title: Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation.
abstract: The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.
authors.affiliation: Molecular Microbiology and Gene Therapy Unit, Institute of Medical Microbiology and Hygiene, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Ludwig
authors.forename: Andreas
authors.initials: A
authors.lastname: Leiherer
authors.forename: Ralf
authors.initials: R
authors.lastname: Wagner
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D011533:Proviruses; D014779:Virus Replication / Q000187:drug effects; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D054301:gag Gene Products, Human Immunodeficiency Virus; C083002:p6 gag protein, Human immunodeficiency virus 1; D016333:HIV Protease