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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

Effects of W94A and W127A on proviral DNA synthesis and integration. (A and B) Analysis of LRT accumulation. (C and D) Analysis of proviral DNA integration. DNA from infected 293T cells was collected at 12 h post-infection for LRT analysis and at 24 h for integration analysis. Infections were performed on 293T cells with HIV[p8.9] (A and C), or on NIH 3T3 cells with MoMLV (B and D). The results reflect the mean RQ ± SD of three independent experiments each performed in quadruplicate. Data were normalized to A2 values.
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gkt527-F3: Effects of W94A and W127A on proviral DNA synthesis and integration. (A and B) Analysis of LRT accumulation. (C and D) Analysis of proviral DNA integration. DNA from infected 293T cells was collected at 12 h post-infection for LRT analysis and at 24 h for integration analysis. Infections were performed on 293T cells with HIV[p8.9] (A and C), or on NIH 3T3 cells with MoMLV (B and D). The results reflect the mean RQ ± SD of three independent experiments each performed in quadruplicate. Data were normalized to A2 values.

Mentions: Retroviruses produced in the presence of A3G display reduced levels of LRT and proviral integration (50). Here, we sought to examine how the W94A and W127A mutations impact LRT accumulation and proviral integration. Results show that neither W94A nor W127A significantly hinder LRT accumulation, whereas wild-type A3G and E259Q reduced these levels by ∼40–60% for both viruses (Figure 3A and B). A3G and E259Q had much more dramatic effects on integration with measured reductions of 94 and 89% for HIV[p8.9] and 92 and 81% for MoMLV, respectively (Figure 3C and D). These results clearly reveal the marginal role of deamination in preventing these two early steps of the infection. On the other hand, W94A had no significant effect on reducing the proviral integration of either MoMLV or HIV[p8.9]. Equally, W127A did not reduce the integration of HIV[p8.9], but appeared to have a slight effect on MoMLV. Inactivation of the deaminase activity of the W94A RNA-binding mutant had no detectable impact on LRT accumulation or integration, which again supports that deamination is not a major contributor in preventing these specific processes.Figure 3.


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 W94A and W127A on proviral DNA synthesis and integration. (A and B) Analysis of LRT accumulation. (C and D) Analysis of proviral DNA integration. DNA from infected 293T cells was collected at 12 h post-infection for LRT analysis and at 24 h for integration analysis. Infections were performed on 293T cells with HIV[p8.9] (A and C), or on NIH 3T3 cells with MoMLV (B and D). The results reflect the mean RQ ± SD of three independent experiments each performed in quadruplicate. Data were normalized to A2 values.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt527-F3: Effects of W94A and W127A on proviral DNA synthesis and integration. (A and B) Analysis of LRT accumulation. (C and D) Analysis of proviral DNA integration. DNA from infected 293T cells was collected at 12 h post-infection for LRT analysis and at 24 h for integration analysis. Infections were performed on 293T cells with HIV[p8.9] (A and C), or on NIH 3T3 cells with MoMLV (B and D). The results reflect the mean RQ ± SD of three independent experiments each performed in quadruplicate. Data were normalized to A2 values.
Mentions: Retroviruses produced in the presence of A3G display reduced levels of LRT and proviral integration (50). Here, we sought to examine how the W94A and W127A mutations impact LRT accumulation and proviral integration. Results show that neither W94A nor W127A significantly hinder LRT accumulation, whereas wild-type A3G and E259Q reduced these levels by ∼40–60% for both viruses (Figure 3A and B). A3G and E259Q had much more dramatic effects on integration with measured reductions of 94 and 89% for HIV[p8.9] and 92 and 81% for MoMLV, respectively (Figure 3C and D). These results clearly reveal the marginal role of deamination in preventing these two early steps of the infection. On the other hand, W94A had no significant effect on reducing the proviral integration of either MoMLV or HIV[p8.9]. Equally, W127A did not reduce the integration of HIV[p8.9], but appeared to have a slight effect on MoMLV. Inactivation of the deaminase activity of the W94A RNA-binding mutant had no detectable impact on LRT accumulation or integration, which again supports that deamination is not a major contributor in preventing these specific processes.Figure 3.

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