Comparing the Accumulation of Active- and Nonactive-Site Mutations in the HIV-1 Protease

Abstract

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active-and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6 54/84 variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystalstructure analysis of the V6 54/84 variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.The development of resistance to protease inhibitors (PI) 1 during treatment of infection by the Human Immunodeficiency Virus (HIV) still poses one of the greatest challenges in the struggle to limit the virus replicative capacity. After initiation of therapy with single or multiple protease inhibitors, resistance mutations in the protease can appear within weeks. Under a constant drug selection pressure, resistant mutations continue to accumulate. It is clear that there is a correlation between the number of resistance mutations and the level of resistance and cross resistance to multiple protease inhibitors. It is of great interest to understand the level of contributions made by active-and nonactive-site mutations to the development of a high level of resistance (1-6). The HIV protease is a symmetric dimer composed of two 99-residue polypeptides (Figure 1). The dimerization region comprises the floor of the active site, which includes two catalytic aspartic acids (Asp25), one provided by each polypeptide. Unlike the human aspartic proteases that have one flap, two flaps completely cap the active site of the HIV protease. A more detailed description of HIV-1 protease structure, inhibitor binding, and resistance can be found in reviews by Wlodawer and Gustchina, Tomasselli and Heinrikson,.In this study, we analyze the effect of adding the nonactive-site mutations M46I and I54V and the active-site mutation I84V to a post...

Analysis of HIV-1 CRF_01 A/E Protease Inhibitor Resistance: Structural Determinants for Maintaining Sensitivity and Developing Resistance to Atazanavir

“…It was originally believed that non-active site mutations were acquired to regain the loss of catalytic activity due to active site mutations. Recent studies from our laboratory and others have shown that many non-active site mutations in HIV-1 subtype-B proteases will contribute to decreasing the binding affinity of inhibitors, while maintaining catalytic efficiency (10,(22)(23)(24)26,27). In this study we provide insights into the effects of natural polymorphisms and the contributions to resistance made by therapy-selected active site and non-active site mutations found in the CRF_01 AE protease.…”

“…The Ile50 residue forms various interactions to lock down the flaps and mutations at this residue may function to destabilize the protease closed conformation (23). The Ile84 residue is located in the center of the active site and we previously suggested that the I84V mutation may function to destabilize the interactions of the core of the inhibitor and its interactions with the flaps (23). Previous studies have also shown the importance of mutations in the flaps in providing a high level of resistance (10,22,23,26), and that the addition of non-active site mutations may decrease binding affinity by destabilizing the flaps in the bound conformation decreasing binding affinity (23).…”

“…The Ile84 residue is located in the center of the active site and we previously suggested that the I84V mutation may function to destabilize the interactions of the core of the inhibitor and its interactions with the flaps (23). Previous studies have also shown the importance of mutations in the flaps in providing a high level of resistance (10,22,23,26), and that the addition of non-active site mutations may decrease binding affinity by destabilizing the flaps in the bound conformation decreasing binding affinity (23). Recent studies by Yanchunas and colleagues, utilizing thermodynamic and modeling techniques, have suggested the importance of ATV P2 and P2′ groups interactions with Ile50 in maintaining binding affinity (33).…”

A Cluster Analysis on the Structural Diversity of Protein Crystals, Exemplified by Human Immunodeficiency Virus Type 1 Protease

“…It should be noted that all of the crystal structures in group 5 were provided by two research groups. [29][30][31] The L63P mutation that is an amino substitution sometimes appeared in drug-resistant viruses 32,33) is seen in every cluster except for group 3.…”

A Conserved Hydrogen-Bonding Network of P2 bis -Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

“…Additionally, mutations at codons 10, 54, 63, 71, 82, and 84 have been known to cause HIV-1 resistance against RTV (32), and PR A02 consists of amino acid substitutions L10I, I54V, L63P, A71V, and V82T. The combination of active-site and non-active-site amino acid substitutions has been shown to cause a significant loss of binding affinity to RTV (42-to 1,330-fold increase in the K i values compared to that for PR WT ) due to loss of direct contacts as well as altered protease-flap dynamics (33,34). As shown in Fig.…”