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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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Effect of A3G on PPT initiation and plus-strand transfer. (A) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α-32P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. (B) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).
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Figure 6: Effect of A3G on PPT initiation and plus-strand transfer. (A) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α-32P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. (B) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).

Mentions: While elongation of minus-strand DNA is being completed, the PPT RNA primer initiates synthesis of a short DNA termed (+) SSDNA (Figure 1, steps 4 and 5) (43). To determine whether A3G interferes with this step, we used a simple oligonucleotide assay that was previously developed in our laboratory (48,57). The 15-nt PPT was heat-annealed to a 35-nt minus-strand DNA template and the hybrid was then incubated with RT, which catalyzed the extension and subsequent removal of the PPT. The amount of 20-nt DNA product formed in reactions with and without A3G was quantified and plotted (Figure 6A).Figure 6.


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)

Effect of A3G on PPT initiation and plus-strand transfer. (A) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α-32P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. (B) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).
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Figure 6: Effect of A3G on PPT initiation and plus-strand transfer. (A) Time course of PPT-primed plus-strand DNA synthesis. The 15-nt PPT RNA was heat-annealed to a 35-nt minus-strand DNA template and was then extended by HIV-1 RT. The 20-nt DNA product was internally labeled with [α-32P]dCTP in the absence (filled circles) and presence (open squares) of A3G (80 nM). The amount of 20-nt DNA was plotted as Relative Extension (%) versus Time (min), where 100% represents the end point value for the ‘no A3G’ reaction. (B) Time course of plus-strand transfer. The percentage of 80-nt plus-strand DNA product was calculated as described in the legend to Figure 5D. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open circles); and +NC/+A3G (open triangles).
Mentions: While elongation of minus-strand DNA is being completed, the PPT RNA primer initiates synthesis of a short DNA termed (+) SSDNA (Figure 1, steps 4 and 5) (43). To determine whether A3G interferes with this step, we used a simple oligonucleotide assay that was previously developed in our laboratory (48,57). The 15-nt PPT was heat-annealed to a 35-nt minus-strand DNA template and the hybrid was then incubated with RT, which catalyzed the extension and subsequent removal of the PPT. The amount of 20-nt DNA product formed in reactions with and without A3G was quantified and plotted (Figure 6A).Figure 6.

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