HIV 1 PROTEASE W/ INHIBITOR ACETYL PEPSTATIN: ISOTHERMAL TITRATION CALORIMETRY

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Human Immunodeficiency Virus (HIV), which causes Acquired Immune Deficiency Syndrome (AIDS), replicates by packaging RNA copies of its genome into infectious particles (or virions) comprised of membrane and at least nine different proteins. This packaging, together with the entry of the virion into a cell, the reverse transcription of the viral RNA into DNA, and the subsequent insertion of this DNA into the host cell's chromosome comprises what might be thought of as the HIV life cycle. Any molecule that is essential to this process could be a potential target for antiretroviral therapy. HIV protease is the enzyme responsible for processing the gag/pol polyprotein into the various structural and enzymatic proteins necessary for the packaging of HIV into virions. If the effect of this enzyme is inhibited, infected cells are not able to produce virions to infect more cells. In clinical settings, there is a well correlated decrease in viral load in peripheral blood lymphocytes in patients taking protease inhibitors. However, HIV has no proofreading mechanism associated with its reverse transcriptase enzyme, and so the reverse transcription process is highly error prone. As a result of this, viral copies arise with mutations in the HIV protease enzyme that confer resistance to inhibitors. In patients this is seen as an increase in the viral load of the resistant viruses, which somehow retain affinity for the polyprotein cleavage sites that act as substrate while losing affinity for the protease inhibitors. We have used isothermal titration calorimetry to characterize the binding of the protease and the acetylated inhibitor pepstatin A. By performing similar characterizations on mutants of the protease with various inhibitors and substrates, we should be able to map the molecular origin of HIV drug resistance, which will be invaluable for the next generation of protease inhibitors.