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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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Retroviral restriction by A3G requires RNA but not a protein co-factor. (A) Cropped field from the homology model of the head-to-head NTD dimer of A3G (see Supplementary Figure S4 for the complete model and details). The W94 and W127 interaction domain at the interface of the monomers is shown, W94 (yellow); W127 (pink). (B) Lysates of 293T cells transfected with the W94A/W127A mutant resolved by non-denaturing velocity sedimentation. (C) Evaluation of the RNA-binding properties of the W94A/W127A mutant. (D and E) Viruses for the complementation assays were produced by co-transfecting a total of 80 ng of APOBEC expression plasmids (40 ng each) for MoMLV restrictions assays, or 150 ng for HIV[p8.9] (75 ng each). Data represent the mean ± SD of triplicate values from three independent experiments.
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gkt527-F7: Retroviral restriction by A3G requires RNA but not a protein co-factor. (A) Cropped field from the homology model of the head-to-head NTD dimer of A3G (see Supplementary Figure S4 for the complete model and details). The W94 and W127 interaction domain at the interface of the monomers is shown, W94 (yellow); W127 (pink). (B) Lysates of 293T cells transfected with the W94A/W127A mutant resolved by non-denaturing velocity sedimentation. (C) Evaluation of the RNA-binding properties of the W94A/W127A mutant. (D and E) Viruses for the complementation assays were produced by co-transfecting a total of 80 ng of APOBEC expression plasmids (40 ng each) for MoMLV restrictions assays, or 150 ng for HIV[p8.9] (75 ng each). Data represent the mean ± SD of triplicate values from three independent experiments.

Mentions: To gain further insight into how W94 and W127 enable A3G to interact with RNA, we conducted homology modeling of the A3G head-to-head NTD dimer (Supplementary Figure S4). In our model, the two A3G NTD monomers make extensive contacts, including the loops connecting the α1-β1 and β4-α4 with the corresponding β4-α4 and α1-β1 loops of the reciprocal protomer. Interestingly, close inspection of the NTD dimer shows that W94 of the first monomer is in close proximity to W127 in the other monomer (Figure 7A and Supplementary Figure S4A), a result also observed by Lavens et al. (53). The structure shows that on dimerization, there is a significant increase in the size of the positively charged patch that extends to the C-terminal end of α6 of the reciprocal dimer’s subunit (Supplementary Figure S4B). Overall, our modeling study suggests that A3G dimerization generates a large surface for RNA binding, and that W94A and W127A substitutions would strongly disfavor the binding of RNA. An A3G mutant carrying a double W94A/W127A substitution should therefore potentiate the RNA-binding defect. To validate this prediction, we generated the double mutant and analyzed its RNA-binding properties. We found that the RNA-dependent oligomerization of W94A/W127A was completely abolished (Figure 7B). Additionally, the double mutant did not significantly bind to any of the RNAs tested (Figure 7C).Figure 7.


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)

Retroviral restriction by A3G requires RNA but not a protein co-factor. (A) Cropped field from the homology model of the head-to-head NTD dimer of A3G (see Supplementary Figure S4 for the complete model and details). The W94 and W127 interaction domain at the interface of the monomers is shown, W94 (yellow); W127 (pink). (B) Lysates of 293T cells transfected with the W94A/W127A mutant resolved by non-denaturing velocity sedimentation. (C) Evaluation of the RNA-binding properties of the W94A/W127A mutant. (D and E) Viruses for the complementation assays were produced by co-transfecting a total of 80 ng of APOBEC expression plasmids (40 ng each) for MoMLV restrictions assays, or 150 ng for HIV[p8.9] (75 ng each). Data represent the mean ± SD of triplicate values from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt527-F7: Retroviral restriction by A3G requires RNA but not a protein co-factor. (A) Cropped field from the homology model of the head-to-head NTD dimer of A3G (see Supplementary Figure S4 for the complete model and details). The W94 and W127 interaction domain at the interface of the monomers is shown, W94 (yellow); W127 (pink). (B) Lysates of 293T cells transfected with the W94A/W127A mutant resolved by non-denaturing velocity sedimentation. (C) Evaluation of the RNA-binding properties of the W94A/W127A mutant. (D and E) Viruses for the complementation assays were produced by co-transfecting a total of 80 ng of APOBEC expression plasmids (40 ng each) for MoMLV restrictions assays, or 150 ng for HIV[p8.9] (75 ng each). Data represent the mean ± SD of triplicate values from three independent experiments.
Mentions: To gain further insight into how W94 and W127 enable A3G to interact with RNA, we conducted homology modeling of the A3G head-to-head NTD dimer (Supplementary Figure S4). In our model, the two A3G NTD monomers make extensive contacts, including the loops connecting the α1-β1 and β4-α4 with the corresponding β4-α4 and α1-β1 loops of the reciprocal protomer. Interestingly, close inspection of the NTD dimer shows that W94 of the first monomer is in close proximity to W127 in the other monomer (Figure 7A and Supplementary Figure S4A), a result also observed by Lavens et al. (53). The structure shows that on dimerization, there is a significant increase in the size of the positively charged patch that extends to the C-terminal end of α6 of the reciprocal dimer’s subunit (Supplementary Figure S4B). Overall, our modeling study suggests that A3G dimerization generates a large surface for RNA binding, and that W94A and W127A substitutions would strongly disfavor the binding of RNA. An A3G mutant carrying a double W94A/W127A substitution should therefore potentiate the RNA-binding defect. To validate this prediction, we generated the double mutant and analyzed its RNA-binding properties. We found that the RNA-dependent oligomerization of W94A/W127A was completely abolished (Figure 7B). Additionally, the double mutant did not significantly bind to any of the RNAs tested (Figure 7C).Figure 7.

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