Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase is a key enzyme for viral replication.
• Understanding the mechanisms of reverse transcriptase inhibition is crucial for developing antiviral therapies.

Purpose of the Study:

• To investigate the use of a succinylfluorescein-labeled dideoxyTTP as a substrate for HIV-1 reverse transcriptase.
• To characterize the interaction of fluorescent chain-terminated primers with HIV-1 reverse transcriptase.
• To elucidate the mechanism of reverse transcriptase inhibition by chain-terminated primers.

Main Methods:

• Utilized succinylfluorescein-labeled dideoxyTTP as a substrate for HIV-1 reverse transcriptase.
• Measured fluorescence yield changes upon nucleotide incorporation and complex dissociation.
• Determined dissociation constants and dissociation rates of enzyme-primer-template complexes.
• Investigated the effect of complementary nucleotide triphosphate concentration on dissociation rates.

Main Results:

• Succinylfluorescein-labeled dideoxyTTP incorporation caused a significant reduction in fluorescence.
• Fluorescent chain-terminated primers formed slowly dissociating complexes with HIV-1 reverse transcriptase in vitro.
• Dissociation constant increased from 0.65 nM to 3 nM upon fluorescent chain terminator incorporation.
• Dissociation rate of the terminated complex was 0.04 s-1, decreased by complementary nucleotide triphosphate.

Conclusions:

• In vitro inhibition of HIV-1 reverse transcriptase by fluorescent chain terminators is primarily due to slowly dissociating complexes.
• This in vitro inhibition mechanism is unlikely to be the primary mode of action in HIV-infected cells.
• Chain termination leading to incomplete transcription is the probable major inhibitory factor in vivo.