HIV-1 Virus-Like Particles Production Figure 2. Gag min in the context of an expression vector fails to produce VLPs. (A) 293T cells were transfected with vectors expressing either Gag WT or Gag min. The mock represents 293T cells transfected with the empty pcDNA3.1 (+) vector (used as a negative control). At 48 h post- transfection, cell lysates (Cl) and cell-free supernatants (Sup) were analyzed on a gradient SDS-PAGE and the protein stained with Coomassie Brilliant Blue. (B) In parallel, an IP assay was performed using an anti-p24 antibody on the same samples (Cl and Sup). Precipitated proteins were run on a gradient SDS-PAGE and Gag proteins were analyzed by western blotting (WB) with a biotinylated anti-p24 antibody. (C) 293T cells expressing Gag WT or Gag min construct 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.g002 distribution pattern of proteins within the cytoplasm (middle panels). Thus, both biochemical and microscopy studies clearly show that proteins expressed upon transfection of the Gag min construct do not assemble properly when inserted within an expression vector. We next determine why this Gag min construct fails to form VLPs in our experimental setting. Considering that it was designed through investigations that almost always used full-length molecular clones [18,19,21], we hypothesized that different sequence stretches may be required for proper assembly of Gag in the absence of all other viral proteins. We thus produced a series of deletion mutants (Fig. 1), to re-examine the necessity of the various Gag domains for producing VLPs, in the context of an expression vector. MA is not required for VLP production, but the basic region allows a more efficient particle release It is now well established that the MA region plays both structural and targeting roles in the virus assembly process, but appears to be somewhat less important in Gag multimerization. The latter phenomenon relies largely on two key sequences. First, the N-terminal myristyl acid has been demonstrated to interact with the inner leaflet of the plasma membrane by hydrophobic interactions [16,52,53,54]. Its absence prevents particle release [22,23,55]. Second, the basic region (BR) (amino acids 18 to 33), which is also located in the N-terminal position, plays a pleiotropic role in the assembly process, notably in plasma membrane targeting and binding. We thus produced two MA mutants differing by the presence (MAD44–132) or the absence of BR (MAD8–132) in order to investigate whether BR is required to allow formation of VLPs. We detected the MA deletant proteins in both the cell lysates and cell-free supernatants. Those proteins were detected either with SDS-PAGE (Fig. 4A) or following immunoprecipitation with an anti-p24 antibody (Fig. 4B). The bands detected at 41 and 46 kDa respectively match the expected molecular weight of MAD8–132 (Mwth = 41.72 kDa) and MAD44–132 (Mwth = 46.02 kDa). However, the efficiency of particle release proved to be different between these two mutants. Indeed, MAD8–132 released about half as many particles as Gag WT, whereas MAD44–132 released around 40% more than Gag WT (Fig. 5). In other words, there is approximately a 3-fold difference between these two mutants. Please note that, for comparison purposes, Fig. 5 shows the relative efficiency of release of VLPs for all mutants assayed in this study. PLoS ONE | www.plosone.org 4 Month 2011 | Volume 6 | Issue 11 | e28314