Immunoneuropathogenesis of HIV-1 clades B and C: role of redox expression and thiol modification

Abstract

Previous studies have shown that, during infection, HIV-1 clade B and clade C differentially contribute to the neuropathogenesis and development of HIV-associated neurocognitive disorders (HANDs). The low-molecular-weight tripeptide glutathione (GSH) alters the redox balance and leads to the generation of reactive oxygen species, which play a significant role in the neuropathogenesis of HANDs. We hypothesized that the HIV-1 clade B and clade C viruses and their respective Tat proteins exert differential effects on monocyte-derived immature dendritic cells (IDCs) and neuroblastoma cells (SK-N-MC) by redox activation, which leads to immunoneuropathogenesis. The GSH/GSSG ratio and mRNA expression levels and protein modification of glutathione synthetase (GSS), glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (SOD1), and catalase (CAT) were analyzed in IDCs infected with HIV-1 clade B or clade C as well as in cells treated with the respective Tat proteins. The results indicated that HIV-1 clade B virus and its Tat protein significantly increased the production of reactive oxygen species and reduced the GSH/GSSG ratio and subsequent downregulation of gene expression and protein modification of GSS, GPx1, SOD1, and CAT compared to infection with the clade C virus or treatment with the clade C Tat protein. Thus, our studies demonstrate that HIV-1 clades B and C exert differential effects of redox expression and thiol modification. HIV-1 clade B potentially induces oxidative stress, leading to more immunoneuropathogenesis than infection with HIV-1 clade C.

Keywords: Dendritic cells; Free radicals; Glutathione; HIV-1 clade B; HIV-1 clade C; Neuron; Oxidative stress.

Fig. 1

The effects of HIV-1 clade B and C viruses on IDC. IDC (1 × 10⁶ cells/ml) were infected with HIV-1 clade B (Bal strain) or HIV-1 clade C (CN54 strain) at 20ng/10⁶ cells (A) or TCID₅₀ (B) for 18 hours. The supernatants were collected at different time points (A) and 6 hr (B) used to estimate the expression of p24 antigen by ELISA. Total RNA was extracted from the infected IDC and then reverse transcribed and subjected to quantitative real time PCR using gene-specific primers to measure the expression of LTR (C), GSS (D) and the housekeeping gene β-actin. The data are expressed as the mean ± SD of the transcript accumulation index (TAI) values of three independent experiments.

Fig. 2

The effects of HIV clade B and C viruses on ROS production and intracellular thiol levels. IDC (1 ×10⁶/ml) were seeded in six-well plates and infected with either HIV-1 clade B (Bal) or C (CN 54) (20 ng/ml) for 18 hours. Next, the cells were washed and maintain for 6 days. The IDC positive cells were stained for CD80 and ROS production by DCFH-DA (A), H₂O₂ by Amplex Red (B) and intracellular thiol content by mBBr (C) and analyzed by flow cytometry. The data are expressed as the mean ± SD of three independent experiments.

Fig. 3

The effects of HIV clade B and C viruses on DC-SIGN expression in IDC. IDC (5×10⁵ cells/ml) were cultured and infected with either HIV-1 clade B (Bal) or C (CN 54) (20 ng/ml) for 18 hours. The cells were then washed to remove unbound virus and cultured for 6 days. At the end of 6^th day, the cells were stained for DC-SIGN, and expression was analyzed by flow cytometry. The data are expressed as the mean ± SD of three independent experiments.

Fig. 4

Differential effect of redox gene expression by HIV-1 clade B and C Tat protein. IDC (1 ×10⁶/ml) were treated with either HIV-1 clade B or clade C Tat protein (50 ng/ml) for 24 h. RNA was extracted and reverse transcribed followed by qRT- PCR for GSS (A), GPx1 (B), SOD1 (C), catalase (D) and the housekeeping gene β-actin. The data are expressed as mean ± SD of the transcript accumulation index (TAI) values of three independent experiments.

Fig. 5

The effects of HIV-1 clade B and C Tat proteins on the GSH/GSSG ratio and intracellular thiol content. IDC (1 × 10⁶ cells/ml) were seeded in six well plates and treated with either HIV-1 clade B or clade C Tat protein (50 ng/ml) for 24 h. The GSH/GSSG ratio was estimated spectrophotometrically (A), and the intracellular expression of thiol was determined by the percent of mBBr-positive cells (B). The data are expressed as the mean ± SD of three independent experiments.

Fig. 6

Differential effect of redox proteins by HIV-1 clade B and C Tat protein. IDC were treated with either HIV-1 clade B or clade C Tat protein (50 ng/ml) for 24 h and analyzed by Western blot to detect GSS (A), GPx1 (B) and SOD1 (C). The data are expressed as the mean ± SD of three independent experiments.

Fig. 7

HIV clade B and clade C Tat sequence alignment and effect on SK-N-MC neuronal cells. Differences in the primary structure of clade B and clade C Tat sequence (A). SK-N-MC cells were cultured in six well plates and separately treated with either HIV-1 clade B Tat or clade C Tat (50 ng/ml) for 24 h. ROS production was analyzed by flow cytometry (B), RNA was extracted, reverse transcribed and then subjected to quantitative real time PCR using GSS-specific primers to measure GSS gene expression (C), and the cell lysates were resolved by western blot using GSS antibody (D). The data are expressed as the mean ± SD of three independent experiments.