HIV Nef Expression Down-modulated GFAP Expression and Altered Glutamate Uptake and Release and Proliferation in Astrocytes

Abstract

HIV infection of astrocytes leads to restricted gene expression and replication but abundant expression of HIV early genes Tat, Nef and Rev. A great deal of neuroHIV research has so far been focused on Tat protein, its effects on astrocytes, and its roles in neuroHIV. In the current study, we aimed to determine effects of Nef expression on astrocytes and their function. Using transfection or infection of VSVG-pseudotyped HIV viruses, we showed that Nef expression down-modulated glial fibrillary acidic protein (GFAP) expression. We then showed that Nef expression also led to decreased GFAP mRNA expression. The transcriptional regulation was further confirmed using a GFAP promoter-driven reporter gene assay. We performed transcription factor profiling array to compare the expression of transcription factors between Nef-intact and Nef-deficient HIV-infected cells and identified eight transcription factors with expression changes of 1.5-fold or higher: three up-regulated by Nef (Stat1, Stat5, and TFIID), and five down-regulated by Nef (AR, GAS/ISRE, HIF, Sp1, and p53). We then demonstrated that removal of the Sp1 binding sites from the GFAP promoter resulted in a much lower level of the promoter activity and reversal of Nef effects on the GFAP promoter, confirming important roles of Sp1 in the GFAP promoter activity and for Nef-induced GFAP expression. Lastly, we showed that Nef expression led to increased glutamate uptake and decreased glutamate release by astrocytes and increased astrocyte proliferation. Taken together, these results indicate that Nef leads to down-modulation of GFAP expression and alteration of glutamate metabolism in astrocytes, and astrocyte proliferation and could be an important contributor to neuroHIV.

Keywords: GFAP; HIV Nef; astrocytes; glutamate uptake and release; proliferation; transcription control.

Figure 1.

Effects of Nef expression on GFAP protein expression in human astrocytes. Human astrocytic cell lines SVGA (A) or U373 (C) were plated in a 6-well plate at a density of 2.5 x 10⁵ cells/well for SVGA or 3.0 x 10⁵ cells/well for U373, transfected with 2.5 μg pcDNA3 plasmid (C3) or pNef.Myc plasmid (Nef). The cell lysates were analyzed by Western blotting (A & C) against an anti-GFAP, Nef, GAPDH, or β-actin antibody. GFAP expression was quantitated, normalized to GAPDH or β-actin, and expressed as the relative values to the control (rel) (B & D). untx: untransfected cells. The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 2.

Effects of Nef expression on GFAP expression in human astrocytes in the context of HIV infection. U373 were infected with VSVG/GAGi and GAGi.Nef- (A-C) or NLGi or NLGi.Nef- (D-F). The cells were cultured for 48 hr, the GFP+ cells were estimated under a fluorescence microscope and expressed as a ratio to the total number of cells (A & D); or the cells were harvested for cell lysates and Western blotting against an anti-GFAP, Nef, GAPDH, or β-actin antibody (B & E). GFAP expression was quantitated, normalized to GAPDH or β-actin, and expressed as the relative values to the control (rel.) (C & F). The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 3.

Effects of Nef on GFAP protein expression in human primary astrocytes in the context of HIV infection. Human primary fetal astrocytes (HPA) were infected with VSVG/GAGi and GAGi.Nef-. The cells were cultured for 48 hr, the GFP+ cells were estimated under a fluorescence microscope and expressed as a ratio to the total number of cells (A); or the cells were harvested for cell lysates and Western blotting against an anti-GFAP, Nef, or β-actin antibody (B). GFAP expression was quantitated, normalized to β-actin, and expressed as the relative values to the control (rel.) (C). The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 4.

Effects of Nef expression on GFAP mRNA expression in astrocytes. U373 (A & B), mouse primary astrocytes (C), human primary fetal astrocytes (D) were transfected with C3 or Nef (A) or infected with VSVG/GAGi and GAGi.Nef- (B-D). Total RNA was extracted and GFAP mRNA were determined by qRT-PCR. Threshold cycle values were normalized to the mRNA level of GFP which was included as the transfection efficiency control (A) or expressed from VSVG/GAGi and GAGi.Nef- and then normalized to the qRT-PCR control GAPDH and expressed as the relative values to the control C3 (A) or VSVG/GAGi (B-D) for infection efficiency and GAPDH mRNA levels. The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 5.

Effects of Nef expression on the GFAP Promoter activity. U373 were transfected with pGFAP.luc plasmid, cultured for 16 hr, split into two equal samples, and infected with VSVG/GAGi and GAGi.Nef-. The cells were cultured for 48 hr and harvested for Western blotting against an anti-Nef, or β-actin antibody (A), or harvested for the luciferase reporter gene assay (B). The relative luciferase activity was normalized to the protein concentration. The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 6.

Effects of Nef expression on expression of transcription factors in astrocytes. U373 were infected with VSVG/GAGi and GAGi.Nef-, cultured for 48 hr, and harvested for subcellular fractionations into cytoplasmic and nuclear extracts, as verified by Western blotting against an anti-Histone 3 (H3) antibody as a marker of nuclear extract, or against an anti-GAPDH antibody as a marker of cytoplasmic extract (A), followed by the transcription factor profiling array using the nuclear extracts, differed in expression by the heat map (B) and identification of eight transcription factors with changes of 1.5-fold or higher (three being down-regulated (open bar, C), or five being up-regulated (closed bar, C).

Figure 7.

Role of Sp1 in Nef-induced GFAP downmodulation. U373 were transfected with C3 or Nef and harvested for Western blotting against an anti-Sp1, Nef, or GAPDH antibody (A), followed by Sp1 quantitation using the loading control GAPDH as the reference (B); or U373 were transfected with pΔSp1-GFAP.luc plasmid, cultured for 16 hr, split into two equal samples, and infected with VSVG/GAGi and GAGi.Nef-. The cells were cultured for 48 hr and harvested for Western blotting against an anti-Nef, or β-actin antibody (C); or harvested for the luciferase reporter gene assay (D). The relative luciferase activity was normalized to the protein concentration. The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 8.

Effects of Nef expression on glutamate uptake into astrocytes. U373 (A & C) or human primary fetal astrocytes (B & D) were transfected with pC3 or Nef (A & B), or infected with VSVG/GAGi and GAGi.Nef- (C & D). The cells were cultured for 24 hr, changed media, pulsed with [³H]-glutamate, and harvested for cell lysates. The cell lysates were subjected to scintillation counting and normalized with protein concentration. GFP was included in the transfection to ensure the comparable transfection efficiency (A & B), or GFP expression from the infected cells was used to ensure the comparable infection efficiency (C & D). The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 9.

Effects of Nef expression on glutamate release from astrocytes. U373 (A & C) or human primary fetal astrocytes (B & D) were transfected with pC3 or Nef (A & B), or infected with VSVG/GAGi and GAGi.Nef- (C & D). The cells were cultured for 24 hr after changing the media and the glutamate level in the culture media was measured using an absorbance assay and normalized with protein concentration. The data were Mean ± SEM and representative of three independent experiments (n=3).

Figure 10.

Effects of Nef on astrocyte proliferation. U373 were transfected with pC3 Nef (A, B, & E), or infected with VSVG/GAGi and GAGi.Nef- (C, D & F) and cultured for 48 hr. Brightfield and FITC images were taken at 20x magnification and cells in the brightfield were counted using Image J software (A -D). The MTT assay was performed, and absorbance was measured at 595 nm (background subtraction at 655nm (E &F). The data were Mean ± SEM and representative of three independent experiments (n=3).