Hydrophilicity dependent budding and secretion of chimeric HIV Gag-V3 virus-like particles

Abstract

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.

Production, Characterization and Assay Application of a Purified, Baculovirus-Expressed, Serogroup Specific Bluetongue Virus Antigen

“…assembly and release) and characteristics (i.e. immunogenicity and antigenicity) by virtue of changing the secondary structure of the protein upstream from this terminal region (Haynes et al., 1986; Clarke et al., 1987; Luo et al., 2007). As binding of these Mabs was dependant on protein conformation, it could be that the immunogenic determinant on the BTV‐1 VP7 protein that induced Mab 3.17.A3 was not completely identical in its structure to the corresponding determinant on BTV‐11 VP7, due to these substitutions, resulting in inferior binding of Mab 3.17.A3 to the BTV‐11 rVP7 protein.…”

Virus-Like Particles Show Promise as Candidates For New Vaccine Strategies

“…Among retro-and lenti-virus-derived VLPs, much insight into immune stimulation and optimum VLP design has been gained from HIV-1 Pr55 gag -derived VLPs [12,[27][28][29], whose immunogenicity can be broadened by making chimeric Gag molecules [30]. However, VLPs incorporating foreign polypeptides fused in frame with Gag assemble with a strongly diminishing efficiency with both the insertion domains [31] and length of exogenous sequences [32].…”