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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 stimulates chemokine secretion of HS-CTLs. (A) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif in the presence or absence of Nevirapine (NVP). At day 4 after infection, viral replication was monitored by FACS. The percentage of infected (Gag+) cells is indicated. (B) Infected cells were then used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (2,500 clones/well). Activation levels reached, using SL9 peptide–loaded cells as positive control, were ∼600 IFN-γ+ spots/well (not depicted). Data are the mean (±SD) of triplicates and are representative of at least three independent experiments. (C) IFN-γ Elispot data are presented as IFN-γ+ spots to percentage of Gag+ cells. (D and G) 2.5 × 105 HIVNLΔnef- and HIVNLΔnefvif-infected cells were co-cultured for 24 h with 5 × 103 EM40-F21 (50/1 ratio). Culture supernatants were collected and the release of the indicated chemokine quantified by Luminex. Background secretions in the absence of HS-CTLs have been subtracted. HIV infection didn’t induce an increase of the background lymphokine productions by target CD4+ T cell alone (not depicted). (E and H) Chemokine quantifications presented as concentration to percentage of Gag+ cells. (F and I) Activated T cells were loaded with SL9 peptide at the indicated concentrations, co-cultured for 24 h with EM40-F21 (50/1 ratio), and chemokine release was measured by Luminex. Luminex data are the mean (±SD) of duplicates and are representative of three independent experiments.
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fig2: Vif-deficient HIV-1 stimulates chemokine secretion of HS-CTLs. (A) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif in the presence or absence of Nevirapine (NVP). At day 4 after infection, viral replication was monitored by FACS. The percentage of infected (Gag+) cells is indicated. (B) Infected cells were then used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (2,500 clones/well). Activation levels reached, using SL9 peptide–loaded cells as positive control, were ∼600 IFN-γ+ spots/well (not depicted). Data are the mean (±SD) of triplicates and are representative of at least three independent experiments. (C) IFN-γ Elispot data are presented as IFN-γ+ spots to percentage of Gag+ cells. (D and G) 2.5 × 105 HIVNLΔnef- and HIVNLΔnefvif-infected cells were co-cultured for 24 h with 5 × 103 EM40-F21 (50/1 ratio). Culture supernatants were collected and the release of the indicated chemokine quantified by Luminex. Background secretions in the absence of HS-CTLs have been subtracted. HIV infection didn’t induce an increase of the background lymphokine productions by target CD4+ T cell alone (not depicted). (E and H) Chemokine quantifications presented as concentration to percentage of Gag+ cells. (F and I) Activated T cells were loaded with SL9 peptide at the indicated concentrations, co-cultured for 24 h with EM40-F21 (50/1 ratio), and chemokine release was measured by Luminex. Luminex data are the mean (±SD) of duplicates and are representative of three independent experiments.

Mentions: CTLs secrete a panel of chemokine and cytokine upon activation (Price et al., 1998; Wagner et al., 1998; Appay et al., 2008). We performed a peptide titration experiment to identify cytokines other than IFN-γ that are secreted in an antigen-specific manner by EM40-F21. Primary CD4+ T cells were loaded with the SL9 peptide, co-cultured with HS-CTLs, and lymphokine production was analyzed in culture supernatants using the luminex technology. As previously reported (Price et al., 1998; Wagner et al., 1998), we observed a dose-dependent secretion of MIP-1α and MIP-1β (Fig. 2, F and I). Thereafter, we examined the effect of Δvif viruses on MIP-1β and MIP-1α secretions by EM40-F21. Primary T cells were infected with VSV-G-HIVΔnef and -HIVΔnefvif, monitored for Gag expression (12 and 1% of Gag+ cells, respectively), and used to activate EM40-F21 (Fig. 2, A, B, D, and G). Using IFN-γ Elispot, we first confirmed the enhancing effect of Δvif on IFN-γ secretion by EM40-F21 (Fig. 2, B and C). We then measured in co-culture supernatants the release of MIP-1α and MIP-1β. Upon co-culture with HIVΔnef- and HIVΔnefvif-infected targets (at a ratio of 1 CTL to 50 CD4+ target cells), EM40-F21 secreted similar levels of MIP-1α (130 and 106 pg/ml, respectively; Fig. 2 D). HIVΔnef- and HIVΔnefvif-infected cells also allowed the secretion of MIP-1β (292 and 167 pg/ml, respectively; Fig. 2 G). These levels were at least three times higher than background secretion induced by uninfected control cells (Fig. 2, D and G). HIV infection did not increase the background lymphokine production by target CD4+ T cells alone (unpublished data). We noticed that the secretion of MIP-1β with Δnefvif viruses was reduced as compared with Δnef (Fig. 2 G), suggesting that the effect of Δvif viruses might vary depending on the lymphokine. Nonetheless, even if very few Gag+ cells were detected, HIVΔnefvif induced a potent secretion of both MIP-1α and MIP-1β by HS-CTLs (Fig. 2, E and H). To gain information on the magnitude of A3G-mediated effect, we used MIP-1α and MIP-1β SL9 peptide titration curves (Fig. 2, F and I) as standards to calculate the relative antigen presentation levels on the surface of infected (Gag+) target cells. Based on MIP-1α secretion, HIVΔnef- and HIVΔnefvif-infected cells presented 6.6 × 10−15 and 4 × 10−14 exogenous peptide equivalent, respectively. Based on MIP-1β secretion, the relative antigen presentation levels were 1.98 × 10−15 and 7.9 × 10−15 exogenous peptide equivalent for HIVΔnef- and HIVΔnefvif Gag+ cells, respectively. Thereafter, cells infected with HIVΔnefvif viruses likely present four to six times more peptide antigen than cells infected with HIVΔnef. This calculation was done using populations harboring different percentages of Gag+ cells. It would have also been informative to confirm these findings by calculating exogenous peptide equivalents in the presence of equal number of infected (Gag+) target cells. The low replicative capacity of HIVΔnefvif in primary CD4+ T cells precluded this possibility. As expected, titrating down the amount of infected cells in the co-culture (1 CTL for 10 CD4+ targets) reduced the overall secretion levels and confirmed that in the absence of Vif, infected cells enhance cytokine production by HS-CTLs (Fig. S1). It is noteworthy that Nevirapine, a reverse transcription inhibitor of viral replication, blocked antigen presentation, indicating that activation of EM40-F21 was not a result of the presentation of incoming HIV antigens (Fig. 2, A and B). Collectively, our results demonstrate that Vif-deficient HIV-1 replicates less efficiently but is a potent activator of HS-CTLs, inducing the secretion of various lymphokines such as IFN-γ, MIP-1α, and MIP-1β. More specifically, Vif impacts the immunogenicity of HIV-infected T cells. In infected cells, Vif targets A3G for proteasomal degradation (Mariani et al., 2003; Yu et al., 2003); therefore, A3G may render HIV-infected cells more able to activate 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 stimulates chemokine secretion of HS-CTLs. (A) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif in the presence or absence of Nevirapine (NVP). At day 4 after infection, viral replication was monitored by FACS. The percentage of infected (Gag+) cells is indicated. (B) Infected cells were then used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (2,500 clones/well). Activation levels reached, using SL9 peptide–loaded cells as positive control, were ∼600 IFN-γ+ spots/well (not depicted). Data are the mean (±SD) of triplicates and are representative of at least three independent experiments. (C) IFN-γ Elispot data are presented as IFN-γ+ spots to percentage of Gag+ cells. (D and G) 2.5 × 105 HIVNLΔnef- and HIVNLΔnefvif-infected cells were co-cultured for 24 h with 5 × 103 EM40-F21 (50/1 ratio). Culture supernatants were collected and the release of the indicated chemokine quantified by Luminex. Background secretions in the absence of HS-CTLs have been subtracted. HIV infection didn’t induce an increase of the background lymphokine productions by target CD4+ T cell alone (not depicted). (E and H) Chemokine quantifications presented as concentration to percentage of Gag+ cells. (F and I) Activated T cells were loaded with SL9 peptide at the indicated concentrations, co-cultured for 24 h with EM40-F21 (50/1 ratio), and chemokine release was measured by Luminex. Luminex data are the mean (±SD) of duplicates and are representative of three independent experiments.
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fig2: Vif-deficient HIV-1 stimulates chemokine secretion of HS-CTLs. (A) Activated primary CD4+ T cells were incubated with VSV-pseudotyped HIVNLΔnef or HIVNLΔnefvif in the presence or absence of Nevirapine (NVP). At day 4 after infection, viral replication was monitored by FACS. The percentage of infected (Gag+) cells is indicated. (B) Infected cells were then used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (2,500 clones/well). Activation levels reached, using SL9 peptide–loaded cells as positive control, were ∼600 IFN-γ+ spots/well (not depicted). Data are the mean (±SD) of triplicates and are representative of at least three independent experiments. (C) IFN-γ Elispot data are presented as IFN-γ+ spots to percentage of Gag+ cells. (D and G) 2.5 × 105 HIVNLΔnef- and HIVNLΔnefvif-infected cells were co-cultured for 24 h with 5 × 103 EM40-F21 (50/1 ratio). Culture supernatants were collected and the release of the indicated chemokine quantified by Luminex. Background secretions in the absence of HS-CTLs have been subtracted. HIV infection didn’t induce an increase of the background lymphokine productions by target CD4+ T cell alone (not depicted). (E and H) Chemokine quantifications presented as concentration to percentage of Gag+ cells. (F and I) Activated T cells were loaded with SL9 peptide at the indicated concentrations, co-cultured for 24 h with EM40-F21 (50/1 ratio), and chemokine release was measured by Luminex. Luminex data are the mean (±SD) of duplicates and are representative of three independent experiments.
Mentions: CTLs secrete a panel of chemokine and cytokine upon activation (Price et al., 1998; Wagner et al., 1998; Appay et al., 2008). We performed a peptide titration experiment to identify cytokines other than IFN-γ that are secreted in an antigen-specific manner by EM40-F21. Primary CD4+ T cells were loaded with the SL9 peptide, co-cultured with HS-CTLs, and lymphokine production was analyzed in culture supernatants using the luminex technology. As previously reported (Price et al., 1998; Wagner et al., 1998), we observed a dose-dependent secretion of MIP-1α and MIP-1β (Fig. 2, F and I). Thereafter, we examined the effect of Δvif viruses on MIP-1β and MIP-1α secretions by EM40-F21. Primary T cells were infected with VSV-G-HIVΔnef and -HIVΔnefvif, monitored for Gag expression (12 and 1% of Gag+ cells, respectively), and used to activate EM40-F21 (Fig. 2, A, B, D, and G). Using IFN-γ Elispot, we first confirmed the enhancing effect of Δvif on IFN-γ secretion by EM40-F21 (Fig. 2, B and C). We then measured in co-culture supernatants the release of MIP-1α and MIP-1β. Upon co-culture with HIVΔnef- and HIVΔnefvif-infected targets (at a ratio of 1 CTL to 50 CD4+ target cells), EM40-F21 secreted similar levels of MIP-1α (130 and 106 pg/ml, respectively; Fig. 2 D). HIVΔnef- and HIVΔnefvif-infected cells also allowed the secretion of MIP-1β (292 and 167 pg/ml, respectively; Fig. 2 G). These levels were at least three times higher than background secretion induced by uninfected control cells (Fig. 2, D and G). HIV infection did not increase the background lymphokine production by target CD4+ T cells alone (unpublished data). We noticed that the secretion of MIP-1β with Δnefvif viruses was reduced as compared with Δnef (Fig. 2 G), suggesting that the effect of Δvif viruses might vary depending on the lymphokine. Nonetheless, even if very few Gag+ cells were detected, HIVΔnefvif induced a potent secretion of both MIP-1α and MIP-1β by HS-CTLs (Fig. 2, E and H). To gain information on the magnitude of A3G-mediated effect, we used MIP-1α and MIP-1β SL9 peptide titration curves (Fig. 2, F and I) as standards to calculate the relative antigen presentation levels on the surface of infected (Gag+) target cells. Based on MIP-1α secretion, HIVΔnef- and HIVΔnefvif-infected cells presented 6.6 × 10−15 and 4 × 10−14 exogenous peptide equivalent, respectively. Based on MIP-1β secretion, the relative antigen presentation levels were 1.98 × 10−15 and 7.9 × 10−15 exogenous peptide equivalent for HIVΔnef- and HIVΔnefvif Gag+ cells, respectively. Thereafter, cells infected with HIVΔnefvif viruses likely present four to six times more peptide antigen than cells infected with HIVΔnef. This calculation was done using populations harboring different percentages of Gag+ cells. It would have also been informative to confirm these findings by calculating exogenous peptide equivalents in the presence of equal number of infected (Gag+) target cells. The low replicative capacity of HIVΔnefvif in primary CD4+ T cells precluded this possibility. As expected, titrating down the amount of infected cells in the co-culture (1 CTL for 10 CD4+ targets) reduced the overall secretion levels and confirmed that in the absence of Vif, infected cells enhance cytokine production by HS-CTLs (Fig. S1). It is noteworthy that Nevirapine, a reverse transcription inhibitor of viral replication, blocked antigen presentation, indicating that activation of EM40-F21 was not a result of the presentation of incoming HIV antigens (Fig. 2, A and B). Collectively, our results demonstrate that Vif-deficient HIV-1 replicates less efficiently but is a potent activator of HS-CTLs, inducing the secretion of various lymphokines such as IFN-γ, MIP-1α, and MIP-1β. More specifically, Vif impacts the immunogenicity of HIV-infected T cells. In infected cells, Vif targets A3G for proteasomal degradation (Mariani et al., 2003; Yu et al., 2003); therefore, A3G may render HIV-infected cells more able to activate 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