M. Moulard et al., KEX2P - A MODEL FOR CELLULAR ENDOPROTEASE PROCESSING HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ENVELOPE GLYCOPROTEIN PRECURSOR, European journal of biochemistry, 225(2), 1994, pp. 565-572
The endoproteolytic cleavage of the envelope glycoprotein precursor (g
p160) 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 endoproteas
e from the yeast Saccharomyces cerevisiae as a processing enzyme model
. Endoproteolytic processing was examined using a synthetic peptide th
at mimics the cleavage site of HIV-1 glycoprotein, and a recombinant g
p160 bearing the entire sequence of the env gene product, including th
e conserved cleavage site Arg508-Glu-Lys-Arg511. Coexpression in BHK-2
1 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 an
d subjected to proteolysis with purified membranes from an S. cerevisi
ae strain overproducing the Kex2p endoprotease. Treatment of recombina
nt gp160, which has an apparent molecular mass of 127 kDa, with Kex2p
and Western blot analysis showed that the precursor was cleaved into t
wo products of about 101 and 34 kDa apparent molecular mass. Amino aci
d sequencing of the NH2-terminus of the 34-kDa product showed that the
cleavage site of recombinant gp160 was between Arg511 and Ala512. Rec
ombinant gp160 mutated at the sequence coding for the potential cleava
ge site, and mature recombinant gp120, however, were not cleaved when
treated with Kex2p. In summary, our results show that Kex2p cleaves bo
th the HIV-1 envelope glycoprotein precursor and a synthetic peptide m
imicking the cleavage site of HIV-1 gp160 at the dibasic site, suggest
ing functional analogy between yeast Kex2p and the cellular protease r
esponsible for the maturation of HIV-1 envelope glycoproteins in infec
ted human cells.