Hydrophilicity dependent budding and secretion of chimeric HIV Gag-V3 virus-like particles.
Virus-like particles (VLPs) of numerous viruses have been considered as possible candidates for vaccine development. We have constructed HIV chimeric genes by coupling the gag gene of HIV-2 with the V3 domain of the gp120 gene of either HIV-1 or HIV-2 and expressed the chimeric genes in SF21 cells using the recombinant baculovirus expression system. Although the level of expression of the chimeric HIV-2 gag gene with the V3 domain of either HIV-1 gp120 (gagC-1V3) or HIV-2 gp120 (gagC-2V3) was high, the VLP assembly and extracellular release of GagC-1V3 was very poor. In contrast, GagC-2V3 chimeric proteins formed VLPs and released efficiently. We have constructed substitution mutants to investigate the effects of the hydrophobic region of the V3 domain of HIV-1 Gp120 (1V3) in VLP assembly and release. The substitution mutant analyses revealed that in replacing the hydrophobic region of the 1V3 in GagC-1V3 with the hydrophilic sequence of the V3 domain of HIV-2 Gp120 (2V3) enhanced the extracellular VLP. We demonstrate here that disruption of the hydrophobic character of the C-terminus of the chimeric protein improves assembly and release of the VLPs. Our results suggest that the poor GagC-1V3 VLP release was attributed to the hydrophobic region in the V3 sequence of the chimeric protein, and that not only the N-terminal myristylation and positively charged domain of the Gag protein functioned as a targeting signal to direct membrane binding, but also that the C-terminal hydrophobic region affected release of chimeric VLPs.
Luo
Lizhong
L
Siebens-Drake Research Institute, The University of Western Ontario, London, ON, Canada N6G 2V4.
Li
Yan
Y
Ha
Soon Duck
SD
Kang
C Yong
CY
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D015498:HIV-2 / Q000235:genetics*; D057927:Hydrophobic and Hydrophilic Interactions; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014771:Virion / Q000737:chemistry / Q000235:genetics*
D015683:Gene Products, gag; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins