title: The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.
abstract: Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.
authors.affiliation: Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Pettit
authors.forename: Sergei
authors.initials: S
authors.lastname: Gulnik
authors.forename: Lori
authors.initials: L
authors.lastname: Everitt
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Kaplan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019281:Dimerization; D004789:Enzyme Activation; D015685:Fusion Proteins, gag-pol / Q000737:chemistry / Q000378:metabolism*; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015685:Fusion Proteins, gag-pol; D011498:Protein Precursors; D016333:HIV Protease