title: Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases.
abstract: The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Laco
authors.forename: M C
authors.initials: MC
authors.lastname: Fitzgerald
authors.forename: G M
authors.initials: GM
authors.lastname: Morris
authors.forename: A J
authors.initials: AJ
authors.lastname: Olson
authors.forename: S B
authors.initials: SB
authors.lastname: Kent
authors.forename: J H
authors.initials: JH
authors.lastname: Elder
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002415:Cats; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology*; D015394:Molecular Structure
chemical_list: D016282:Aspartic Acid Endopeptidases; C095555:FIV protease