Active Site and Non-Active Mutations in HIV-1 Protease.

Ben M. Dunn, Jose C. Clemente, Reena Hemrajani, Lisa E. Blum, and Maureen M. Goodenow. University of Florida College of Medicine, Gainesville, FL (USA).

Background: Secondary mutations in the sequence of the HIV-1 protease can provide an advantage for the emergence of the primary mutations that reduce inhibitor binding. We have explored this in two series of variants of HIV-1 protease with enzymatic and structural analyses. Material and Methods: We have expressed HIV-LAI with D30N, M36I, and A71V in single, double and the triple combination. We have also expressed variants of HIV-1 protease derived from patient samples following anti-retroviral therapy. We have studied cleavage of a synthetic substrate and inhibitor binding to derive Ki values for five inhibitors. We have co-crystallized a variant and solved the structure to provide information on the effects of the mutations. Results: D30N is a known mutation that arises when patients are treated with nelfinavir. This form of HIV-1 protease has a reduced level of catalytic efficiency, as does the M36I and A71V point mutants and the double mutants of the three variants. In all cases, inhibitor binding is reduced, especially for nelfinavir and less so for ritonavir, indinavir and two newer synthetic inhibitors. However, when all three mutations are combined, the inhibitor binding remains affected, but the catalytic efficiency returns to wild-type levels. Variant V6 has been isolated from a patient and has the following mutations: K20R, V32I, L33F, M36I, L63P, A71V, V82A, and L90M. We have added the following mutations singly, in double combination and as the triple combination: M46I, I54V, and I84V. These changes were made to assess the influence of non-active site mutations on the properties of variants with active site mutations. All variants were studied with respect to catalytic efficiency and binding of inhibitors. All mutations increase the resistance of the enzyme to the effects of the inhibitors Ritonavir, Indinavir, and Nelfinavir. Variant V6-06, with I54V and I84V added to the eight mutations present in variant V6, has been crystallized with ritonavir and data collected to 2.4 Ang resolution. The structure will be reported. Conclusion: Non-active site mutations on residues that do not contact bound inhibitors can have major effects upon the catalytic efficiency and Ki values for inhibitor binding. We are pursuing a systematic study of the influence of multiple non-active site changes on the properties of HIV-1 protease.

Supported by NIH grant AI-28571 to BMD and MMG.