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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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Related in: MedlinePlus

Effect of deamination on MoMLV particle release. (A) Diagram of the experimental method. (B) Relative infection of NIH 3T3 cells by MoMLV measured after 24 h. Viral particles were produced in presence of a 1:10 A3G (80 ng)-to-MoMLV (800 ng) proviral plasmid ratio for W94A and W94A/E259Q, and increasing amounts of wild-type A3G as indicated. (C) p30 levels in cell supernatants were measured by enzyme-linked immunosorbent assay 72 h after infection. Data represent the mean ± SD of three independent protein measurements.
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gkt527-F4: Effect of deamination on MoMLV particle release. (A) Diagram of the experimental method. (B) Relative infection of NIH 3T3 cells by MoMLV measured after 24 h. Viral particles were produced in presence of a 1:10 A3G (80 ng)-to-MoMLV (800 ng) proviral plasmid ratio for W94A and W94A/E259Q, and increasing amounts of wild-type A3G as indicated. (C) p30 levels in cell supernatants were measured by enzyme-linked immunosorbent assay 72 h after infection. Data represent the mean ± SD of three independent protein measurements.

Mentions: We were curious to determine whether viral particle release was affected by the DNA mutator activity of the RNA-binding mutant W94A. Because W94A is not adequately packaged into HIVΔVif particles, we performed this experiment on MoMLV. Deamination-induced damage that can affect particle release includes: mutational damage to the retroviral promoter, loss of protein function or localization, or the generation of stop codons that halt protein synthesis. A3G variants and MoMLV expression plasmids were co-transfected at increasing A3G-to-virus ratios into 293T cells, and NIH 3T3 target cells were infected with MoMLV particles at an MOI of 0.5. Virus-containing supernatants were then collected 72 h later, and p30 levels were measured by enzyme-linked immunosorbent assay (Figure 4A).Figure 4.


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)

Effect of deamination on MoMLV particle release. (A) Diagram of the experimental method. (B) Relative infection of NIH 3T3 cells by MoMLV measured after 24 h. Viral particles were produced in presence of a 1:10 A3G (80 ng)-to-MoMLV (800 ng) proviral plasmid ratio for W94A and W94A/E259Q, and increasing amounts of wild-type A3G as indicated. (C) p30 levels in cell supernatants were measured by enzyme-linked immunosorbent assay 72 h after infection. Data represent the mean ± SD of three independent protein measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt527-F4: Effect of deamination on MoMLV particle release. (A) Diagram of the experimental method. (B) Relative infection of NIH 3T3 cells by MoMLV measured after 24 h. Viral particles were produced in presence of a 1:10 A3G (80 ng)-to-MoMLV (800 ng) proviral plasmid ratio for W94A and W94A/E259Q, and increasing amounts of wild-type A3G as indicated. (C) p30 levels in cell supernatants were measured by enzyme-linked immunosorbent assay 72 h after infection. Data represent the mean ± SD of three independent protein measurements.
Mentions: We were curious to determine whether viral particle release was affected by the DNA mutator activity of the RNA-binding mutant W94A. Because W94A is not adequately packaged into HIVΔVif particles, we performed this experiment on MoMLV. Deamination-induced damage that can affect particle release includes: mutational damage to the retroviral promoter, loss of protein function or localization, or the generation of stop codons that halt protein synthesis. A3G variants and MoMLV expression plasmids were co-transfected at increasing A3G-to-virus ratios into 293T cells, and NIH 3T3 target cells were infected with MoMLV particles at an MOI of 0.5. Virus-containing supernatants were then collected 72 h later, and p30 levels were measured by enzyme-linked immunosorbent assay (Figure 4A).Figure 4.

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