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, 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-1(MN) 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. [References: 55]

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