title: HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag.
abstract: During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se. Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.
authors.affiliation: Department of Global Health, University of Washington, 1616 Eastlake Ave. East, Suite 305, Seattle, WA 98102, USA.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Klein
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Reed
authors.forename: Motoko
authors.initials: M
authors.lastname: Tanaka
authors.forename: Veronica T
authors.initials: VT
authors.lastname: Nguyen
authors.forename: Samina
authors.initials: S
authors.lastname: Giri
authors.forename: Jaisri R
authors.initials: JR
authors.lastname: Lingappa
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D012260:Ribonucleases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D015683:Gene Products, gag; D012260:Ribonucleases