Purification and characterization of a Ca2+-independent endoprotease activity from peripheral blood lymphocytes: involvement in HIV-1 gp160 maturation.
We have analyzed the calcium requirement for HIV-1 gp160 processing in cultured nonlymphoid (CV-1 and HeLa-CD4) and human-lymphoid [Jurkat, Molt-4 and peripheral blood lymphocytes (PBMCs)] cells. The processing of gp160 in these cells, infected with recombinant vaccinia virus encoding the gp160 gene, was only partially affected by intracellular calcium depletion induced by the calcium ionophore A23187 and calcium chelator EGTA. These observations prompted us to purify the Ca(2+)-independent gp160 processing enzyme from natural targets of HIV-1 PBMCs. The endoprotease was purified to homogeneity by the use of four chromatography fractionation steps and the constant detection of the Ca(2+)-independent activity at each one of them. The enzyme was believed to be a membrane-associated heteromeric 120-kDa protein composed of three subunits of 66, 32, and 24 kDa. It was found to be specifically inhibited by substrate analogues, decanoyl-RVKR-chloromethyl ketone, and serine protease inhibitors including diisopropyl fluorophosphate (DFP) and TLCK. In contrast, no effect was observed with reducing agents including 2-beta-mercaptoethanol, N-ethylmaleimide, L-cysteine, and dithiothreitol. There were significant similarities between inhibition profiles of the purified enzyme in vitro and those of the endogenous endoprotease(s) in cell culture experiments. Therefore, the selectivity of purified endoprotease for the gp160 cleavage site, its requirement for additional residues around this consensus sequence, and its isolation from natural targets of HIV-1, made it a good candidate in the gp160 maturation process. We provide more direct and supporting evidence that HIV-1 gp160 maturation may involve at least two families of divergent endoproteases according to calcium dependence.
Bendjennat
M
M
Laboratoire d'immuno-virologie, EA 30-38 Université Paul Sabatier, UFR/SVT, 31062 Toulouse, France.
Bahbouhi
B
B
Bahraoui
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D015726:Giant Cells / Q000201:enzymology / Q000821:virology; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000201:enzymology* / Q000821:virology; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured
D019302:HIV Envelope Protein gp160; D010450:Endopeptidases; C096883:calcium-independent processing protease; C081780:gp160 processing protease; D002118:Calcium