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Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G.

Iwatani Y, Chan DS, Wang F, Maynard KS, Sugiura W, Gronenborn AM, Rouzina I, Williams MC, Musier-Forsyth K, Levin JG - Nucleic Acids Res. (2007)

Bottom Line: We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage.In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC.These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (-) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

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Examples of lambda DNA stretching (continuous) and relaxation (dashed) curves. (A) A3G. 0 nM (black); 150 nM (red). (B) NC. 0 nM (black); 10 nM (red). All stretching experiments were conducted at 20°C in 10 mM HEPES, pH 7.5 and 50 mM Na+.
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Figure 7: Examples of lambda DNA stretching (continuous) and relaxation (dashed) curves. (A) A3G. 0 nM (black); 150 nM (red). (B) NC. 0 nM (black); 10 nM (red). All stretching experiments were conducted at 20°C in 10 mM HEPES, pH 7.5 and 50 mM Na+.

Mentions: When optical tweezers are used to stretch single DNA molecules by applying forces approaching 60 pN (Figure 7), a force-induced melting transition occurs, in which dsDNA is converted to ssDNA (59). In the absence of protein, this transition occurs over a very narrow force range due to the cooperative melting of DNA. Saturating levels of HIV-1 NC result in a significant increase in the width of the force-induced melting transition (46), which correlates with NC's relatively efficient chaperone activity. In addition, the reversibility of DNA stretch/relax curves in the presence of NC (i.e., DNA stretch curves show very little hysteresis), suggests that NC has a fast nucleic acid binding on/off rate and is therefore capable of rapidly switching between dsDNA and ssDNA bound states (60).Figure 7.


Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G.

Iwatani Y, Chan DS, Wang F, Maynard KS, Sugiura W, Gronenborn AM, Rouzina I, Williams MC, Musier-Forsyth K, Levin JG - Nucleic Acids Res. (2007)

Examples of lambda DNA stretching (continuous) and relaxation (dashed) curves. (A) A3G. 0 nM (black); 150 nM (red). (B) NC. 0 nM (black); 10 nM (red). All stretching experiments were conducted at 20°C in 10 mM HEPES, pH 7.5 and 50 mM Na+.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175344&req=5

Figure 7: Examples of lambda DNA stretching (continuous) and relaxation (dashed) curves. (A) A3G. 0 nM (black); 150 nM (red). (B) NC. 0 nM (black); 10 nM (red). All stretching experiments were conducted at 20°C in 10 mM HEPES, pH 7.5 and 50 mM Na+.
Mentions: When optical tweezers are used to stretch single DNA molecules by applying forces approaching 60 pN (Figure 7), a force-induced melting transition occurs, in which dsDNA is converted to ssDNA (59). In the absence of protein, this transition occurs over a very narrow force range due to the cooperative melting of DNA. Saturating levels of HIV-1 NC result in a significant increase in the width of the force-induced melting transition (46), which correlates with NC's relatively efficient chaperone activity. In addition, the reversibility of DNA stretch/relax curves in the presence of NC (i.e., DNA stretch curves show very little hysteresis), suggests that NC has a fast nucleic acid binding on/off rate and is therefore capable of rapidly switching between dsDNA and ssDNA bound states (60).Figure 7.

Bottom Line: We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage.In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC.These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of (-) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

Show MeSH
Related in: MedlinePlus