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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 -primed (−) SSDNA synthesis. (A) Time course of  annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. (B) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). (C) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of (−) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.
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Figure 2: Effect of A3G on -primed (−) SSDNA synthesis. (A) Time course of annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. (B) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). (C) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of (−) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.

Mentions: Purified from human placenta was obtained from Bio S&T (Lachine, Quebec, Canada). DNA and RNA oligonucleotides were purchased from Lofstrand (Gaithersburg, MD), Integrated DNA Technologies (Coralville, IA), Oligos Etc., Inc. (Wilsonville, OR). [γ-32P]ATP (3000 Ci/mmol) and [α-32P]dCTP (6000 Ci/mmol) were purchased from GE Healthcare (Piscataway, NJ). HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Calf intestinal phosphatase, T4 polynucleotide kinase, and Vent DNA polymerase were obtained from New England Biolabs (Beverly, MA). SUPERaseIn, an RNase inhibitor, was purchased from Ambion, Inc. (Austin, TX). Recombinant wild-type HIV-1 NC (55-amino-acid form) was a generous gift from Dr Robert Gorelick and was prepared as described previously (52,53). Recombinant enzymatically active A3G and the deaminase-deficient A3G mutant (C291S) were expressed in a baculovirus expression system and purified as previously described (12). A3G preparations were confirmed to be free from contamination with RNases (data not shown) and no RNA degradation was apparent in any of the experiments (e.g. see Figure 2A).


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 -primed (−) SSDNA synthesis. (A) Time course of  annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. (B) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). (C) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of (−) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.
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Figure 2: Effect of A3G on -primed (−) SSDNA synthesis. (A) Time course of annealing to RNA UL244. Reactions were performed in the absence or presence of NC and A3G, as indicated by the headings at the top of the gel. The positions of the RNA UL244 template and the annealed RNA duplex are shown on the right. (B) The percentage of annealed product was calculated by dividing the amount of annealed RNA by the sum of annealed plus unannealed RNA, multiplied by 100. Symbols: no NC/no A3G (filled circles); +NC/no A3G (open squares); +NC/+hdA3G (open circles); and +NC/+A3G (open triangles). (C) A /RNA 244 complex was extended by HIV-1 RT in the absence (lane 1) or presence of hdA3G (lanes 2–4) or A3G (lanes 5–7). The positions of (−) SSDNA and initial pause products at bases +1, +3 and +5 are shown on the right. A3G concentrations: lane 1, 0 nM; lanes 2 and 5, 20 nM; lanes 3 and 6, 40 nM; lanes 4 and 7, 80 nM.
Mentions: Purified from human placenta was obtained from Bio S&T (Lachine, Quebec, Canada). DNA and RNA oligonucleotides were purchased from Lofstrand (Gaithersburg, MD), Integrated DNA Technologies (Coralville, IA), Oligos Etc., Inc. (Wilsonville, OR). [γ-32P]ATP (3000 Ci/mmol) and [α-32P]dCTP (6000 Ci/mmol) were purchased from GE Healthcare (Piscataway, NJ). HIV-1 RT was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Calf intestinal phosphatase, T4 polynucleotide kinase, and Vent DNA polymerase were obtained from New England Biolabs (Beverly, MA). SUPERaseIn, an RNase inhibitor, was purchased from Ambion, Inc. (Austin, TX). Recombinant wild-type HIV-1 NC (55-amino-acid form) was a generous gift from Dr Robert Gorelick and was prepared as described previously (52,53). Recombinant enzymatically active A3G and the deaminase-deficient A3G mutant (C291S) were expressed in a baculovirus expression system and purified as previously described (12). A3G preparations were confirmed to be free from contamination with RNases (data not shown) and no RNA degradation was apparent in any of the experiments (e.g. see Figure 2A).

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