Abstract
Previous studies have suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity. Here, we examined diglutathionylated wild type protease (Cys-67-SSG, Cys-95-SSG) and the monoglutathionylated protease mutants (C67A, Cys-95-SSG and C95A, Cys-67-SSG) as potential substrates for thioltransferase (glutaredoxin). Time-dependent changes in the extent of deglutathionylation of each protein were assayed by reverse phase-high performance liquid chromatography. Glutathione alone was not an effective reductant, whereas thioltransferase displayed differential catalysis toward the Cys-95-SSG and Cys-67-SSG sites. At low thioltransferase concentrations (5 nM), deglutathionylation occurred almost exclusively at Cys-95-SSG. With substantially more thioltransferase (100 nM) Cys-67-SSG was partially deglutathionylated but only at 20% of the rate of Cys-95-SSG reduction. Treatment of the diglutathionylated protease with thioltransferase not only restored protease activity but generated an enzyme preparation that had a 3- to 5-fold greater specific activity relative to the fully reduced form. Immunoblot analysis of HIV-1MN virus with an antibody to thioltransferase detected a band co-migrating with recombinant thioltransferase that persisted following subtilisin treatment, indicating the presence of thioltransferase within HIV-1. Our results implicate thioltransferase in the regulation and/or maintenance of protease activity in HIV-1 infected cells.
Highlights
Previous studies have suggested that the two conserved cysteines of the Human immunodeficiency virus type 1 (HIV-1) protease may be involved in regulating protease activity
Cysteine residues in proteins serve a number of important roles, including catalysis [33], protein folding [34], and DNA binding [35]
The data demonstrate that thioltransferase preferentially catalyzes the deglutathionylation of cysteine 95 from each of the diglutathionylated subunits of the dimeric HIV-1 wild type protease
Summary
25935–25940, 1997 Printed in U.S.A. Thioltransferase (Glutaredoxin) Is Detected Within HIV-1 and Can Regulate the Activity of Glutathionylated HIV-1 Protease in Vitro*. Previous studies have suggested that the two conserved cysteines of the HIV-1 protease may be involved in regulating protease activity. Human immunodeficiency virus type 1 (HIV-1) encodes for an aspartyl protease which is required for viral maturation This enzyme is synthesized as part of the Gag-Pol polyprotein precursor and is active as a tightly associated dimer [1]. Additional studies, utilizing protease mutants produced by site-directed mutagenesis, demonstrated that mixed disulfides between the cysteine residues and glutathione (glutathionylation) have dramatic effects on protease activity. The reversible nature of glutathionylation and its effects on HIV-1 protease activity led us to investigate the possible involvement of human thioltransferase in regulating the redox status of these cysteine residues. We report, for the first time, the detection of this mammalian protein within HIV-1 virions
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