Purification and Characterization of a Ca²⁺-Independent Endoprotease Activity from Peripheral Blood Lymphocytes:  Involvement in HIV-1 gp160 Maturation

Abstract

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.

Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

“…In contrast, treatment with 10 mM NH 4 Cl retarded exocytic transport, as a predominantly Endo H-sensitive form of Hendra F 0 was identified (Fig. 6C, lane 8).…”

“…6) strongly argues that the proteolytic event occurs intracellularly. Previous studies examining proteolytic processing of proalbumin (50) and precursor complement protein (proC3) (51) used weak basic amines such as chloroquine and NH 4 Cl to establish that maturation occurred in an acidic intracellular compartment, namely the Golgi complex, where furin is now recognized as the processing enzyme (45). Early virus entry studies used chloroquine to determine whether virus entry was a pHdependent process or an independent process (9,14).…”

“…Chloroquine and NH 4 Cl were present throughout the starvation, label, and chase periods, whereas bafilomycin A1 and concanamycin A were added only to the chase medium. Endo H digestion.…”

“…It was therefore of interest to examine whether manipulations of intracellular pH affected cleavage of Hendra F. Weak basic amines such as chloroquine and NH 4 Cl (32, 50), as well as inhibitors of the vacuolar H ϩ V-ATPase, bafilomycin A1 and concanamycin A (18, 41), have been shown to raise intracellular pH. Vero cells transiently expressing Hendra F and SV5 F were metabolically labeled in the presence of increasing concentrations of chloroquine (1 to 100 M), NH 4 Cl (1 to 20 mM), or bafilomycin A1 or concanamycin A (10 to 100 nM). Furin is known to be proteolytically active in the TGN and at the cell surface, as well as in endocytic vesicles, and has been characterized to be active over a broad pH range (pH 6 to 8) (62).…”

“…The arrows on the right designate the positions of the F 0 , F 1 , and F 2 proteins. (C) pCAGGSHendra F-transfected Vero cells were metabolically labeled and chased as before in the absence (Ϫ) or presence (ϩ) of 10 M chloroquine, 10 mM NH 4 Cl, 50 nM bafilomycin A1, or 10 nM concanamycin A. Chloroquine and NH 4 Cl were present in the starvation, label, and chase media, and bafilomycin A1 and concanamycin A were present only in the chase medium. Samples were lysed, immunopreciptated, and either mock treated (Ϫ) or treated with 2 mU of Endo H (ϩ) for 27 h at 37°C prior to analysis by SDS-10% PAGE and the STORM imaging system.…”