HIV-1 entry and trans-infection of astrocytes involves CD81 vesicles

Abstract

Astrocytes are extensively infected with HIV-1 in vivo and play a significant role in the development of HIV-1-associated neurocognitive disorders. Despite their extensive infection, little is known about how astrocytes become infected, since they lack cell surface CD4 expression. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. Astrocytes were found to bind and harbor virus followed by biphasic decay, with HIV-1 detectable out to 72 hours. HIV-1 was observed to associate with CD81-lined vesicle structures. shRNA silencing of CD81 resulted in less cell-associated virus but no loss of co-localization between HIV-1 and CD81. Astrocytes supported trans-infection of HIV-1 to T-cells without de novo virus production, and the virus-containing compartment required 37°C to form, and was trypsin-resistant. The CD81 compartment observed herein, has been shown in other cell types to be a relatively protective compartment. Within astrocytes, this compartment may be actively involved in virus entry and/or spread. The ability of astrocytes to transfer virus, without de novo viral synthesis suggests they are capable of sequestering and protecting virus and thus, they could potentially facilitate viral dissemination in the CNS.

Figure 1. Astrocytes harbor short-term HIV-1 viral reservoirs.

Virus half-life assays were performed using HIV-1 BaL and the immortalised human foetal astrocyte cell line, SVG. Cell-associated HIV-1 p24 was quantified via ELISA over a time course from 0 to 72 h. The curve of best fit is shown in black. The initial virus half-life of 1.2 h is shown via a dotted dark gray line and the slower rate of 9.5 h is shown via a dotted light gray line. Dot points and error bars denote the mean and standard deviation, respectively. Data is representative of three independent experiments.

Figure 2. HIV-1 colocalizes with CD81-lined vesicle compartments in astrocytes.

(A) SVG astrocyte cells were infected with an EGFP content-labelled HIV-1 YU2 virus (green) and samples were harvested at 135 mins post-infection. Cells were stained with the vesicle/endosomal markers CD81, EEA1, CD63 or CD107b (red) and nuclei counterstained with DAPI (blue). Panels represent the individual fluorescent channels with the overlay (OL) on the far right. Scale bars are 10 μm. Images are representative of multiple fields of view and three independent experiments. (B) Quantification of colocalization of vesicle/endosomal markers with HIV-1 using IMARIS software. Bar graphs and error bars represent the mean and standard deviation, respectively. Data are a compilation of multiple fields of view and three independent experiments.

Figure 3. CD81-HIV-1 association is maintained when CD81 expression is reduced.

(A) CD81 expression levels of the parental SVG cell line, SVG-scramble (cells treated with shRNA-scramble) and SVG-lowCD81 (cells treated with shRNA-CD81) cells as determined via FACS analysis. The data are expressed as mean values and error bars represent standard deviation. (B) SVG-scramble and SVG-CD81 cells were infected with an EGFP content-labelled HIV-1 YU2 virus (green) and samples were harvested at 135 mins post-infection. Cells were stained with the vesicle marker CD81 (red). Panels represent the individual fluorescent channels with the overlay (OL) on the far right. Scale bars are 10 μm. Images are representative of multiple fields of view and three independent experiments. (C) Quantification of colocalization of CD81 with HIV-1 using IMARIS software. The data are expressed as mean values and error bars represent standard deviation. Data are a compilation of multiple fields of view and three independent experiments. P values were calculated using a parametric unpaired t test; ****, P < 0.0001.

Figure 4. Astrocytes are capable of trans-infection.

FACS analysis of EGFP expression in JLTRG cells following co-culture with SVG cells loaded with virus under different conditions (37°C, 37°C followed by trypsin treatment, or 4°C). Media treated cells were included as a negative control for background fluorescence in the JLTRG cells. The data are expressed as mean values and error bars represent standard deviation. Data are a compilation of three independent experiments. P values were calculated using a parametric unpaired t test; ***, P = 0.0005; **, P = 0.001.