title: Reassessing the role of region A in Pit1-mediated viral entry.
abstract: The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.
authors.affiliation: Unit on Molecular Virology, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Karen B
authors.initials: KB
authors.lastname: Farrell
authors.forename: Jill L
authors.initials: JL
authors.lastname: Russ
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Murthy
authors.forename: Maribeth V
authors.initials: MV
authors.lastname: Eiden
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D005256:Leukemia Virus, Feline / Q000378:metabolism / Q000472:pathogenicity*; D017989:Leukemia Virus, Gibbon Ape / Q000378:metabolism / Q000472:pathogenicity*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011991:Receptors, Virus / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011991:Receptors, Virus; C067981:leukemia virus receptor, gibbon ape