Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression

Abstract

Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.

Figure 1

METH inhibits HIV-1 replication in primary CD4⁺ T cells. A: Primary CD4⁺ T cells were isolated by negative selection from human PBMCs. After isolation, the purity of CD4⁺ T cells was determined by FACS. CD4⁺ T cells with a purity >95% were activated by PHA for 48 to 72 hours, infected with HIV-1 LAI by spinoculation, and cultured in the presence or absence of METH. Productive infection was measured by detecting intracellular viral p24 protein 3 days after infection by FACS. B: Data from six different donors with percentage inhibitory activity of METH calculated from intracellular p24 expression in infected CD4⁺ T cells. C: Measurement of extracellular p24 levels in the supernatants of infected primary CD4⁺ T cells by ELISA assay. D: Percentage inhibition of p24 released from infected primary CD4⁺ T cells isolated from three different donors. Results are expressed as means ± SD from three independent experiments. Statistical analysis was performed by analysis of variance. ^∗P < 0.05.

Figure 2

METH inhibits replication of R5 tropic HIV-1 in CD4⁺ T cells. A: Activated primary CD4⁺ T cells were infected with HIV-1 BAL virions by spinoculation and cultured in the presence or absence of METH. Productive infection was measured by detecting intracellular viral p24 protein 3 to 4 days after infection by FACS. B: Data from three different donors with relative inhibition in intracellular p24 expression in the presence of METH. Results are expressed as means ± SD from three independent experiments. Statistical analysis was performed by analysis of variance. ^∗P < 0.05.

Figure 3

Effects of METH on HIV-1 LTR-driven transcription and cytotoxicity. A: SupT1 cells and primary CD4⁺ T cells were transfected with HIV-1 LTR-GFP reporter construct and treated with METH for 24 hours. HIV-1 LTR-driven GFP expression was measured by FACS and expressed as relative GFP expression. B: MTT-based cytotoxicity assay using SupT1 and primary CD4⁺ T cells. Cells were treated with METH, and cytotoxicity was measured by MTT assay after 24 hours. Cellular apoptosis was measured by AV and PI staining using FACS in SupT1 cells (C and D) and primary CD4⁺ T cells (E and F) from three different donors. Results are expressed as means ± SD from three independent experiments. Statistical analysis was performed by analysis of variance.

Figure 4

METH up-regulates anti–HIV-1 miRNAs in CD4⁺ T cells. METH up-regulates anti–HIV-1 miRNA expression in primary CD4⁺ T cells (n = 6) (A) and CD4⁺ T-cell model SupT1 cells (C). Cells were treated with METH for 48 hours, and expression of cellular miRNAs was analyzed by real-time RT-PCR using RNA isolated from METH-treated cells. miRNA expression levels were determined by miRNA-specific primers and normalized to 5s-rRNA. B: Expression of miR-296-5p in primary CD4⁺ T cells (n = 3) as a positive control. D: Knockdown experiments were conducted in SupT1 cells using anti-miRs. Anti-miRs or scrambled control oligos were transfected into SupT1 cells using the Neon Transfection System. These cells were then infected with VSV-G–pseudotyped HIV-1 luciferase reporter virus. Infection was determined by measuring luciferase activity in the cellular lysates. Luciferase activity was normalized to total protein content of the lysate. Knockdown of miR-125b and miR-28-5p resulted in increased luciferase activity. Results are expressed as means ± SD from three independent experiments. Statistical analysis was performed by analysis of variance. ^∗P < 0.05.