.e. Dp6, DNCp6, p6449LD and NCp6378LZLD). At 48 h post-transfection, cell lysates and cell-free supernatants were analyzed on gradient SDS-PAGE and protein stained with Coomassie Brilliant Blue. (B) In parallel, an IP assay was performed using the anti-p24 antibody on the same samples. Precipitated proteins were run on SDS-PAGE and Gag proteins were analyzed by WB. (C) Transfected 293T cells were analyzed by TEM (magnification: 6 50,000). The Biochemical experiments shown are representative of at least three independent experiments and two for the microphotographs. doi:10.1371/journal.pone.0028314.g007 studies. Nevertheless, the chimera protein is expressed inside the cells, because we were able to detect a signal by western blot and indirect immunofluorescence microscopy (Figs. 2 and 3). In addition, we detected the mRNA by RT-PCR in cell extracts (data not shown). The failure seems to happen early in the assembly process, as illustrated by the very diffuse signal appearing HIV-1 Virus-Like Particles Production in microphotographs (Fig. 3). It could consist in a major preassembly default. This sharply contrasts with the Gag WT proteins that display a punctate staining pattern, a phenotype in accordance with previous studies [57,58,59,60]. These sites of intense Gag localization presumably represent active centers of virus assembly. Characterization of such sites will provide additional insights into the mechanism of Gag trafficking and virus assembly. Our results though do not necessarily contradict those of Accola and colleagues [18], as the noted discrepancies can be explained by numerous factors. First, the authors worked with a different expression system, which was based on the HXBH10-PR- molecular clone, and, as stated above, the presence of all virus proteins may influence the action of the Gag protein during VLP production. Second, we used a different dimerization domain since we substituted the previously used GCN4 sequence by the hCREB leucine zipper domain. Although this sequence had already proven to efficiently replace NC in a previous work [43], we cannot rule out the possibility that hCREB and GCN4 differently affect VLP formation. However, it must be noted that the substitution of NC by hCREB did not modify VLP production in our assays. Third, Accola and co-workers used only biochemical approaches in their study. They did not provide any electron microscopic studies in their work. Therefore, a direct visual comparison with our observations is lacking. Most importantly, it is our TEM studies that allowed us to detect a default in assembly and budding of the CADNTD mutant, whereas biochemical approaches indicated VLP production. Fourth, the authors used different methods for detecting VLPs in cell lysates and in supernatants; for the latter, they used 35[S] methionine metabolic labelling, a method well- known for its sensitivity, whereas they performed a classical WB on cell lysates. On the other hand, we revealed the Gag protein with a biotinylated monoclonal antibody against p24 and the primary antibody was detected with Cy-3-conjugated streptavidin technol- ogy both for cell lysates and VLP with the aim of keeping the same detection method for the two types of samples. In our quest to determine the reason(s) why Gag min does not produce VLPs, we designed a series of mutants/deletants derived from Gag WT and Gag min in order to find out which domain(s) appears to be essential to produce VLPs in our conditions. We confirmed that, at least under our experimental conditions, the MA domain is dispensable for VLP formation as long as two fundamental elements are present: (1) the myristyl acid in the MA N-terminal position that interacts with the inner leaflet of the plasma membrane by hydrophobic interactions [16,52,53,61]. The absence of this myristyl acid prevents particle release [22,23,55]; (2) the basic region (BR: 18–33) in the MA N-terminal position, that fulfills multiple functions in assembly process, like plasma membrane targeting and binding [54,59,61,62]. Indeed, MAD44–132 is efficiently produced like depicted in Fig. 4 and is as efficiently released as Gag WT. In comparison, the MAD8–132 mutant, in which almost the totality of MA was deleted, is still able to produce VLPs, but less efficiently than Gag WT or MAD44–132. We observed through microscopy that this mutant lacking the BR region presents partial virus assembly and that assembly is redirected from the plasma membrane to intracellular membranes (Fig. 6) [54,61,63]. More precisely, we can suggest here that, like Ono et al. have already reported [33], assembly of BR-deficient mutant partially takes place in Golgi or post Golgi vesicles. Concerning the NC and p6 substitutions, we are in agreement with previous studies that demonstrated the importance of these two domains for VLP production. It seems clear that the deletion of p6 results in severe defaults in VLP release, which is in PLoS ONE | www.plosone.org 8 Month 2011 | Volume 6 | Issue 11 | e28314