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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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Virus-dependent packaging of W94A and W127A, and retroviral restriction analysis. (A–C) Viral particles were purified from cell supernatants, lysed and assayed for the presence of FLAG-tagged APOBEC proteins by western blot analysis (top panels). Lysates of virus-producing cells are shown in the bottom panels. (D–F) Restriction analysis of virus infectivity as measured by eGFP expression in target cells infected with HIVΔVif (D), HIV[p8.9] (E) or MoMLV (F). Results represent the mean ± SD of triplicate values from at least three independent transfection experiments. (G and H) Analysis of mutations in proviral DNA. Histograms depict the proportion of total sequences containing the indicated number of mutations. The total number of clones sequenced is indicated in the chart legend. Sequences were compiled from one experiment for A3G and W127A and from two independent experiments for W94A.
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gkt527-F2: Virus-dependent packaging of W94A and W127A, and retroviral restriction analysis. (A–C) Viral particles were purified from cell supernatants, lysed and assayed for the presence of FLAG-tagged APOBEC proteins by western blot analysis (top panels). Lysates of virus-producing cells are shown in the bottom panels. (D–F) Restriction analysis of virus infectivity as measured by eGFP expression in target cells infected with HIVΔVif (D), HIV[p8.9] (E) or MoMLV (F). Results represent the mean ± SD of triplicate values from at least three independent transfection experiments. (G and H) Analysis of mutations in proviral DNA. Histograms depict the proportion of total sequences containing the indicated number of mutations. The total number of clones sequenced is indicated in the chart legend. Sequences were compiled from one experiment for A3G and W127A and from two independent experiments for W94A.

Mentions: Here, we compared the virion packaging efficiency of the wild-type A3G protein to that of W94A, W127A, an inactive catalytic mutant E259Q, and corresponding W94A or W127A compound mutants: W94A/E259Q and W127A/E259Q. Three retroviruses were tested: HIVΔVif (NL4-3-derived with the env gene substituted for eGFP), HIV[p.8.9] (a self-inactivating HIV-derived pseudovirus expressing eGFP from an internal SFFV promoter) and replicative ecotropic MoMLV expressing an Env-eGFP fusion protein (see ‘Materials and Methods’ section for details). As previously described by others, we found that W94A and W127A were poorly packaged into HIVΔVif particles (Figure 2A). Surprisingly, all A3G variants were packaged efficiently into HIV[p8.9] and MoMLV virions (Figure 2B and C). These results indicate that the factors that govern virion encapsidation are different for HIV-1ΔVif and MoMLV. Our reasoning as to why the mutants proteins are packaged efficiently into HIV[p8.9] virions is presented in the discussion.Figure 2.


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)

Virus-dependent packaging of W94A and W127A, and retroviral restriction analysis. (A–C) Viral particles were purified from cell supernatants, lysed and assayed for the presence of FLAG-tagged APOBEC proteins by western blot analysis (top panels). Lysates of virus-producing cells are shown in the bottom panels. (D–F) Restriction analysis of virus infectivity as measured by eGFP expression in target cells infected with HIVΔVif (D), HIV[p8.9] (E) or MoMLV (F). Results represent the mean ± SD of triplicate values from at least three independent transfection experiments. (G and H) Analysis of mutations in proviral DNA. Histograms depict the proportion of total sequences containing the indicated number of mutations. The total number of clones sequenced is indicated in the chart legend. Sequences were compiled from one experiment for A3G and W127A and from two independent experiments for W94A.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt527-F2: Virus-dependent packaging of W94A and W127A, and retroviral restriction analysis. (A–C) Viral particles were purified from cell supernatants, lysed and assayed for the presence of FLAG-tagged APOBEC proteins by western blot analysis (top panels). Lysates of virus-producing cells are shown in the bottom panels. (D–F) Restriction analysis of virus infectivity as measured by eGFP expression in target cells infected with HIVΔVif (D), HIV[p8.9] (E) or MoMLV (F). Results represent the mean ± SD of triplicate values from at least three independent transfection experiments. (G and H) Analysis of mutations in proviral DNA. Histograms depict the proportion of total sequences containing the indicated number of mutations. The total number of clones sequenced is indicated in the chart legend. Sequences were compiled from one experiment for A3G and W127A and from two independent experiments for W94A.
Mentions: Here, we compared the virion packaging efficiency of the wild-type A3G protein to that of W94A, W127A, an inactive catalytic mutant E259Q, and corresponding W94A or W127A compound mutants: W94A/E259Q and W127A/E259Q. Three retroviruses were tested: HIVΔVif (NL4-3-derived with the env gene substituted for eGFP), HIV[p.8.9] (a self-inactivating HIV-derived pseudovirus expressing eGFP from an internal SFFV promoter) and replicative ecotropic MoMLV expressing an Env-eGFP fusion protein (see ‘Materials and Methods’ section for details). As previously described by others, we found that W94A and W127A were poorly packaged into HIVΔVif particles (Figure 2A). Surprisingly, all A3G variants were packaged efficiently into HIV[p8.9] and MoMLV virions (Figure 2B and C). These results indicate that the factors that govern virion encapsidation are different for HIV-1ΔVif and MoMLV. Our reasoning as to why the mutants proteins are packaged efficiently into HIV[p8.9] virions is presented in the discussion.Figure 2.

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