title: Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins.
abstract: The extensive diversity of human immunodeficiency virus type 1 (HIV-1) and its capacity to mutate and escape host immune responses are major challenges for AIDS vaccine development. Ancestral sequences, which minimize the genetic distance to circulating strains, provide an opportunity to design immunogens with the potential to elicit broad recognition of HIV epitopes. We developed a phylogenetics-informed algorithm to reconstruct ancestral HIV sequences, called Center of Tree (COT). COT sequences have potentially significant benefits over isolate-based strategies, as they minimize the evolutionary distances to circulating strains. COT sequences are designed to surmount the potential pitfalls stemming from sampling bias with the consensus method and outlier bias with the most-recent-common-ancestor approach. We computationally derived COT sequences from circulating HIV-1 subtype B sequences for the genes encoding the major viral structural protein (Gag) and two regulatory proteins, Tat and Nef. COT genes were synthesized de novo and expressed in mammalian cells, and the proteins were characterized. COT Gag was shown to generate virus-like particles, while COT Tat transactivated gene expression from the HIV-1 long terminal repeat and COT Nef mediated downregulation of cell surface major histocompatibility complex class I. Thus, retrodicted ancestral COT proteins can retain the biological functions of extant HIV-1 proteins. Additionally, COT proteins were immunogenic, as they elicited antigen-specific cytotoxic T-lymphocyte responses in mice. These data support the utility of the COT approach to create novel and biologically active ancestral proteins as a starting point for studies of the structure, function, and biological fitness of highly variable genes, as well as for the rational design of globally relevant vaccine candidates.
authors.affiliation: Department of Microbiology SC-42, University of Washington, Seattle, WA 98195-8070, USA.
authors.forename: Morgane
authors.initials: M
authors.lastname: Rolland
authors.forename: Mark A
authors.initials: MA
authors.lastname: Jensen
authors.forename: David C
authors.initials: DC
authors.lastname: Nickle
authors.forename: Jian
authors.initials: J
authors.lastname: Yan
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Learn
authors.forename: Laura
authors.initials: L
authors.lastname: Heath
authors.forename: David
authors.initials: D
authors.lastname: Weiner
authors.forename: James I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D016915:AIDS Vaccines / Q000235:genetics / Q000276:immunology*; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005260:Female; D015683:Gene Products, gag / Q000145:classification / Q000235:genetics / Q000276:immunology; D015693:Gene Products, nef / Q000145:classification / Q000235:genetics / Q000276:immunology; D015696:Gene Products, tat / Q000145:classification / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010802:Phylogeny; D054311:nef Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D016915:AIDS Vaccines; D000956:Antigens, Viral; D000939:Epitopes; D015683:Gene Products, gag; D015693:Gene Products, nef; D015696:Gene Products, tat; D054311:nef Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus