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The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

Casartelli N, Guivel-Benhassine F, Bouziat R, Brandler S, Schwartz O, Moris A - J. Exp. Med. (2009)

Bottom Line: The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs.Enzymatically inactive A3G mutants failed to enhance CTL activation.Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité Virus et Immunité, Institut Pasteur, 75724 Paris cedex 15, France.

ABSTRACT
The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

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Vif-deficient HIV-1 is a potent activator of HS-CTLs. (A) Viruses were produced by transfection of 293T cells (that do not express endogenous A3G). Viruses were then used to infect PHA-activated primary CD4+ T cells. Endogenous A3G (red) is incorporated into the progeny virions and exerts its editing activity starting from the second cycle of replication. (B) Activated T cells were incubated with HIVNL or HIVNLΔvif (100 ng/ml p24) and the kinetic of viral infection analyzed by intracellular Gagp24 FACS staining. (C) At day 5 after infection, infected cells were collected and used to stimulate HS-CTL clone EM40-F21 (2,500 clone/well) in an IFN-γ Elispot. Background IFN-γ production induced by uninfected cells is shown. Background IFN-γ productions by target cells alone have been subtracted and are at least three times lower than with HS-CTLs. Activation levels reached using SL9 peptide–loaded cells as positive control were ∼600 IFN-γ+ spots/well (not depicted). Data are mean (±SD) of triplicates. (D) Data from six experiments performed as in B and C, using primary CD4+ cells from three donors, are presented as IFN-γ+ spots to percentage of Gag+ cells (as measured by FACS) on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIV-1 are more efficient than WT HIV-infected cells in activating HS-CTL (P = 0.032, Wilcoxon rank-sum test). Each symbol corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio based on six experiments. (E) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif and kinetic of viral infection monitored (as in A). (F) Infected cells were collected and used to stimulate EM40-F21 (as in C). Data are the mean (±SD) of triplicates. (G) Similar results were obtained with cells from eight donors infected at various inocula. Data are presented as IFN-γ+ spots to percentage of Gag+ cells on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIVΔnef are more efficient than HIVΔnef-infected cells in activating HS-CTLs (P < 0.0005, Wilcoxon rank-sum test). Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio. NI, noninfected cells; p.i., post infection.
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fig1: Vif-deficient HIV-1 is a potent activator of HS-CTLs. (A) Viruses were produced by transfection of 293T cells (that do not express endogenous A3G). Viruses were then used to infect PHA-activated primary CD4+ T cells. Endogenous A3G (red) is incorporated into the progeny virions and exerts its editing activity starting from the second cycle of replication. (B) Activated T cells were incubated with HIVNL or HIVNLΔvif (100 ng/ml p24) and the kinetic of viral infection analyzed by intracellular Gagp24 FACS staining. (C) At day 5 after infection, infected cells were collected and used to stimulate HS-CTL clone EM40-F21 (2,500 clone/well) in an IFN-γ Elispot. Background IFN-γ production induced by uninfected cells is shown. Background IFN-γ productions by target cells alone have been subtracted and are at least three times lower than with HS-CTLs. Activation levels reached using SL9 peptide–loaded cells as positive control were ∼600 IFN-γ+ spots/well (not depicted). Data are mean (±SD) of triplicates. (D) Data from six experiments performed as in B and C, using primary CD4+ cells from three donors, are presented as IFN-γ+ spots to percentage of Gag+ cells (as measured by FACS) on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIV-1 are more efficient than WT HIV-infected cells in activating HS-CTL (P = 0.032, Wilcoxon rank-sum test). Each symbol corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio based on six experiments. (E) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif and kinetic of viral infection monitored (as in A). (F) Infected cells were collected and used to stimulate EM40-F21 (as in C). Data are the mean (±SD) of triplicates. (G) Similar results were obtained with cells from eight donors infected at various inocula. Data are presented as IFN-γ+ spots to percentage of Gag+ cells on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIVΔnef are more efficient than HIVΔnef-infected cells in activating HS-CTLs (P < 0.0005, Wilcoxon rank-sum test). Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio. NI, noninfected cells; p.i., post infection.

Mentions: We first analyzed how T cells infected with WT or Δvif HIV stimulate HS-CTLs. To this end, primary CD4+ T cells harboring endogenous A3G were infected with HIV particles produced in the absence of A3G (Fig. 1 A). From the literature, we anticipated that after CD4+ T infection, A3G will be incorporated in the progeny virions, thus exerting its editing activity starting from the second cycle of replication. Viral propagation was monitored by flow cytometry (intracellular Gag staining; Fig. 1 B). As expected, replication of HIVΔvif was markedly reduced compared with HIV, with 3 and 20% Gag+ cells at day 5 after infection, respectively. We compared the ability of HIV and HIVΔvif-infected CD4+ T cells to activate EM40-F21, an HS CD8+ CTL clone (Fig. 1 C). EM40-F21 was derived from an HIV-infected patient and recognizes a well-characterized immunodominant epitope of Gag p17 (SL9) presented by HLA-A*0201 (Moris et al., 2004). Surprisingly, HIVΔvif-infected CD4+ T cells activated EM40-F21 to a greater extent than cells infected with the WT virus (Fig. 1 C). Similar results were obtained with cells from three different donors infected with various HIV inocula (Fig. 1 D). When expressed as the number of IFN-γ–producing CTL/Gag+ CD4 cells, CD4+ T cells infected with HIVΔvif were found to be two to three times more efficient at activating HS-CTLs than HIV-infected cells (Wilcoxon rank-sum test: P = 0.032; Fig. 1 D). These data strongly suggest that there is dissociation between the capacity of HIVΔvif to infect CD4+ T cells and to activate HS-CTLs.


The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells.

Casartelli N, Guivel-Benhassine F, Bouziat R, Brandler S, Schwartz O, Moris A - J. Exp. Med. (2009)

Vif-deficient HIV-1 is a potent activator of HS-CTLs. (A) Viruses were produced by transfection of 293T cells (that do not express endogenous A3G). Viruses were then used to infect PHA-activated primary CD4+ T cells. Endogenous A3G (red) is incorporated into the progeny virions and exerts its editing activity starting from the second cycle of replication. (B) Activated T cells were incubated with HIVNL or HIVNLΔvif (100 ng/ml p24) and the kinetic of viral infection analyzed by intracellular Gagp24 FACS staining. (C) At day 5 after infection, infected cells were collected and used to stimulate HS-CTL clone EM40-F21 (2,500 clone/well) in an IFN-γ Elispot. Background IFN-γ production induced by uninfected cells is shown. Background IFN-γ productions by target cells alone have been subtracted and are at least three times lower than with HS-CTLs. Activation levels reached using SL9 peptide–loaded cells as positive control were ∼600 IFN-γ+ spots/well (not depicted). Data are mean (±SD) of triplicates. (D) Data from six experiments performed as in B and C, using primary CD4+ cells from three donors, are presented as IFN-γ+ spots to percentage of Gag+ cells (as measured by FACS) on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIV-1 are more efficient than WT HIV-infected cells in activating HS-CTL (P = 0.032, Wilcoxon rank-sum test). Each symbol corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio based on six experiments. (E) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif and kinetic of viral infection monitored (as in A). (F) Infected cells were collected and used to stimulate EM40-F21 (as in C). Data are the mean (±SD) of triplicates. (G) Similar results were obtained with cells from eight donors infected at various inocula. Data are presented as IFN-γ+ spots to percentage of Gag+ cells on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIVΔnef are more efficient than HIVΔnef-infected cells in activating HS-CTLs (P < 0.0005, Wilcoxon rank-sum test). Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio. NI, noninfected cells; p.i., post infection.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig1: Vif-deficient HIV-1 is a potent activator of HS-CTLs. (A) Viruses were produced by transfection of 293T cells (that do not express endogenous A3G). Viruses were then used to infect PHA-activated primary CD4+ T cells. Endogenous A3G (red) is incorporated into the progeny virions and exerts its editing activity starting from the second cycle of replication. (B) Activated T cells were incubated with HIVNL or HIVNLΔvif (100 ng/ml p24) and the kinetic of viral infection analyzed by intracellular Gagp24 FACS staining. (C) At day 5 after infection, infected cells were collected and used to stimulate HS-CTL clone EM40-F21 (2,500 clone/well) in an IFN-γ Elispot. Background IFN-γ production induced by uninfected cells is shown. Background IFN-γ productions by target cells alone have been subtracted and are at least three times lower than with HS-CTLs. Activation levels reached using SL9 peptide–loaded cells as positive control were ∼600 IFN-γ+ spots/well (not depicted). Data are mean (±SD) of triplicates. (D) Data from six experiments performed as in B and C, using primary CD4+ cells from three donors, are presented as IFN-γ+ spots to percentage of Gag+ cells (as measured by FACS) on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIV-1 are more efficient than WT HIV-infected cells in activating HS-CTL (P = 0.032, Wilcoxon rank-sum test). Each symbol corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio based on six experiments. (E) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif and kinetic of viral infection monitored (as in A). (F) Infected cells were collected and used to stimulate EM40-F21 (as in C). Data are the mean (±SD) of triplicates. (G) Similar results were obtained with cells from eight donors infected at various inocula. Data are presented as IFN-γ+ spots to percentage of Gag+ cells on a logarithmic scale. CD4+ T cells infected with Vif-deficient HIVΔnef are more efficient than HIVΔnef-infected cells in activating HS-CTLs (P < 0.0005, Wilcoxon rank-sum test). Horizontal bars indicate the mean IFN-γ/Gag+ cell ratio. NI, noninfected cells; p.i., post infection.
Mentions: We first analyzed how T cells infected with WT or Δvif HIV stimulate HS-CTLs. To this end, primary CD4+ T cells harboring endogenous A3G were infected with HIV particles produced in the absence of A3G (Fig. 1 A). From the literature, we anticipated that after CD4+ T infection, A3G will be incorporated in the progeny virions, thus exerting its editing activity starting from the second cycle of replication. Viral propagation was monitored by flow cytometry (intracellular Gag staining; Fig. 1 B). As expected, replication of HIVΔvif was markedly reduced compared with HIV, with 3 and 20% Gag+ cells at day 5 after infection, respectively. We compared the ability of HIV and HIVΔvif-infected CD4+ T cells to activate EM40-F21, an HS CD8+ CTL clone (Fig. 1 C). EM40-F21 was derived from an HIV-infected patient and recognizes a well-characterized immunodominant epitope of Gag p17 (SL9) presented by HLA-A*0201 (Moris et al., 2004). Surprisingly, HIVΔvif-infected CD4+ T cells activated EM40-F21 to a greater extent than cells infected with the WT virus (Fig. 1 C). Similar results were obtained with cells from three different donors infected with various HIV inocula (Fig. 1 D). When expressed as the number of IFN-γ–producing CTL/Gag+ CD4 cells, CD4+ T cells infected with HIVΔvif were found to be two to three times more efficient at activating HS-CTLs than HIV-infected cells (Wilcoxon rank-sum test: P = 0.032; Fig. 1 D). These data strongly suggest that there is dissociation between the capacity of HIVΔvif to infect CD4+ T cells and to activate HS-CTLs.

Bottom Line: The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs.Enzymatically inactive A3G mutants failed to enhance CTL activation.Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité Virus et Immunité, Institut Pasteur, 75724 Paris cedex 15, France.

ABSTRACT
The cytidine deaminase APOBEC3G (A3G) enzyme exerts an intrinsic anti-human immunodeficiency virus (HIV) defense by introducing lethal G-to-A hypermutations in the viral genome. The HIV-1 viral infectivity factor (Vif) protein triggers degradation of A3G and counteracts this antiviral effect. The impact of A3G on the adaptive cellular immune response has not been characterized. We examined whether A3G-edited defective viruses, which are known to express truncated or misfolded viral proteins, activate HIV-1-specific (HS) CD8+ cytotoxic T lymphocytes (CTLs). To this end, we compared the immunogenicity of cells infected with wild-type or Vif-deleted viruses in the presence or absence of the cytidine deaminase. The inhibitory effect of A3G on HIV replication was associated with a strong activation of cocultivated HS-CTLs. CTL activation was particularly marked with Vif-deleted HIV and with viruses harboring A3G. Enzymatically inactive A3G mutants failed to enhance CTL activation. We also engineered proviruses bearing premature stop codons in their genome as scars of A3G editing. These viruses were not infectious but potently activated HS-CTLs. Therefore, the pool of defective viruses generated by A3G represents an underestimated source of viral antigens. Our results reveal a novel function for A3G, acting not only as an intrinsic antiviral factor but also as an inducer of the adaptive immune system.

Show MeSH
Related in: MedlinePlus