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.