title: Critical role of helix 4 of HIV-1 capsid C-terminal domain in interactions with human lysyl-tRNA synthetase.
abstract: Human tRNALys3 is used as the primer for human immunodeficiency virus type 1 (HIV-1) reverse transcription. HIV-1 Gag and GagPol, as well as host cell factor lysyl-tRNA synthetase (LysRS), are required for specific packaging of tRNALys into virions. Gag alone is sufficient for packaging of LysRS, and these two proteins have been shown to interact in vitro with an equilibrium binding constant of approximately 310 nM. The capsid (CA) domain of Gag binds to LysRS with a similar affinity as full-length Gag. In this work, we report further characterization of the interaction between HIV-1 CA and human LysRS using truncation constructs and point mutations in the putative interaction helices. Fluorescence anisotropy binding measurements reveal that a LysRS variant lacking the N-terminal 219 residues still displays high affinity binding to CA. The CA C-terminal domain (CTD) is also sufficient for binding to LysRS. Nuclear magnetic resonance spectroscopy studies using 15N-labeled CA-CTD reveal chemical shift perturbations of residues in and proximal to helix 4 of CA-CTD upon LysRS binding. A synthetic peptide that includes helix 4 binds to LysRS with high affinity, whereas peptides derived from the other three helical domains of CA-CTD do not. Alanine-scanning mutagenesis studies targeting residues in the helix 4 region support a direct interaction between this domain of CA-CTD and LysRS. The high resolution mapping studies reported here will facilitate future work aimed at disrupting the Gag-LysRS interaction, which represents a novel anti-viral strategy.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA, and Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Brandie J
authors.initials: BJ
authors.lastname: Kovaleski
authors.forename: Robert
authors.initials: R
authors.lastname: Kennedy
authors.forename: Ahmad
authors.initials: A
authors.lastname: Khorchid
authors.forename: Lawrence
authors.initials: L
authors.lastname: Kleiman
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuo
authors.forename: Karin
authors.initials: K
authors.lastname: Musier-Forsyth
mesh_terms: D000595:Amino Acid Sequence; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus; D008250:Lysine-tRNA Ligase