Mutations that alter the activity of the Rous sarcoma virus protease.

Area of Science:

• Structural biology
• Enzymology
• Virology

Background:

• Rous sarcoma virus (RSV) protease (PR) and human immunodeficiency virus (HIV)-1 PR share structural similarities.
• Understanding protease function is crucial for antiviral drug development.
• Crystal structure analysis provides a basis for rational enzyme engineering.

Purpose of the Study:

• To investigate the functional impact of specific mutations in RSV PR based on structural models.
• To correlate structural changes with alterations in enzymatic activity against RSV and HIV-1 substrates.
• To explore the roles of the substrate binding pocket, flaps, and surface loops in RSV PR function.

Main Methods:

• Site-directed mutagenesis of RSV PR based on structural comparisons with HIV-1 PR.
• Expression and purification of mutant RSV PR in Escherichia coli.
• Enzymatic assays measuring cleavage of precursor polypeptides and synthetic peptide substrates in vitro.

Main Results:

• Mutations in the substrate binding pocket yielded varied results, with some (e.g., H65G) inactivating the enzyme, while others (e.g., R105P,G106V) enhanced activity against HIV-1 substrates.
• Alterations to catalytic residues (D37S, A40T) or flap regions (G69L, G70L) generally resulted in inactive proteases.
• Shortening surface loops to mimic HIV-1 PR dimensions significantly reduced enzyme activity.

Conclusions:

• Structural predictions regarding the importance of specific residues and regions in RSV PR activity were partially confirmed.
• Mutations can modulate substrate specificity, demonstrating potential for engineering protease function.
• Unexpected functional roles and interactions were uncovered, highlighting the complexity of protease mechanisms.