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-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.
Davis
D A
DA
HIV and AIDS Malignancy Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Newcomb
F M
FM
Starke
D W
DW
Ott
D E
DE
Mieyal
J J
JJ
Yarchoan
R
R
D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D010088:Oxidoreductases; D011506:Proteins / Q000032:analysis*; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism
D054477:Glutaredoxins; D011506:Proteins; D010088:Oxidoreductases; D013381:Subtilisins; D016333:HIV Protease; D005978:Glutathione; D003545:Cysteine