Attenuation of DNA replication by HIV-1 reverse transcriptase near the central termination sequence

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Equine infectious anemia virus-1 reverse transcriptase (EIAV RT) studies showed DNA central termination sequence (CTS) effects on polymerization.
• CTS reduced burst amplitude and increased termination products during EIAV RT DNA synthesis.

Purpose of the Study:

• To investigate the molecular mechanisms behind reduced burst amplitudes in human immunodeficiency virus reverse transcriptase (HIV RT) using pre-steady-state kinetics.
• To compare HIV RT's response to CTS-containing DNA with random sequence DNA substrates.

Main Methods:

• Pre-steady-state kinetic techniques were employed to study HIV RT activity.
• Experiments involved varying primer/template length, binding orientation, and protein concentration.
• Gel mobility shift assays, enzyme activity, and fluorescence monitored RT-DNA complex dissociation kinetics.

Main Results:

• Substoichiometric burst amplitudes with normal substrates were attributed to reversible dissociation of RT dimer, confirmed by gel mobility shift assays.
• HIV RT exhibited lower burst amplitudes with CTS-containing DNA substrates compared to random sequences, similar to EIAV RT.
• Biphasic dissociation kinetics were observed for both random sequence and CTS-containing DNA complexes, indicating two forms of the RT-DNA complex.

Conclusions:

• Reversible RT dimer dissociation explains substoichiometric burst amplitudes in HIV RT.
• A proposed mechanism accounts for reduced burst amplitude with CTS-containing DNA, consistent with single nucleotide addition and dissociation data.
• The two RT-DNA complex forms may reflect primer/template partitioning between P- and N-sites on HIV RT.