Interaction between a membrane‐associated serine proteinase of U‐937 monocytes and peptides from the V3 loop of the human immunodeficiency virus type 1 (HIV‐1) gp120 envelope glycoprotein

Abstract

A trypsin-like proteinase which is inhibited by recombinant gp120 and by synthetic peptides of various lengths spanning the conserved sequence of the V3 loop has been purified and partially characterized from a U-937 cell membrane extract. V3 loop peptides behave as competitive inhibitors of the enzyme, while gp120 exerts a tight-binding inhibition, reacting in stoichiometric amounts with the proteinase to provide significant inhibition. Though the properties of the U-937 membrane proteinase towards gp120 and synthetic peptides of the V3 loop resemble those of the Molt-4 T-cell tryptase TL2, these two proteinases differ by their physicochemical properties and their susceptibility to other inhibitors of serine proteinases. These results give support to the concept of a membrane-associated proteinase as a complementary or alternative receptor to the CD4, for allowing virus to enter host cells and thus spreading HIV infection.

Inhibition of U‐937 membrane‐associated cathepsin G by GP 120 (IIIB) and V3 loop‐derived peptides from several strains of HIV‐1

“…Tryptase TL2 from Molt4 lymphocytes, and membrane cathepsin G from U-937 cells both have a combined trypsin-like and chymotrypsin-like specificity, and both are inhibited by recombinant gp120 via a V3 loop-dependent mechanism [9,14]. Supporting these observations, peptides reproducing the sequence at the tip of the V3 loop and including the conserved GPGRAF sequence inhibited tryptase TL2 and cathepsin G [9,11]. There was no indication however, that the GPGRAF sequence is involved in the interaction, or that a proteolytic cleavage within the V3 loop sequence occurs as a result of the interaction.…”

“…Membrane-associated cathepsin G was purified from U-937 monocyte-like cells as previously described [11,14] [27] using an automated Applied Biosystems 431 A peptide synthesizer. The cleaved and deprotected peptides were purified on an Aquapore RP300 CB column by high pressure liquid chromatography using a linear (0-60%) gradient of acetonitrile in 0.07% trifluoroacetic acid.…”

“…This peculiar feature explains why a membrane associated proteinase was suggested to participate in HI¥-I binding at the surface of sensitive cells [9]. Since then, several proteinases that may interact with peptides mimicking V3 loop sequences, have been identified, but there is as yet no proof that the interaction with a proteinase is a prerequisite for virus binding and fusion [8][9][10][11][12][13][14]. The proteolytic cleavage of the V3 loop by a membrane or endosomal proteinase also remains to be demonstrated.…”

Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase

“…This is illustrated by the HIV replication cycle that requires the involvement of several cellular catalysts, from the early steps of the intracellular biosynthetic pathway through particle assembly and release to the ultimate fusion of the virus and cellular membranes (2,3,11,12,15,17,30,35,36). We investigated here the relationship of Env with two major cellular partners involved in Env folding and hence in establishing its disulfide network, namely CNX, which functions early during biosynthesis (2, 3), and PDI, which functions both during biosynthesis (12) and following virus binding at the cell surface to produce the fusogenic conformation of the viral envelope (18 -20).…”

“…We and others have shown that enzymatic activities associated with the lymphoid cell surface play a key role probably by catalyzing the receptor-mediated conformational changes that occur within Env and eventually trigger fusion (15,16). Among these reactions, cleavage of the Env disulfides by a lymphocyte surface-associated PDI activity, which acts as a reductase in the redox potential conditions of the extracellular environment, is necessary for entry (17)(18)(19)(20)(21).…”