Figure 4. MA region is not required for VLP production. (A) 293T cells were transfected either with Gag WT, MA mutants (i.e. MAD8–132 and MAD44–132) or pcDNA3.1 (+) vector. At 48 h post-transfection, both the cell lysates and cell-free supernatants were analyzed on a gradient SDS-PAGE with Coomassie Brilliant Blue staining. (B) In parallel, an IP assay was performed using the anti-p24 antibody on the same samples. Precipitated proteins were run on gradient SDS-PAGE and Gag proteins were analyzed by Western Blotting (WB). The Biochemical experiments shown are representative of at least three independent experiments. doi:10.1371/journal.pone.0028314.g004 The above results confirm that p6 and NC are necessary for our VLP model. Moreover, we evaluated if heterologous sequences were able to efficiently substitute NC and p6 domains, like they can when Gag is expressed in a molecular clone [18]. We designed 2 substitution mutants, p6449LD and NCp6378LZLD. Concerning p6449LD, this construct differs from the Gag WT only by the substitution of the p6 domain with the RSV p2b sequence (see introduction section for more details). Overall, p6449LD was expressed intracellularly and produced VLPs to a similar extent as the Gag WT (release efficiency of about 1.4 fold compared to Gag WT) (Fig. 5). The p6449LD protein signal was detected both by SDS-PAGE and western blot assays (Figs. 7A and 7B) with a 50 kDa molecular weight (Mwth = 51.18 kDa). TEM data showed that the morphology of p6449LD VLPs (Fig. 7C, upper right panel) is comparable to that of the Gag WT (data not shown). Next, to get closer to the structure of the Gag min construct, we performed a double substitution where, in addition to replacing p6 with p2b, we replaced NC and SP2 by hCREB-1. As expected, the HIV-1 Virus-Like Particles Production resulting NCp6378LZLD mutant was able to generate VLPs, which were released efficiently from cells (Figs. 7A and 7 B). This was confirmed by TEM (Fig. 7C, bottom right) (1.4 fold compared to Gag WT) (Fig. 5). Taken together, the above substitutions and deletions in our VLP model confirm that both NC and p6 are required for particle release. In addition, their substitution with heterologous sequences does not diminish the efficiency of the Gag plasmid. N-terminal domain of CA is necessary for VLP formation The last domain remaining to be investigated was the CA protein. We focused our attention on the NTD because the CTD has been recognized as essential to the particle assembly process. On the other hand, controversial results have been obtained with NTD, as explained in the introduction section. Thus, we designed a CA NTD mutant where the complete NTD region (amino acids 133 to 277) was deleted like in the Gag min mutant. This new mutant named CADNTD at first sight appeared to be able to produce VLPs efficiently as shown both by SDS-PAGE and IP/ western blot (Figs. 8A and 8B). However, in contrast to previous studies, the morphological characterization failed to confirm that our protein gel results correspond to bona fide VLP. In fact, this mutant showed a severe default in the assembly and budding process (Fig. 8C). Nevertheless, this finding is consistent with previous observations made in baculovirus-infected cells by Chazal and colleagues where a Gag insertion mutant at position 209 resulted in a very similar cellular membrane phenotype [28]. Our CADNTD mutant actually shows very large electron-dense patches of the Gag mutant protein at deformed areas of the plasma membrane. In conclusion, the CADNTD mutant was unable to form VLPs in our hands. We hypothesize that the supernatant protein signals detected biochemically may correspond partially to cell-free patches of cell plasma membrane containing densely-aggregated Gag mutant proteins. Discussion In the present work, we designed a series of Pr55Gag mutants with the aim of defining, in the context of a mammalian expression vector, the intrinsic role of the Gag protein in the assembly and release of immature VLPs from human producer cells. Past investigations almost always used full-length molecular clones in which a point mutation inactivated the viral protease gene in order to produce immature HIV-1 particles [18,19,21]. However, using these molecular constructs, one cannot exclude the influence on the assembly process of other viral gene components like ENV or accessory molecules. Those viral accessory proteins are not desirable when investigating Gag auto-assembly. Also, working with mammalian expression plasmids presents a number of other advantages, as compared to a full-length molecular clone. For example, smaller plasmids are easier to handle. More importantly, the particles produced have no possibility of replication and cannot revert to an infectious virus. We thus studied VLP formation in the complete absence of the other viral proteins, in the perspective of gathering valuable information for future vaccine design. To achieve this goal, we expressed the precursor Pr55Gag polyprotein under a promoter different from the HIV-1 LTR in a mammalian expression vector. On the basis of biochemical and microscopical data, we found that, in contrast to previous published observations [18], the minimal Gag chimera protein failed to produce VLPs. In fact, we were unable to detect viral protein in the supernatant, nor any budding structures in TEM PLoS ONE | www.plosone.org 6 Month 2011 | Volume 6 | Issue 11 | e28314