Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is crucial for viral replication, converting the viral RNA genome into DNA.
• The precise mechanism of nucleotide addition by HIV-1 RT, despite its essential role, remains incompletely understood.

Purpose of the Study:

• To elucidate the mechanism of DNA polymerization by HIV-1 RT.
• To integrate structural, genetic, and biochemical data to understand nucleotide addition and translocation.

Main Methods:

• X-ray crystallography of HIV-1 RT complexed with dsDNA template-primer.
• DNA binding affinity measurements.
• Molecular modeling of nucleotide triphosphate binding.
• Biochemical assays measuring nucleotide incorporation efficiency (kcat/Km).

Main Results:

• DNA binding induces a significant conformational change in HIV-1 RT, involving the p66 thumb subdomain.
• Specific regions within the fingers subdomain (beta 3 and beta 4) interact with single-stranded template regions.
• Nucleotide incorporation efficiency is independent of template overhang length.
• Molecular modeling suggests specific secondary structural elements (alpha C-beta 6, alpha E, beta 11b, beta 9-beta 10) define the dNTP binding site.
• Nucleotide incorporation is coupled with protein conformational changes and potentially metal-mediated catalysis involving Asp185.
• Translocation involves rotational and translational motions, potentially powered by dNTP hydrolysis and nucleic acid conformational changes.

Conclusions:

• HIV-1 RT undergoes significant conformational changes upon DNA and nucleotide binding, facilitating catalysis.
• Key structural elements and residues are identified that are critical for substrate binding, nucleotide incorporation, and translocation.
• Comparison with other polymerases highlights features contributing to processivity.