The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.The protease encoded by human immunodeficiency virus type 1 (HIV-1) plays an essential role in the retroviral life cycle by processing the viral p55Gag and p160 Gag-Pol polyprotein precursors into structural proteins and enzymes. The activity of the protease is required for conformational rearrangement of the immature virion and production of infectious virus particles, thus providing an attractive target for development of antiviral agents to treat AIDS and related disorders (29). Several potent HIV-1 protease inhibitors are widely used in clinics (7, 10). However, the constant emergence of resistant strains due to the additive effect of multiple amino acid substitutions within and outside the catalytic site motivates the continuous development of new protease inhibitors (26). The availability of reliable and convenient assays for protease activity is, in this context, of great importance.The majority of assays available today are based on trans-or autocatalytic cleavage of reporter proteins in bacteria, in yeasts, or in vitro (1,8,17,24) or on the in vitro hydrolysis of synthetic peptides encompassing the scissile bonds in p55 Gag and p160 14,23). However, none of these assays allows probing of all the native HIV-1 protease specificity sites under physiologic conditions, a situation for which a human cell environment would be required. An important reason for the lack of convenient mammalian cell-based assays is the cytotoxicity observed upon expression of the protease in cells. Thus, while this retroviral aspartic protease possesses unique structural and functional properties that distinguish it from its cellular counterparts (6), several cellular proteins are efficient substrates of the protease. Among those are cytoskeletal proteins such as vimentin, actin, troponin, and tropomyosin (20, 22), microtubule-associated proteins (30), bcl-2 (25), and precursors of NF-B (19) providing a likely explanation for the capacity of the protease to induce apoptosis.We report here on the dev...
All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be ∼10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.Keywords: Retroviral protease; dimerization; HIV protease; fireman's grip; kinetic assay; fluorescence; pressure Retroviral proteases (PRs) belong to the family of retropepsins (Dunn 1998), which are dimeric aspartic proteases. The two monomers are interconnected by their C and N termini, which form an antiparallel -sheet that is primarily responsible for the dimer stability (Wlodawer et al. 1989). The proteases are expressed as parts of the viral polyproteins Gag or Gag-Pol, from which they cleave themselves before cleaving the rest of the polyproteins to yield the structural proteins and replication enzymes. The Gag-Pol polyprotein is a product of ribosomal frame-shifting or readthrough suppression, which takes place with 5% to 10% frequency. In contrast, in the case of alpharetroviruses (e.g., myeloblastosis-associated virus [MAV]), the protease is a part of the Gag polyprotein and is thus expressed in equimolar ratio with its Gag substrate. In the case of betaretroviruses (Mason-Pfizer monkey virus [MPMV]) the protease also is expressed by frame-shifting event. It was observed previously that the activities of the alpharetroviral proteases are con-
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