title: Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles.
abstract: HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from approximately 100 nm for Pr55Gag to approximately 250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines.
authors.affiliation: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, PO Observatory 7925, Cape Town, South Africa.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Halsey
authors.forename: Fiona L
authors.initials: FL
authors.lastname: Tanzer
authors.forename: Ann
authors.initials: A
authors.lastname: Meyers
authors.forename: Sirika
authors.initials: S
authors.lastname: Pillay
authors.forename: Alisson
authors.initials: A
authors.lastname: Lynch
authors.forename: Enid
authors.initials: E
authors.lastname: Shephard
authors.forename: Anna-Lise
authors.initials: AL
authors.lastname: Williamson
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage / Q000276:immunology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D005260:Female; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020815:Polyproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018411:Spodoptera / Q000821:virology; D013601:T-Lymphocytes / Q000276:immunology; D014771:Virion / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D016915:AIDS Vaccines; D015683:Gene Products, gag; D010455:Peptides; D020815:Polyproteins; D011993:Recombinant Fusion Proteins