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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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Fusion to the RNA-binding defective Vpr14–86 polyprotein [Vpr(ΔRNA)] does not restore the restriction potential of the W94A and W127A mutants. (A) Analysis of the packaging of all Vpr(ΔRNA)-A3G fusion proteins into HIVΔVif virions. (B) Antiretroviral activities of Vpr(ΔRNA)-A3G fusion proteins on HIVΔVif. (C) Evaluation of the RNA-binding properties of VprΔRNA) fusion proteins. Data represent the mean ± SD of triplicate values from three independent experiments. (D) Effect of Vpr(ΔRNA) fusion proteins on HIVΔVif proviral integration. The results reflect the mean RQ of one experiment performed in quadruplicate. Data were normalized to Vpr-A2 values.
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gkt527-F6: Fusion to the RNA-binding defective Vpr14–86 polyprotein [Vpr(ΔRNA)] does not restore the restriction potential of the W94A and W127A mutants. (A) Analysis of the packaging of all Vpr(ΔRNA)-A3G fusion proteins into HIVΔVif virions. (B) Antiretroviral activities of Vpr(ΔRNA)-A3G fusion proteins on HIVΔVif. (C) Evaluation of the RNA-binding properties of VprΔRNA) fusion proteins. Data represent the mean ± SD of triplicate values from three independent experiments. (D) Effect of Vpr(ΔRNA) fusion proteins on HIVΔVif proviral integration. The results reflect the mean RQ of one experiment performed in quadruplicate. Data were normalized to Vpr-A2 values.

Mentions: To ensure that the observed phenotype of the Vpr fusion proteins was conferred specifically by the RNA-binding properties of Vpr, we deleted amino acids 87 and 88 of the Vpr14–88 polypeptide that have been previously been shown to mediate RNA binding (52) and repeated experiments depicted in Figure 5. We found that W94A and W127A fusions with the Vpr14–86 polypeptide defective in RNA binding, Vpr(ΔRNA), were efficiently packaged into HIVΔVif virions (Figure 6A), did not restrict the infection (Figure 6B), were unable to inhibit proviral integration (Figure 6C) and displayed RNA-binding defects (Figure 6D). In summary, these data show that RNA binding is an essential property for A3G to be able to restrict Vif-deficient HIV-1 infection.Figure 6.


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)

Fusion to the RNA-binding defective Vpr14–86 polyprotein [Vpr(ΔRNA)] does not restore the restriction potential of the W94A and W127A mutants. (A) Analysis of the packaging of all Vpr(ΔRNA)-A3G fusion proteins into HIVΔVif virions. (B) Antiretroviral activities of Vpr(ΔRNA)-A3G fusion proteins on HIVΔVif. (C) Evaluation of the RNA-binding properties of VprΔRNA) fusion proteins. Data represent the mean ± SD of triplicate values from three independent experiments. (D) Effect of Vpr(ΔRNA) fusion proteins on HIVΔVif proviral integration. The results reflect the mean RQ of one experiment performed in quadruplicate. Data were normalized to Vpr-A2 values.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt527-F6: Fusion to the RNA-binding defective Vpr14–86 polyprotein [Vpr(ΔRNA)] does not restore the restriction potential of the W94A and W127A mutants. (A) Analysis of the packaging of all Vpr(ΔRNA)-A3G fusion proteins into HIVΔVif virions. (B) Antiretroviral activities of Vpr(ΔRNA)-A3G fusion proteins on HIVΔVif. (C) Evaluation of the RNA-binding properties of VprΔRNA) fusion proteins. Data represent the mean ± SD of triplicate values from three independent experiments. (D) Effect of Vpr(ΔRNA) fusion proteins on HIVΔVif proviral integration. The results reflect the mean RQ of one experiment performed in quadruplicate. Data were normalized to Vpr-A2 values.
Mentions: To ensure that the observed phenotype of the Vpr fusion proteins was conferred specifically by the RNA-binding properties of Vpr, we deleted amino acids 87 and 88 of the Vpr14–88 polypeptide that have been previously been shown to mediate RNA binding (52) and repeated experiments depicted in Figure 5. We found that W94A and W127A fusions with the Vpr14–86 polypeptide defective in RNA binding, Vpr(ΔRNA), were efficiently packaged into HIVΔVif virions (Figure 6A), did not restrict the infection (Figure 6B), were unable to inhibit proviral integration (Figure 6C) and displayed RNA-binding defects (Figure 6D). In summary, these data show that RNA binding is an essential property for A3G to be able to restrict Vif-deficient HIV-1 infection.Figure 6.

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