title: Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein.
abstract: A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: T G
authors.initials: TG
authors.lastname: Edwards
authors.forename: T L
authors.initials: TL
authors.lastname: Hoffman
authors.forename: F
authors.initials: F
authors.lastname: Baribaud
authors.forename: S
authors.initials: S
authors.lastname: Wyss
authors.forename: C C
authors.initials: CC
authors.lastname: LaBranche
authors.forename: J
authors.initials: J
authors.lastname: Romano
authors.forename: J
authors.initials: J
authors.lastname: Adkinson
authors.forename: M
authors.initials: M
authors.lastname: Sharron
authors.forename: J A
authors.initials: JA
authors.lastname: Hoxie
authors.forename: R W
authors.initials: RW
authors.lastname: Doms
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D001798:Blood Proteins / Q000494:pharmacology; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002459:Cell Fusion; D002460:Cell Line; D000939:Epitopes / Q000378:metabolism; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011487:Protein Conformation; D011784:Quail; D019718:Receptors, CXCR4 / Q000378:metabolism; D014162:Transfection; D014760:Viral Fusion Proteins / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D015704:CD4 Antigens; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D019718:Receptors, CXCR4; D014760:Viral Fusion Proteins