Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor

Abstract

The endoproteolytic cleavage of the envelope glycoprotein precursor (gpl60) of the human immunodeficiency virus type 1 (HIV-1) by a cellular protease is required for full activation of the virus. In this study, processing of gp160 was analyzed in v i m using the Kex2p endoprotease from the yeast Succharomyces cerevisiae as a processing enzyme model. Endoproteolytic processing was examined using a synthetic peptide that mimics the cleavage site of HIV-1 glycoprotein, and a recombinant gp160 bearing the entire sequence of the env gene product, including the conserved cleavage site Arg508-Glu-Lys-Arg511. Coexpression in BHK-21 of Kex2p and gp160 by recombinant vaccinia viruses demonstrates that Kex2p can correctly process the HIV-1 glycoprotein to gp120 and gp41. Furthermore, recombinant gp160 and peptide were used as substrates and subjected to proteolysis with purified membranes from an S. cerevisiae strain overproducing the Kex2p endoprotease. Treatment of recombinant gp160, which has an apparent molecular mass of 127 kDa, with Kex2p and Western blot analysis showed that the precursor was cleaved into two products of about 101 and 34 kDa apparent molecular mass. Amino acid sequencing of the NH,-terminus of the 34-kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and AlaS12. Recombinant gp160 mutated at the sequence coding for the potential cleavage site, and mature recombinant gp120, however, were not cleaved when treated with Kex2p. In summary, our results show that Kex2p cleaves both the HIV-1 envelope glycoprotein precursor and a synthetic peptide mimicking the cleavage site of HIV-1 gp160 at the dibasic site, suggesting functional analogy between yeast Kex2p and the cellular protease responsible for the maturation of HIV-1 envelope glycoproteins in infected human cells.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

“…Using genetic and biochemical approaches, the yeast Kex2 protease was shown to be both necessary and sufficient for correctly processing gp160 (33,34). That work supported other studies implicating a mammalian member(s) of the SPC protease family in HIV-1 gpl60 processing.…”

“…A number of studies have implicated the SPC proteases in HIV-1 gpl60 processing to the mature gp120 and gp41 subunits in virally infected human T cells (33,34,39,(62)(63)(64)(65)). An earlier study suggested that hPC1 was capable of processing gpl60 in vitro, and that the hPC1 gene was expressed in human H9 T cells (39).…”

Effect of alpha-1 antitrypsin Portland variant (α1-PDX) on HIV-1 replication

“…However, the fact that gp160 maturation can be obtained in LoVo and CHO-RPE40 cells which are deficient in furin activity [28,29] supports the hypothesis that other cellular endoprotease(s) could participate in the maturation of HIV-1 viral glycoproteins [25,[30][31][32][33]. In addition, our group has shown previously that the HIV-1 gp160 precursor is processed at the same Arg-Glu-Lys-Arg site by Kex2p [34], a yeast serine protease belonging to the family of the subtilisin-like endoproteases differing from furin by the fact that a basic amino acid in position P4 is not required [17]. We have also shown that the lymphocyte endoprotease responsible for gp160 maturation seems to be calcium-dependent [35].…”

The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

“…Gp160 is rarely incorporated into virions, probably because so little reaches the cell surface (Duensing et al, 1995;Pal et al, 1991;Pfeiffer et al, 1997;Willey et al, 1988). HIV-1 gp160 is cleaved between residues 518 R and 519 A by the subtilisin/kexin-like Ca 2+ -dependent convertases such as furin, PACE4, PC5/6-B, and PC1 (Morikawa et al, 1993;Moulard et al, 1994;Vollenweider et al, 1996). Gp160 is probably cleaved by more than one cellular protease.…”