Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides.

Abstract

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Eseherichia coli, to =90% purity. The purified enzyme at a concentration of "=20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 ,uM.The capsid and nonstructural proteins of all retroviruses, including human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that are proteolytically processed to the mature viral proteins by a virus-encoded, virus-associated proteinase (for a review see ref. 1). Viral proteinases (PR; for the new nomenclature for common retroviral proteins see ref.2) have been purified from virions and biochemically characterized for a number of avian (3, 4) and mammalian (5-7) retroviruses. These enzymes share limited amino acid sequence homology with members of the aspartic proteinases (8,9) and invariably contain the sequence Asp-Thr(Ser)-Gly corresponding to the catalytic center of cellular aspartic proteinases. However, retroviral enzymes are much smaller than cellular aspartic proteinases and contain only a single homologous catalytic center.

Ordered Processing of the Human Immunodeficiency Virus Type 1 GagPol Precursor Is Influenced by the Context of the Embedded Viral Protease

“…Several studies have examined the order of cleavage for the different sites within the HIV-1 Gag precursor, and there is general agreement that the first cleavage occurs between the p2 spacer peptide and the NC protein (11,15,23,28,35,36,54). Intermediate cleavages occur at the MA/CA and p1/p6 sites (36,54).…”

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

“…Cleavage of the Gag precursor at these multiple sites also appears to be highly regulated. Peptide substrates of these different sites are cleaved by the viral protease at different rates, and cleavage of the intact Gag precursor in vitro goes through a distinct series of intermediates that are generated due to different rates of cleavage at the various sites (4,7,9,24,30,36,38,43,52). The pattern of intermediates seen in infected cells and in recently budded virus particles is consistent with the ordered cleavage of Gag that has been observed in vitro (11,12,29,36,56,58).…”

“…The pattern of intermediates seen in infected cells and in recently budded virus particles is consistent with the ordered cleavage of Gag that has been observed in vitro (11,12,29,36,56,58). In the in vitro studies the initial site of cleavage occurs at the site that defines the p2/NC boundary to generate N-terminal and C-terminal intermediates of Gag (9,24,36). These intermediates are cleaved at an approximately 10-fold-lower rate, at the MA/CA site in the N-terminal intermediate and at the p1/p6 site in the C-terminal intermediate (36).…”

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties

“…HIV‐1 protease plays a crucial role in viral replication13 and a number of specific inhibitors are available. As aspartic proteases, both HIV‐1 protease and pepsin are also efficiently inhibited by the peptidic inhibitor pepstatin A 14…”