The Human Immunodeficiency Virus Type 1 (HIV-1) Protease (PR) has no direct involvement in the early steps of HIV-1 replication. Nonetheless, it is the timely and ordered processing of the viral structural proteins by the HIV-1 PR during virion maturation that facilitates the successful completion of virus entry, reverse transcription, and integration. Though a considerable amount of research has been devoted to deciphering how the enzyme prepares a virus particle for infection, the mechanisms regulating its activities continue to remain incompletely defined. RNA serves as one putative regulatory factor, since efficient processing of the maturation intermediate p15NC requires RNA in vitro. Though previously believed relevant to only p15NC cleavage, I demonstrate that RNA enhances HIV-1 proteolysis reactions in a substrate-independent manner. The increased catalytic activity of the HIV-1 PR results from a direct interaction between RNA and the enzyme, with the magnitude of the effect dependent upon the size of the RNA molecule. Large (>400 base) RNAs accelerated proteolytic processing by over 100-fold under near-physiological conditions. This considerable change stemmed from both improved substrate recognition (Km) and turnover rate (kcat). Variability in amino acid sequence also guides HIV-1 PR activity. However, the absence of any overt patterns across HIV-1 cleavage sites has complicated the delineation of why these differences result in diverse processing efficiencies. To address this question, I generated the largest-to-date dataset of globular proteins cleaved by the HIV-1 PR in near-physiological conditions. From these data, I unravel a number of site-specific processing requirements, and identify potentially important relationships shared between multiple cleavage sites. These results additionally enabled the formation of a preliminary conceptual model for explaining processing site amino acid composition. Advisors/Committee Members: Potempa, Marc, Swanstrom, Ronald, Lemon, Stanley, Margolis, David, De Paris, Kristina, Carter, Charles.