HIV-1 Virus-Like Particles Production Figure 8. 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.g008 agreement with the literature [44]. Although the Dp6 mutant displays a classical VLP morphology, assembling at the plasma membrane, the particles appear unable to pinch off the cell membrane. They seem to remain tethered to the cell surface, which implies the necessity of a late domain that is able to interact with ESCRT complexes for efficiently budding out of the cells [8,9,46]. On the other hand, we obtained for the DNCp6 mutant results somewhat different from previously published ones. As depicted in Fig. 7C, Gag seems to assemble directly underneath the plasma membrane, in contrast to the formation of large patches of Gag already described [42]. Considering that the DNCp6 mutant consists in MA, CA and SP1, it is not surprising that the targeting of the protein to the plasma membrane is occurring. The CA region, along with the adjoining SP1 14 amino acids, may adopt a hypothetical a-helix conformation. This region is well known to mediate strong Gag-Gag interactions, leading to higher-order multimerization critical for particle assembly. Therefore, the morphology observed in this TEM study for the DNCp6 mutant agrees with its expected properties. Now, concerning the double substitution mutant NCp6378LZLD, it is released efficiently from HEK293T cells as confirmed by the TEM results. This agrees with previous substitutions of p6 with the RSV p2b region [48], and of NC with a heterologous LZ domain [43]. It is noteworthy that for replacing NC, instead of the GCN4 yeast sequence used in Gag min by Accola et al. we selected the hCREB sequence, a sequence successfully used previously for the same purpose [43]. Indeed, in the perspective of the VLPs being used in vaccine preparations, we preferred to avoid including an exogenous domain, which can interfere with the immune VLP specific response. Moreover, to the best of our knowledge, this is the first such double substitution of NC and p6, in an otherwise intact Gag. Finally, the most noteworthy mutant in this study is CADNTD, in which the N-terminal domain is entirely deleted. With biochemical methods, we were able to detect, as expected, a protein signal for CADNTD, both in the cell lysate and supernatant, indicating that this mutant was able to produce VLPs. Remarkably however, electron microscopy showed a total absence of VLP formation, either intracellularly or extracellularly (Fig. 8C). We suggest that the supernatant protein signals detected biochemically may correspond partially to cell-free patches of cell plasma membrane containing densely-aggregated Gag mutant proteins and not bona fide VLPs. However the cellular release of microvesicles and/or exosomes cannot be totally excluded even though this scenario is not supported by our TEM data. Additional studies are warranted to validate this postulate. This PLoS ONE | www.plosone.org 9 Month 2011 | Volume 6 | Issue 11 | e28314