HIV-1 Virus-Like Particles Production Figure 1. Schematic representation of the different HIV-1 Pr55Gag-derived constructs used in this study. The various molecular constructs, derived from the HXB2 Pr55Gag sequence (designated here as Gag WT), were made by PCR as described under the Materials and Methods section. All constructs contain the wild-type myristic acid post-translational sequence in the N-terminal position and all were subcloned into the pcDNA3.1 (+) eukaryotic expression vector. Wild-type HIV-1 sequences are denoted in black, and deleted virus sequences correspond to the dotted line. Numbers above correspond to amino acids in the wild-type sequence. BR, basic region of MA; NTD, N-terminal domain; CTD, C-terminal domain. doi:10.1371/journal.pone.0028314.g001 anti-p24 antibody, followed by western blot detection. With this technical strategy, we obtained for Gag min a specific signal at the predicted 18.2 kDa in the cell lysate (Fig. 2B). However, we were still unable to detect a signal in the cell-free supernatant, whereas we detected a distinctive band for the positive Gag WT control. At this point, we hypothesized that the Gag min protein was being expressed by the transfected cell, but could not give rise to VLP production due, for example, to a malfunction in the assembly process. To explore this possibility, we analyzed our transiently-transfected HEK293T cells by transmission electron microscopy (TEM). For Gag min, we did not observe any VLP in the process of budding through the cell membrane, or in the supernatant close to the cell, in accordance with our previous biochemical experiments (Fig. 2C). Moreover, there was no VLP, whether incomplete or fully formed, in the cytoplasm of the transiently transfected cells. This is in sharp contrast to Gag WT that showed a characteristic VLP spherical morphology (average size around 100 nm), similar to that of immature HIV-1 particles [51]. To further investigate the intracellular location of the Gag min proteins within the transfected cells, indirect immunofluorescence studies were performed with an anti-p24 antibody. As illustrated in Fig. 3 (upper panel), Gag WT proteins were detected throughout the cytoplasm of transfected cells, organized in dotted structures suggesting they were undergoing VLP assembly. In contrast, cells transfected with the Gag min vector showed a more diffuse PLoS ONE | www.plosone.org 3 Month 2011 | Volume 6 | Issue 11 | e28314