HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Recombinant HIV-1 reverse transcriptase (RT) is expressed in bacteria.
• RT functions as a homodimer or heterodimer.
• Heterodimer formation involves cleavage of a 66,000 Mr subunit.

Purpose of the Study:

• To investigate the structural and functional properties of bacterially expressed HIV-1 RT.
• To characterize the role of proteolytic cleavage in RT activity.
• To understand the relationship between RT structure and its enzymatic function.

Main Methods:

• Bacterial expression and purification of recombinant HIV-1 RT.
• Analysis of RT subunit composition using SDS-PAGE.
• Limited proteolysis assays with alpha-chymotrypsin and trypsin.
• Enzyme kinetics studies measuring Vmax and Km for dTTP.

Main Results:

• Purified RT preparations contained both 66,000 Mr and 51,000 Mr polypeptides.
• Proteolytic digestion generated stable 66,000/51,000 Mr mixtures, suggesting a protease-sensitive linker.
• Trypsin cleavage yielded specific peptides, indicating cleavage sites within the RT molecule.
• Proteolysis increased RT activity by enhancing Vmax without significantly altering Km for dTTP.

Conclusions:

• HIV-1 RT exists in active dimeric forms, including a heterodimer generated by proteolytic cleavage.
• A protease-sensitive linker region is likely present in the RT molecule.
• Proteolytic processing enhances reverse transcriptase activity, suggesting a regulatory mechanism.