The HIV-1 vpr R77Q Mutant Induces Apoptosis, G2 Cell Cycle Arrest, and Lower Production of Pro-Inflammatory Cytokines in Human CD4+ T Cells

Abstract

Acquired immunodeficiency syndrome (AIDS) occurs when HIV depletes CD4+ helper T cells. Some patients develop AIDS slowly or not at all, and are termed long-term non-progressors (LTNP), and while mutations in the HIV-1 Viral Protein R (vpr) gene such as R77Q are associated with LTNP, mechanisms for this correlation are unclear. This study examines the induction of apoptosis, cell cycle arrest, and pro-inflammatory cytokine release in the HUT78 T cell line following infection with replication-competent wild-type strain NL4-3, the R77Q mutant, or a vpr Null mutant. Our results show a significant enhancement of apoptosis and G₂ cell cycle arrest in HUT78 cells infected with R77Q, but not with WT NL4-3 or the vpr Null strain. Conversely, HUT78 cells infected with the WT virus show higher levels of necrosis. We also detected lower TNF and IL-6 release after infection with R77Q vs. WT. The apoptotic phenotype was also seen in the CEM cell line and in primary CD4+ T cells. Protein expression of the R77Q vpr variant was low compared to WT vpr, but expression levels alone cannot explain these phenotypes because the Null virus did not show apoptosis or G₂ arrest. These results suggest that R77Q triggers a non-inflammatory apoptotic pathway that attenuates inflammation, possibly contributing to LTNP.

Keywords: AIDS; HIV; apoptosis; immune activation; inflammation; necrosis; vpr.

Figure 1

(A) Nucleotide and amino acid differences in the vpr gene for various strains used in this study. (B) vpr mutations have minimal impact on the replication capacity of HIV-1 in HUT78 cells. WT, R77Q, or Null mutants were used to infect HUT78 cells at MOI 0.01. At 3, 5, and 7 dpi, supernatants were analyzed for viral load through Q-RT-PCR. See Supplementary Material S1 for complete statistical analysis.

Figure 2

Differences in nucleotide and amino acid sequences between vpr mutants and confirmation of gene expression by immunoblot. (A) Immunoblotting confirmed vpr expression in HUT78 cells infected with WT virus or the R77Q mutant, while no vpr expression was detected in the Null mutant or in uninfected cells. GAPDH was used as a loading control. (B) p24 was also used as a loading control for differing viral replication kinetics and/or protein production. (C) Bar graph indicating the quantitative analysis of the relative western blot bands expressed as a ratio to the uninfected control. Data are representative of three independent experiments, and error bars indicate SE. Asterisk * indicates that there was a statistically significant difference when compared to all other conditions (p ≤ 0.05) whereas n.s. signifies no statistical significance (p > 0.05).

Figure 3

The R77Q vpr mutant triggers different death pathways in HUT78 cells than WT and Null. WT, R77Q, and Null HIV vpr mutants were used to infect HUT78 cells at MOI 0.01, and cell samples were analyzed at 3, 5, and 7 dpi. Apoptosis was detected by Annexin V staining and fixable viability dye (FVD) was used to detect dead cells via penetration of the dye across a porous plasma membrane. Thus, all FVD+ cells are expected to also bind Annexin V but not due to phosphatidylserine flipping to the outer membrane. (A) Representative dot plots from samples collected at 7 dpi. (B) Apoptotic cells (positive for Annexin V only). (C) Dead cells (all cells positive for FVD). At 3 dpi, no statistical differences were detected between any samples. At 5 dpi and 7 dpi, all samples were statistically different from each other, with the sole exceptions being Null and uninfected at 5 dpi and R77Q and Null at 7 dpi. Data are representative of three independent experiments, and error bars indicate SE. ** p-value ≤ 0.01; statistics shown focus on 7 dpi. See Supplementary Material S2 for complete datasets and statistical analysis. For the flow cytometry dot plots, the x-axis runs from 10⁰ to 10⁷ in increments of 10, while the y-axis runs from 10⁰ to 10⁸ in increments of 10.

Figure 4

HUT78 cells productively infected with R77Q virus are highly apoptotic, but non-productively infected cells also showed increased apoptosis. WT, R77Q, and Null HIV vpr mutants were used to infect HUT78 cells at MOI 0.01, and cell samples were analyzed at 7 dpi. Apoptosis was detected by Annexin V staining, fixable viability dye (FVD) was used to detect dead cells, and an antibody to the HIV-1 p24 protein was used to detect productively-infected cells. (A) Representative dot plots from samples collected on 7 dpi, with gating first performed on the p24+ or p24− populations. (B) Apoptotic cells (positive for Annexin V only, after first gating on p24+ or p24− populations). (C) Dead cells (all cells positive for FVD, after first gating on p24+ or p24− populations). Error bars indicate SE. * p-value ≤ 0.05. See Supplementary Dataset S9 for complete datasets and statistical analysis. ^a indicates that the labeled population was significantly different than all other virus strains when comparing within the p24+ populations; similarly, ^b indicates that the labeled population was significantly different than all other virus strains within the p24− populations. For the flow cytometry dot plots, the x-axis runs from 10⁰ to 10⁷ in increments of 10, while the y-axis runs from 10⁰ to 10⁸ in increments of 10.

Figure 5

Primary CD4+ T cells become apoptotic after infection with the R77Q mutant. CBMC-derived CD4+ T cells were infected with WT NL4-3, R77Q, or Null HIV vpr mutants in triplicate at MOI 0.01 and analyzed for apoptosis at 7 dpi using the Annexin V/Fixable viability dye staining method. Shown are results from a representative sample from a non-Caucasian CBMC donor (Patient 2). Similar results were obtained from three additional donors (two Caucasian and one non-Caucasian). See Supplementary Material S7 for results from additional patients and for statistical analysis. (A) Representative dot plots of Annexin V/FVD for uninfected and WT NL4-3, R77Q, and Null HIV vpr-infected CBMC-derived CD4+ T cells. (B) Bar graph representing the proportion of apoptotic cells (Annexin V+ only) for patient 2 alone. CD4+ T cells were gated from CBMCs, and singlet discrimination was performed before gating on Annexin V/FVD guided by single color controls and an unstained control (Supplementary Figure S2). R77Q showed a statistically significant difference when compared to all other groups. All groups were statistically different from each other, except for the comparison between Null and uninfected (p = 0.085) and Null and WT (p = 0.063). (C) Bar graph representing the proportion of dead cells (FVD+ and Annexin V+). The gating strategy was performed as described in B above. WT and uninfected were statistically different when compared to all other groups. All other groups were statistically different from each other except for R77Q compared to Null (p = 0.466). Error bars indicate standard error while * indicates significant difference compared to all other groups (p < 0.05). For the flow cytometry dot plots, the x-axis runs from 10⁰ to 10⁶ in increments of 10, while the y-axis runs from 10⁰ to 10⁶ in increments of 10.

Figure 6

R77Q-enhanced apoptosis confirmed by TUNEL stain. WT, R77Q, and Null HIV vpr mutants were used to infect HUT78 cells at MOI 0.01, and cell samples were stained by the TUNEL assay and analyzed at 5 dpi. (A) Representative dot plots from 5 dpi. (B) Bar graph representing the TUNEL-positive cells of each sample. Asterisks show statistical differences between R77Q and all the other samples. Data are representative of three independent experiments, and error bars indicate SE. ** p-value ≤ 0.01. See Supplementary Material S3 for complete statistical analysis.

Figure 7

Infection of HUT78 cells with the R77Q vpr mutant enhances G₂ cell cycle arrest. WT, R77Q, and Null HIV vpr mutants were used to infect HUT78 cells at MOI 0.1, then stained with propidium iodide to detect relative DNA content, and analyzed by flow cytometry. Cells were first gated on p24+ populations prior to cell cycle analysis in order to focus on productively infected populations. (A) Representative histograms from 7 dpi. (B) Percentage of p24+ cells in G₂ phase. The asterisk shows a statistical difference between R77Q and all other samples. Data are representative of three independent experiments, and error bars indicate SE. * p-value ≤ 0.05. See Supplementary Material S4 and S5 for complete statistical analysis. Colors in the bar graphs match the various populations in the histograms above. The x-axis ends at 21M for each sample. The y-axis ends at 1.4K for Uninfected, at 500 for WT, at 560 for R77Q, and at 640 for Null.

Figure 8

Expression of TNF and IL-6 are strongly upregulated after infection with WT virus, but not with the R77Q mutant. WT, R77Q, and Null viruses were used to infect HUT78 cells at MOI 0.01, and then at 5 dpi, supernatants were collected and assayed for TNF (A) and IL-6 (B) production by cytometric bead array. Data are representative of three independent experiments, and error bars indicate SE. See Supplementary Material S8 for complete statistical analysis.