Identification of the Paired Basic Convertases Implicated in HIV gp160 Processing Based on in Vitro Assays and Expression in CD4+ Cell Lines

Abstract

The human immunodeficiency virus HIV envelope glycoprotein gp160 is synthesized as an inactive precursor, which is processed into its fusiogenic form gp120/gp41 by host cell proteinases during its intracellular trafficking. Kexin/subtilisin-related endoproteases have been proposed to be enzyme candidates for this maturation process. In the present study, 1) we examined the ability of partially purified precursor convertases and their isoforms to cleave gp160 in vitro. The data demonstrate that all the convertases tested specifically cleave the HIV envelope glycoprotein into gp120 and gp41. 2) We demonstrated that a 19-amino acid model peptide spanning the gp120/gp41 junction is cleaved by all convertases at the same gp160 site as that recognized in HIVinfected cells. 3) In an effort to evaluate specific convertase inhibitors, we showed that the ␣ 1 -antitrypsin variant, ␣ 1 -PDX, inhibits equally well the ability of the tested convertases to cleave gp160 in vitro. 4) Three lymphocyte cell lines were screened by reverse transcription polymerase chain reaction in an effort to identify which are the convertases expressed in the most common HIV target, the CD4 ؉ lymphocytes. The data demonstrate that furin, PC5/6, and the newly cloned PC7 are the main transcribed convertases, suggesting that these proteinases are the major gp160-converting enzymes in T4 lymphocytes.

Processing and trafficking of a prohormone convertase 2 active site mutant

“…With regard to the identification of this enzyme activity, furin is known to be quite active at neutral pH; it exhibits greater than 50% of maximal activity between pH 6.0 and 8.5 [20]. The convertases PC6 and PACE4 have a broad pH optimum with peak activity at neutral to weakly basic pHs (7.0-8.0 for PC6; 7.0-8.5 for PACE4) [21]. In contrast to these other convertases, PC1 active forms are active within a much narrower pH range; 87 kDa PC1 has an optimum pH between pH 5.5 and 6.5 [22] and 74/66 kDa PC1 displays an even narrower pH optimum (between 5.0 and 5.5) with significantly reduced activity at neutral pH [23].…”

“…Our ability to purify the intact zymogen from PC2-S383A-producing CHO-K1 cells co-expressing 21 kDa 7B2 should lead to a better understanding of its molecular structure including its interaction with 7B2, its complex activation mechanism, and the development of specific and potent PC2 inhibitors for therapeutic use. A. Sequence of the PC2 propeptide; Three potential cleavage sites within the proPC2 propeptide are shown in bold; the primary and secondary sites known to be cleaved [21] are indicated. B. Purified PC2 and PC2-S383A were incubated at pH 5.0 for the indicated times and were immunoblotted with PC2 antiserum.…”

Comparative processing of bovine leukemia virus envelope glycoprotein gp72 by subtilisin/kexin‐like mammalian convertases

“…So far, a total of seven mammalian subtilisin-like proteases have been identified: furin (also called PACE), PCI (PC3), PC2, PACE4, PC4, PC5-A (PC6-A) and its isoform PC5-B (PC6-B) and the newly discovered PC7 (PC8, LPC or SPC7) (for reviews and updates [6][7][8][9]). While furin is ubiquitously expressed, PC7, PACE4 and PC5 exhibit a widespread tissue distribution, and all catalyze the processing of precursors within the constitutive secretory pathway [6][7][8][9][10][11]. In addition, we recently showed that immune cells express large amounts of both furin and PC7 [9][10][11].…”

“…Thus, in cellular coexpression experiments, furin was shown to catalyze the proteolytic processing of Newcastle disease virus F protein [16], parainfluenza virus type 3 HA protein [17] and cytomegalovirus gpUL55 protein [18], and furin, PACE4 [19,20] and PC7 [10] cleave HIV-1 gpl60. In vitro experiments have demonstrated that furin, PACE4, PC5 as well as PCI can cleave the HIV-1 gpl60 into gpl20/gp41 [10,11,20,21]. Although these results suggest that a certain redundancy exists in the ability of mammalian convertases to process viral envelope glycoproteins, quantitation of the level of expression of the PCs in virus-infectable host cells should be considered.…”

“…While furin is ubiquitously expressed, PC7, PACE4 and PC5 exhibit a widespread tissue distribution, and all catalyze the processing of precursors within the constitutive secretory pathway [6][7][8][9][10][11]. In addition, we recently showed that immune cells express large amounts of both furin and PC7 [9][10][11]. In contrast, PCI, PC2 [9,12,13] and possibly PC5-A [14] are responsible for precursor processing within the regulated secretory pathway, mainly in endocrine and neural cells.…”