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

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Abstract
The endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) 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 vitro using the Kex2p endoprotease from the yeast Saccharomyces 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 NH2‐terminus of the 34‐kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and Ala512. 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.