Hydroxyl radical footprint analysis of human immunodeficiency virus reverse transcriptase-template.primer complexes

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for viral replication.
• Understanding the interaction between HIV-1 RT and its DNA template-primer is crucial for developing antiviral therapies.

Purpose of the Study:

• To investigate the protective role of HIV-1 RT against hydroxyl radical attack on DNA.
• To determine the conformation of DNA when bound to HIV-1 RT.
• To elucidate the role of the RNase H domain in DNA conformational changes.

Main Methods:

• Hydroxyl radical footprinting assays were used to probe DNA accessibility.
• Replication complexes were analyzed after varying degrees of primer extension.
• Conformational analysis of DNA-template primer complexes was performed.

Main Results:

• HIV-1 RT protects DNA sugar moieties from positions +3 to -15.
• A protected DNA region of approximately 18 nucleotides migrates with the enzyme.
• DNA adopts an A-form conformation when bound to HIV-1 RT.
• Increased DNA accessibility near position -15 suggests enzyme-induced conformational changes.

Conclusions:

• HIV-1 RT actively protects the DNA template-primer during reverse transcription.
• The enzyme-bound DNA likely adopts an A-conformation.
• The RNase H domain of p66 RT induces conformational changes in the DNA template.