Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Area of Science:

• Structural Biology
• Virology
• Biochemistry

Background:

• HIV-1 replication relies on reverse transcription of its RNA genome into DNA by reverse transcriptase (RT).
• Existing structural data for RT lacks detailed insights into the dynamic intermediate states of DNA synthesis.

Purpose of the Study:

• To elucidate the intermediate structural states during dATP addition by HIV-1 RT.
• To understand the mechanism of nucleotide incorporation and its role in viral replication.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) with catalytically active substrates.
• Captured 11 distinct structural states (reactant, intermediate, product) of dATP addition at high resolution (2.2–3.0 Å).

Main Results:

• Detailed the role of Mg2+ ions in dATP binding and activation.
• Observed a previously uncharacterized dATP conformer aligning for nucleophilic attack.
• Characterized the product state, including dAMP incorporation and pyrophosphate release.

Conclusions:

• Provides unprecedented structural snapshots of HIV-1 RT's DNA synthesis mechanism.
• Highlights the dynamic interplay of substrates, metal ions, and enzyme active site.
• Identifies K220 mutations' impact on dNTP incorporation and viral replication capacity.