discovery disagrees with the commonly accepted notion that NTD is dispensable for VLP production [18,19], but rather plays a role in the rearrangement of different Gag components during the maturation process. We are currently investigating the reasons why the CA NTD appears to be important in VLP production at least under our experimental settings. It may be that the NTD region allows weak interactions with the CTD during virus assembly as suggested by a previous study [31]. The authors clearly demonstrated that point mutations in helices 4–6 greatly reduce viral production. It is also possible that these mutations disrupt the overall folding of CA. The structure of full-length HIV- 1 CA has been previously depicted by Ganser-Pornillos and colleagues [10,64] and it was shown that NTD-NTD forms the hexamerisation interface, whilst CTD-CTD forms the dimeriza- tion interface, and that an additional interface exists between NTD and CTD in a mature CA context. This might help to explain why deletion of the CA NTD prevents VLP formation. In conclusion, the observed requirement of NTD for Gag-Gag interaction and consequently for Gag assembly may explain the failure of Gag min to lead to VLP formation in our experimental setting. Therefore, we believe that a better understanding of mechanisms underlying VLP production will help the scientific community in future design of VLP-based HIV-1 vaccines. Methods Pr55Gag molecular constructs Gag sequences were derived from the hGag-TAP construct (i.e. HXB2-based vector) (GenBank K03455) in which codons were humanized [65]. Gag fragments were PCR amplified with Taq Hi-Fi (Invitrogen, Burlington, Canada) from the hGag-TAP construct and cloned into the pcDNA3.1 (+) vector (Invitrogen, Burlington, Canada) using HindIII (forward) and EcoRI (reverse) restriction sites present in respective PCR primers (restriction sites are underlined in the following primer sequences). We constructed a series of mutant Pr55Gag expression plasmids as depicted in Fig. 1. In brief, the Gag wild-type (Gag WT) sequence was amplified using forward primer 59-CCC AAG CTT ATG GGC GCC CGC GCC AGC and reverse primer 59-CCG GAA TTC TCA TTG TGA CGA GGG GTC GCT GC. The MAD8–132 construct involved deletion of the MA domain except for the myristylation signal sequence included in the forward primer (shown in italic): 59- CCC AAG CTT ATG GGC GCC CGC GCC AGC GTG CCC ATC GTG CAG AAC ATC CAG. The reverse primer was the same one as for the Gag WT construct. In the p6449LD construct, the p6 domain was replaced by a LD sequence derived from the RSV p2b domain [48], which consists of a TASAPPPPYVG motif in the expressed protein. The corresponding motif sequence was introduced in the reverse primer (shown in italic). The forward primer used was the same as for Gag WT construct, and the reverse primer was 59- CCG GAA TTC TCA GCC CAC GTA GGG AGG TGG AGG GGC GCT GGC GGT AAA ATT CCC TGG CCT TCC CTT G. For the MAD44–132 construct, fragment 1 was amplified using the forward primer used for the Gag WT construct. The reverse primer contained the sequences corresponding to the beginning of the CA domain (underlined in the text) and to the end of the basic region sequence (shown in italic): 59- CTG GAT GTT CTG CAC GAT GGG GCG CTC CAG CTC GCG GCT G. For fragment 2, the forward primer contained the end of the basic region domain sequence (shown in italic) and the beginning of the CA domain sequence (underlined in the text): 59- C AGC CGC GAG CTG GAG CGC CCC ATC GTG CAG AAC ATC CAG. The reverse primer was the same one as for the Gag WT construct. The two fragments HIV-1 Virus-Like Particles Production were combined in a fusion PCR reaction (see Dp6 construct below) using forward and reverse primers used for the Gag WT construct. For the NCp6378LZLD construct, fragment 1 was PCR amplified with the forward primer used for Gag WT construct. The reverse primer contained the sequence corresponding to the beginning of the LZ domain (underlined in text) from the CREB gene and the end of the SP1 spacer (shown in italic): 59- TTT CTT CTT TCT ACG ACA CTC TCG CAT GAT GGT GGC GCT GTT GGT C. For fragment 2, the DNA template used was the plasmid KCREB1 provided generously by Dr. R. H. Goodman (Vollum Institute, Portland, USA). The forward primer contained the end of the SP1 sequence (shown in italic) and the beginning of the LZ sequence (underlined in the text): 59- G ACC AAC AGC GCC ACC ATC ATG CGA GAG TGT CGT AGA AAG AAG AAA. The reverse primer contained the sequence corresponding to the end of LZ region (underlined in the text), a sequence coding for LD (shown in italic) humanized according to a previously published code [50] and the restriction site for EcoRI (underlined in the text): 59- CCG GAA TTC TCA GCC CAC GTA GGG AGG TGG AGG GGC GCT GGC GGT ATC TGA TTT GTG GCA GTA AAG G. The two fragments were combined in a fusion PCR (see Dp6 construct below) using the forward primer used for fragment 1 amplification and the reverse primer used for fragment 2 amplification. For the CADNTD construct, fragment 1 was amplified using the forward primer used for the Gag WT construct. The reverse primer contained the sequences corre- sponding to the beginning of the CA MHR domain (underlined in the text) and to the end of the MA domain (shown in italic): 59- GTC CAG GAT GCT GGT GGG GCT GTA GTT CTG GCT CAC CTG GTT G. For fragment 2, the forward primer contained the end of the MA domain (shown in italic) and the beginning of the CA MHR domain (underlined in the text): 59- C AAC CAG GTG AGC CAG AAC TAC AGC CCC ACC AGC ATC CTG GAC. The reverse primer was the one used for the Gag WT construct. The 2 fragments were combined in a fusion PCR reaction (see Dp6 construct below) using forward and reverse primers used for the Gag WT construct. The Gag min construct was based on published work describing the minimal Gag sequence required to generate VLPs [18]. Fragment 1 was amplified with a forward primer containing a HindIII restriction site (underlined in the text), a myristylation site (shown in italic) and the beginning of the CA MHR domain (underlined in the text): 59- CCC AAG CTT ATG GGC GCC CGC GCC AGC GTG AGC CCC ACC AGC ATC CTG GAC. The reverse primer contained the sequences corresponding to the beginning of LZ domain (underlined in the text) and to the end of the SP1 sequence (shown in italic): 59- TTT CTT CTT TCT ACG ACA CTC TCG CAT GAT GGT GGC GCT GTT GGT C. For fragment 2, the template DNA used was the KCREB1 plasmid. The forward primer contained sequences corresponding to the end of SP1 (underlined in the text) and to the beginning of the LZ domain (shown in italic): 59- G ACC AAC AGC GCC ACC ATC ATG CGA GAG TGT CGT AGA AAG AAG AAA. The reverse primer was the same one as for the NCp6378LZLD construct. The 2 fragments were combined in a fusion PCR using the forward primer used for fragment 1 and the reverse primer used for fragment 2. For both Dp6 and DNCp6 constructs, the forward primer was the same one as for the Gag WT construct. The reverse primer used for the Dp6 construct was 59- CCG GAA TTC TCA AAA ATT CCC TGG CCT TCC CTT G. The reverse primer used for the DNCp6 construct was 59- CCG GAA TTC TCA CAT GAT GGT GGC GCT GTT GG. Chimeric sequences were generated by fusion PCR. Briefly, two fragments were PCR amplified using PLoS ONE | www.plosone.org 10 Month 2011 | Volume 6 | Issue 11 | e28314