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 ␤-branched amino acid or the P1 amino acid with lysine. Cleavage-resistant PRs which had K m 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.

Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

“…Urea allows the unfolding of hydrophobic regions of proteins including β-sheet and extended conformations, but not α-helical regions [29]. Urea has been utilized in vitro to test the stability of other enzymes in which the N-termini were modified [30]. As a result, the enzymatic stability of Top68W/A was tested in a supercoiled DNA relaxation assay in the presence of increasing concentrations of urea (Experimental).…”

Kinetic Characterization of Newly Discovered Inhibitors of Various Constructs of Human T-Cell Leukemia Virus-1 (HTLV-1) Protease and Their Effect on HTLV-1-Infected Cells

“…Cultures were inoculated with 4% (v/v) starter culture, which was grown overnight for approximately 16 h. When the optical density reached 0.8, gene expression was induced with final concentration of 1 mM isopropyl b-d-thiogalactopyranoside. The cells were allowed to grow for an additional 3-4 h and then pelleted by centrifugation at 10,000×g for 10 min at 0ºC [11]. Cell pellets were thawed and resuspended in buffer A (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5 M urea) and DNase I was added to a final concentration of 0.1 mg/ml.…”

“…The flow through from the column was collected and adjusted to pH 4.0 with acetic acid. The pH adjusted solution was then loaded onto a Sepharose Fast Flow SP column (Pharmacia) equilibrated in buffer D (6 M urea, 20 mM sodium acetate pH 5.0, 5 mM EDTA) [11]. The PR that bound to the column was eluted with 0.4 M NaCl.…”

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

“…1B). Similarly to simian immunodeficiency virus (SIV) and HIV-1 proteases, autoproteolysis of FIV protease is observed in vitro (19). Despite this similarity, FIV protease is specific to its respective substrates, and the most potent inhibitors of HIV-1 protease do not inhibit FIV protease (8,35,49).…”