domain (CTD) linked by a short flexible sequence [10,26,27]. The NTD consists of five long helices forming a stable coiled-coil structure, two more short helices, two hairpins, and a proline-rich loop. NTD is considered to play a structural role in core formation rather than actively driving particle production [18,19]. Indeed, Gag lacking this entire domain was shown to assemble and bud from the host cell [18,19]. However, previous studies have shown that Gag constructs bearing certain specific mutations or in-frame insertions in the N-terminal domain gave rise to a dramatically reduced particle production even in the absence of the other viral components, such as genomic RNA, envelope glycoproteins, enzymes and auxillary proteins [28,29,30,31]. The authors identified a previously unrecognized contiguous CA domain located beneath the cyclophilin-A binding loop that participates in Gag assembly. In contrast, CTD is recognized as being fundamental for VLP formation. This specific region contains four conserved helices and a highly conserved sequence among retroviruses known as the major homology region (MHR) [11]. The MHR determines the Gag conformation required for efficient protein-protein and protein-membrane interactions during virus assembly [31,32,33]. Moreover, the hypothetical a-helix formed by the last residues of CTD and SP1 participates in the assembly process [34]. Mutations within the 14-amino acid peptide that separates CA from NC or SP1 have been reported to induce strongly aberrant budding structures and reduce the number of released extracellular particles [31,35,36,37]. NC contains two zinc finger domains that bind RNA, and an interaction domain (I domain), consisting in a stretch of basic residues, that mediates Gag-Gag interactions [7,38]. Interestingly, the zinc fingers are not absolutely necessary for the function of the I domain [38,39,40]. Nevertheless, the role of RNA in assembly is so vital that HIV-1 will recruit other cellular RNAs by default if viral RNA is not available [41]. As far as the assembly process is concerned though, NC can be efficiently replaced by a heterologous leucine zipper (LZ) domain (i.e. a coiled-coil sequence capable of forming parallel homodimers) [18,42,43]. The p6 domain contains two late domains (LDs), which are required for pinching off the newly assembled virions from the host membrane. The first one, located in N-terminal position, is the principal LD. It contains a proline-rich PTAP motif, which appears to play a critical role in exocytosis of assembled particles [44]. PTAP interacts directly with Tsg101 [45], which is a host component of the endosomal sorting complex required for transport type-I (ESCRT-I) [46]. The second LD region located in C-terminal contains a YPLTSL motif that interacts with another cellular machinery molecule, i.e. AIP-1/ALIX (link between ESCRT-I, and –II) [47]. It is well established that, in some conditions, p6 can be replaced with a heterologous LD sequence, notably with p2b, a small spacer peptide from another retrovirus, i.e. Rous Sarcoma Virus (RSV), containing the PPPY motif [18,48,49]. Although there are numerous bacterial, yeast and insect systems to produce HIV-1-based VLP [1,2,15], the vast majority of VLP studies using a mammalian system have been performed in a provirus context [19,21,43]. Very few studies used solely the Pr55Gag polyprotein under the control of another promoter than LTR, and obviously in the absence of virus auxiliary proteins. The latter conditions moreover correspond to the common setup for vaccine studies. Now, Accola and colleagues previously described a minimal sequence of Pr55Gag able to produce VLPs as efficiently as wild type virus [18]. This chimera protein, expressed as part of a molecular clone of HIV-1, was shown to be efficiently released from host cells. In the present study, we show that a similar minimal sequence can result in different experimental observations HIV-1 Virus-Like Particles Production when inserted in a mammalian expression vector instead of being part of a molecular clone. Given this discrepancy, we investigated further and, to do so, designed a series of Pr55Gag mutants with the aim of identifying which region(s) of Pr55Gag are necessary to VLP formation in our experimental approach. We examined expression and VLP formation of these mutants by biochemical and electron microscopy approaches and, in agreement with Accola and co- workers, we demonstrate that MA is not necessary, and that NC and p6 are dispensable when heterologous sequences like hCREB and p2b are used. However, we observed that, in the context of a mammalian expression vector, VLP formation requires the presence of the NTD of the CA domain (amino acids 133 to 277) for assembly, budding and release in human human embryonic kidney 293T (HEK293T) cells. Results We tested in this study the VLP-producing ability of a minimal Pr55Gag sequence inserted in a strong mammalian expression vector, namely pcDNA3.1 (+). One of the major advantages of a plasmid over a molecular clone for producing VLPs is that, most evidently, the particles produced do not replicate, cannot revert to an infectious virus, and are thus much safer. We decided to evaluate a minimal Gag construct (called Gag min hereinafter) that had already been reported in the literature by Accola and colleagues [18]. This mutant was derived from the HXBH10-PR- molecular clone, which is identical to HXBH10, a Vpu-positive variant of the HXB2 HIV-1 molecular clone, except for a point mutation that inactivates the protease (PR) [18]. The chimera protein was shown to be efficiently released from cells. It comprises only 6 amino acids of the myristoylation signal in MA, only residues 146 to 231 of CA, the 14 amino acids of SP1, and heterologous sequences that replace NC and p6. As illustrated in Fig. 1 (bottom line), our Gag min construct is identical to the one previously described [18], except for the heterologous NC sequence, which in our case consists in the hCREB-1 sequence. This sequence forms a coiled-coil structure capable of forming parallel homodimers. We decided to use this basic LZ domain (bZIP) sequence instead of the yeast-derived GNC4 sequence used previously [18] because, when formulating a vaccine, one should avoid including foreign sequences in order not to induce non-specific cross-immune reactions. Moreover, the hCREB-1 sequence was successfully used in the past for the same purpose [43]. Finally, to improve the efficiency of protein translation in HEK293T cells, codons were humanized [50]. Gag min fails to produce VLPs To determine how Gag min behaves in our VLP experimental model system, this vector was first transiently transfected in HEK293T cells as well Gag WT (used as a positive control). At 48 h post-transfection, cell-free supernatants containing VLPs were harvested and transfected cells were collected as well, and prepared as described in Methods section. Both VLPs and cell lysates were analyzed on a gradient SDS-PAGE, and the gel was next stained with Coomassie blue. As depicted in Fig. 2A, a major band at a molecular weight (MW) of 55 kDa was observed for Gag WT and, as expected, no similar protein signal appeared in the cell-free supernatant from cells transfected with an empty control vector (called mock). However, we failed to detect a band at the predicted MW of Gag min (theoretical MW (MWth) = 18.2 kDa) in the cell-free supernatant. Too many bands were present in cell lysis samples to allow identifying the different proteins of interest. To achieve better sensibility and specificity, we performed immunoprecipitation (IP) assays on the same samples with an PLoS ONE | www.plosone.org 2 Month 2011 | Volume 6 | Issue 11 | e28314