We are proposing to investigate the effects of non-nucleoside inhibitor-resistance (NNRTI-R) mutations on HIV-1 replication and reverse transcriptase function. We have demonstrated that 4 NNRTI-R mutants of HIV- 1 (K103N, V106A, Y181C, and P236L) alter RNase H activity of HIV-1 RT. NNRTI-R mutants with more extensive reductions in RNase H activity impair replication fitness in cell culture, and are less likely to appear during clinical failure of NNRTIs. These studies suggest that development of an active, bioavailable RNase H inhibitor will be effective clinically, and that combining NNRTI treatment with an RNase H inhibitor may be an effective strategy to prevent the emergence of NNRTI-R variants of HIV-1. Further study of these and other NNRTI-R mutants could lead to a better understanding of (a) the pathogenic consequences of selection for NNRTI-R mutants, (b) which steps in the RNase H catalytic cycle are affected by NNRTI-R mutations (c) which steps in reverse transcription are affected by specific RNase H abnormalities and their contribution to viral replication fitness, (d) how the accumulation of NNRTI-resistance mutations affects HIV-1 replication fitness during treatment failure, and (e) how RNase H cleavages are modulated by residues in RT. We propose to address these clinically relevant, pathogenic questions with the following specific aims: 1. Characterize further the effects of NNRTI-R mutants on RNase H and reverse transcription. 2. Determine the factors that contribute to selection for primary resistance mutations early in efavirenz failure. 3. Determine the factors which select for secondary mutations later in efavirenz failure. 4. Identify and characterize mutations that modulate the effects of NNRTI-R mutants. In order to accomplish these specific aims, we will utilize a broad array of experimental approaches, ranging from studies of RT structure and function in vitro, to analyses of the replication characteristics and biochemical function of HIV-1 from patient samples.