Mechanism of HIV Reverse Transcriptase Inhibition by Zinc

• Published in: The Journal of biological chemistry 2011-11, Vol.286 (47), p.40433-40442
• Main Authors: Fenstermacher, Katherine J., DeStefano, Jeffrey J.
• Format: Article
• Language: English
• Subjects: Calcium; HIV; Manganese; Reverse Transcription; Zinc
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.289850

Several physiologically relevant cations including Ca2+, Mn2+, and Zn2+ have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg2+ ions bound to RT. We analyzed the effects of Zn2+ on reverse transcription and compared them to Ca2+ and Mn2+. Using nucleotide extension efficiency as a readout, Zn2+ showed significant inhibition of reactions with 2 mm Mg2+, even when present at only ∼5 μm. Mn2+ and Ca2+ were also inhibitory but at higher concentrations. Both Mn2+ and Zn2+ (but not Ca2+) supported RT incorporation in the absence of Mg2+ with Mn2+ being much more efficient. The maximum extension rates with Zn2+, Mn2+, and Mg2+ were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 μm, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn2+ and Mg2+, and single RT molecules were able to continue extension in the presence of Zn2+ for several hours on the same template. Consistent with this result, the half-life for RT-Zn2+-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg2+, indicating ∼130-fold more stable binding with Zn2+. Essentially, the presence of Zn2+ promotes the formation of a highly stable slowly progressing RT-(primer-template) complex. Background: HIV-RT uses Mg2+ for efficient nucleotide incorporation but is inhibited by other divalent cations. Results: Zn2+ supports HIV-RT catalysis but with profoundly reduced kinetics. Conclusion: Zn2+ inhibition is not due to catalysis blockage but to the formation of a highly stable, kinetically diminished complex. Significance: HIV-RT is inhibited by low Zn2+ concentrations presenting an avenue for future drug research.