Vif-deficient HIV-1 stimulates chemokine secretion of HS-CTLs. (A) Activated primary CD4⁺ T cells were incubated with VSV-pseudotyped HIV_NLΔnef or HIV_NLΔnefvif 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 HIV_NLΔnef- and HIV_NLΔnefvif-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.

The editing activity of A3G is required to enhance HS-CTL activation. (A) CEM-A2⁺ cells were infected with HIV_NLΔnef produced without A3G or in the presence of WT and C-terminal domain mutants (H257R and C288S) of A3G (triangles, p24 doses: 20 and 10 ng/ml). 48 h after infection, viral infection was analyzed by Gagp24 staining (data are representative of four independent experiments) and infected cells were used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (10,000 clones/well) (B). Background IFN-γ production induced by uninfected cells has been subtracted. Background IFN-γ productions by CEM-A2⁺ cells alone were close to zero. Activation levels reached using SL9 peptide–loaded cells as positive control were ∼600 IFN-γ⁺ spots/well (not depicted). (C) Data are presented as a ratio of IFN-γ Elispot to percentage of Gag⁺ cells. Impairing the editing activity of A3G abrogates A3G-mediated enhancement of CTL recognition. Data are the mean (±SD) of triplicates and are representative of four independent experiments.

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 HIV_NL or HIV_NLΔvif (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 HIV_NLΔnef or HIV_NLΔnefvif 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 HIV_Δnef are more efficient than HIV_Δnef-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.

A3G-mediated viral restriction enhances HS-CTL activation. (A) Viruses were produced upon cotransfection in 293T cells of HIV genome and plasmids encoding for A3G. CEM-A2⁺ cells were then infected. CEM-A2⁺ cells do not express A3G (blue); hence, exogenous A3G exerts its editing activity exclusively during the first cycle of replication. (B) CEM-A2⁺ cells were incubated with HIV_SF2Δnef or HIV_SF2Δnef + A3G (100 ng/ml p24) and the kinetic of viral infection analyzed by p24 FACS staining. (C) 48 h after infection, infected cells were collected and used to stimulate HS-CTL clone EM40-F21 in an IFN-γ Elispot (10,000 clones/well). Background IFN-γ production induced by uninfected cells is shown. Background IFN-γ productions by CEM-A2⁺ cells alone were close to zero. Activation levels with SL9 peptide–loaded cells were around 600 IFN-γ⁺ spots/well (not depicted). Data are the mean (±SD) of triplicates. (D) Data from nine independent experiments performed as in B and C using two HIV isolates (NLΔnef and SF2Δnef) are presented as IFN-γ⁺ spots to percentage of Gag⁺ cells on a logarithmic scale. CEM-A2⁺ cells infected with HIV_Δnef + A3G are more efficient than HIV_Δnef-infected cells in activating HS-CTL (P < 0.0005, Wilcoxon rank-sum test). Each symbol corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ/gag⁺ cell ratio based on the nine independent experiments. (E) To reach similar numbers of Gag⁺ cells, CEM-A2⁺ cells were infected with 5–10× more HIV_SF2Δnef + A3G than HIV_SF2Δnef (doses ranging from 20 to 100 ng p24/ml). The percentage of Gag⁺ cells achieved at 48 h after infection is indicated for six independent experiments. Using these experimental conditions, there is no difference in terms of gag⁺ cells between HIV_SF2Δnef or HIV_SF2Δnef + A3G-infected cells (P = 0.29, Wilcoxon rank-sum test). Horizontal bars indicate the mean percentage of Gag⁺ cells based on the six independent experiments. (F) Infected cells were then used in Elispot assays to stimulate EM40-F21 (10,000 clones/well). Results of six independent experiments are summarized. In the presence of A3G, there was a stronger CTL activation (P = 0.015, Wilcoxon rank-sum test). Each symbol in E and F corresponds to an independent experiment. Horizontal bars indicate the mean IFN-γ Elispot response based on the six independent experiments. (G) T1-B7 cells were incubated with HIV_SF2 or HIV_SF2 + A3G (100 ng/ml p24). 48 h after infection, cells were submitted to an IFN-γ Elispot assay to activate HIV Nef-specific CTLs restricted by HLA-B*0702 (10⁴ CTL/well). HIV Nef-specific CTL lines were generated in three different HLA-B*0702 transgenic mice and used in two independent experiments. One representative experiment with one CTL line is shown. (H) As in F, HIV-infected cells were used to activate a control CTL line specific for CMV. Data are the mean (±SD) of triplicates.

Defective viruses with premature STOP codons are efficiently recognized by HS-CTLs. (A) Schematic representation of the strategy used to construct defective HIV (HIV_NLΔnefP2 and HIV_NLΔnefP3) mimicking A3G-mediated editing. (Top) Nucleotide and amino acid sequences of a fraction of gag gene (gagP2) targeted for mutagenesis. The G-to-A transition introduced by mutagenesis is underlined and the resulting amino acid change (W₂₁₂ to STOP) is in bold. Nucleotide numbering is according to HIV_LAI sequence. (Bottom) Schematic representation of Gag full length and truncated Gag proteins (GagP2 and GagP3) generated by mutagenesis. The SL9 peptide amino acid sequence within p17 is indicated. HeLa-A2⁺ cells were transfected with HIV, HIV_NLΔnefP2, and HIV_NLΔnefP3 proviruses. (B and C) Gag expression profiles were analyzed 2 d after transfection using anti-p17 antibody and Western blotting (B) or anti-p24 antibody and FACS (C). Data are representative of three independent experiments. For Western blotting, transfected cells were pelleted, lyzed, loaded on SDS-PAGE, and transferred to nitrocellulose. Membranes were then incubated with antibodies to p17 and actine. (D) HeLa-A2⁺ cells were transfected with the indicated doses of provirus. At 36 h after transfection, cells were used to activate HS-CTL clone EM40-F21 in an IFN-γ Elispot assay (10,000 clones/well). Data are the mean (±SD) of triplicates and are representative of three independent experiments. (E) Hela-A2 cells transfected with 1 µg of indicated HIV DNA were mock or Epoxomicin treated (1 µM) for 6 h prior to co-culture with HS-CTLs. Epoxomicin, a proteasome inhibitor, diminishes CTL activation. Background IFN-γ production induced by untransfected 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 ∼400 IFN-γ⁺ spots/well (not depicted). Data are mean (±SD) of triplicates and are representative of two independent experiments.