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Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses.

Bélanger K, Savoie M, Rosales Gerpe MC, Couture JF, Langlois MA - Nucleic Acids Res. (2013)

Bottom Line: This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity.We did not find that deaminase activity made a significant contribution to the restriction of any of these processes.In summary, this work reveals that there is a direct correlation between A3G's capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, Emerging Pathogens Research Centre, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 and Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.

ABSTRACT
APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G's capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

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Effects of the W94A and W127A mutations on HMM complex assembly, subcellular distribution, RNA-binding and DNA deaminase activity. (A) Lysates of transfected 293T cells, treated (right panels) or untreated (left panels) with RNase, were resolved by velocity sedimentation over a non-denaturing 5–40% sucrose gradient and analyzed by western blot using anti-FLAG and anti-β-tubulin antibodies. (B) Fluorescent imaging of the subcellular localization of 293T cells expressing eGFP-A3G, eGFP-W94A and eGFP-W127A. (C) Schematic representation of the location of important residues and binding domains in the A3G protein sequence. (D) Binding of FLAG-tagged A2, A3G, W94A and W127A to 7SL, Alu, hY1 and hY3 RNAs were determined by qPCR. Relative binding to A3G is depicted. Results represent the mean ± SD of triplicate values from three independent transfection experiments. (E) Evaluation of the intrinsic DNA cytidine deaminase activity using a bacterial mutator assay. Each point represents the mutation frequency (RifR mutants per 107 viable cells) of an independent bacterial culture; median values are indicated.
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gkt527-F1: Effects of the W94A and W127A mutations on HMM complex assembly, subcellular distribution, RNA-binding and DNA deaminase activity. (A) Lysates of transfected 293T cells, treated (right panels) or untreated (left panels) with RNase, were resolved by velocity sedimentation over a non-denaturing 5–40% sucrose gradient and analyzed by western blot using anti-FLAG and anti-β-tubulin antibodies. (B) Fluorescent imaging of the subcellular localization of 293T cells expressing eGFP-A3G, eGFP-W94A and eGFP-W127A. (C) Schematic representation of the location of important residues and binding domains in the A3G protein sequence. (D) Binding of FLAG-tagged A2, A3G, W94A and W127A to 7SL, Alu, hY1 and hY3 RNAs were determined by qPCR. Relative binding to A3G is depicted. Results represent the mean ± SD of triplicate values from three independent transfection experiments. (E) Evaluation of the intrinsic DNA cytidine deaminase activity using a bacterial mutator assay. Each point represents the mutation frequency (RifR mutants per 107 viable cells) of an independent bacterial culture; median values are indicated.

Mentions: During the course of a screen to identify the amino acids of A3G that govern its assembly into HMM complexes, we discovered that mutation of tryptophans 94 and 127 to alanine (W94A; W127A) prevented the formation of these complexes (Figure 1A and Supplementary Figure S2). Despite the absence of HMM complexes in fractions 8 and 9, RNA-dependent LMM oligomeric complexes were present throughout the middle fractions of the sucrose gradient (fractions 4–7). Pretreatment of the extracts with RNase resulted in a complete shift toward the top of the gradient populated by the monomeric, dimeric and tetrameric forms of the A3G protein (fractions 1–3). These particular features of the W94A and W127A mutants were not observed with any of the other A3G point mutants that were tested (Supplementary Figure S2).Figure 1.


Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses.

Bélanger K, Savoie M, Rosales Gerpe MC, Couture JF, Langlois MA - Nucleic Acids Res. (2013)

Effects of the W94A and W127A mutations on HMM complex assembly, subcellular distribution, RNA-binding and DNA deaminase activity. (A) Lysates of transfected 293T cells, treated (right panels) or untreated (left panels) with RNase, were resolved by velocity sedimentation over a non-denaturing 5–40% sucrose gradient and analyzed by western blot using anti-FLAG and anti-β-tubulin antibodies. (B) Fluorescent imaging of the subcellular localization of 293T cells expressing eGFP-A3G, eGFP-W94A and eGFP-W127A. (C) Schematic representation of the location of important residues and binding domains in the A3G protein sequence. (D) Binding of FLAG-tagged A2, A3G, W94A and W127A to 7SL, Alu, hY1 and hY3 RNAs were determined by qPCR. Relative binding to A3G is depicted. Results represent the mean ± SD of triplicate values from three independent transfection experiments. (E) Evaluation of the intrinsic DNA cytidine deaminase activity using a bacterial mutator assay. Each point represents the mutation frequency (RifR mutants per 107 viable cells) of an independent bacterial culture; median values are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753645&req=5

gkt527-F1: Effects of the W94A and W127A mutations on HMM complex assembly, subcellular distribution, RNA-binding and DNA deaminase activity. (A) Lysates of transfected 293T cells, treated (right panels) or untreated (left panels) with RNase, were resolved by velocity sedimentation over a non-denaturing 5–40% sucrose gradient and analyzed by western blot using anti-FLAG and anti-β-tubulin antibodies. (B) Fluorescent imaging of the subcellular localization of 293T cells expressing eGFP-A3G, eGFP-W94A and eGFP-W127A. (C) Schematic representation of the location of important residues and binding domains in the A3G protein sequence. (D) Binding of FLAG-tagged A2, A3G, W94A and W127A to 7SL, Alu, hY1 and hY3 RNAs were determined by qPCR. Relative binding to A3G is depicted. Results represent the mean ± SD of triplicate values from three independent transfection experiments. (E) Evaluation of the intrinsic DNA cytidine deaminase activity using a bacterial mutator assay. Each point represents the mutation frequency (RifR mutants per 107 viable cells) of an independent bacterial culture; median values are indicated.
Mentions: During the course of a screen to identify the amino acids of A3G that govern its assembly into HMM complexes, we discovered that mutation of tryptophans 94 and 127 to alanine (W94A; W127A) prevented the formation of these complexes (Figure 1A and Supplementary Figure S2). Despite the absence of HMM complexes in fractions 8 and 9, RNA-dependent LMM oligomeric complexes were present throughout the middle fractions of the sucrose gradient (fractions 4–7). Pretreatment of the extracts with RNase resulted in a complete shift toward the top of the gradient populated by the monomeric, dimeric and tetrameric forms of the A3G protein (fractions 1–3). These particular features of the W94A and W127A mutants were not observed with any of the other A3G point mutants that were tested (Supplementary Figure S2).Figure 1.

Bottom Line: This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity.We did not find that deaminase activity made a significant contribution to the restriction of any of these processes.In summary, this work reveals that there is a direct correlation between A3G's capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, Emerging Pathogens Research Centre, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 and Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.

ABSTRACT
APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G's capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

Show MeSH
Related in: MedlinePlus