CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca(2+)-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.

FIG. 1.

Interaction of HIV-1 with astrocytes. Human primary astrocytes (A) and U87.CD4.CCR5 cells (B) were plated in a six-well plate at a density of 5 × 10⁵ cells/well 1 day before infection. On the day of infection, the cells were incubated with anti-CD4 Leu3A antibody (20 μg/ml), anti-GalCer antibody (20 μg/ml), anti-CCR5 antibody (20 μg/ml), or 500 ng of MIP-1β/ml for 3 h. The cells were then infected with 100 ng of gag^p24 HIV-Luc reporter viruses pseudotyped with YU-2 envelope protein and allowed to grow for 48 h before harvest for the Luc activity assay. Compared to human primary astrocytes, only one-tenth of the lysates from U87.CD4.CCR5 cells was used in the Luc assay. (C) For HIV-1 gp120 binding, human primary astrocytes were incubated with ¹²⁵I-labeled gp120 protein (•) or ¹²⁵I-labeled BSA (○) at the indicated concentrations on ice for 30 min. Unbound proteins were removed, followed by extensive washes with prechilled regular cell culture medium. The cells were then harvested and processed for measuring gp120 binding by using a gamma scintillation counter

FIG. 2.

Expression of hMR in human primary astrocytes and in astrocytes of human normal brain tissues. Human primary astrocytes (A to C), human normal brain tissues (D to G), and U87.MG cells (H to J) were stained with primary antibodies against astrocyte-specific GFAP and hMR, followed by appropriate secondary antibodies. Expression of hMR and GFAP was determined by confocal microscopy. (A, E and H) GFAP staining; (B, F, and I) MR staining; (D) DAPI counterstaining for nuclei; (C and J) colocalization of GFAP and MR staining; (G) colocalization of GFAP, MR, and nuclei in brain tissues. Parallel staining containing isotype-matched IgGs was also performed, and no nonspecific staining was observed.

FIG. 3.

Expression and functionality of hMR in U87.MR cells. (A) hMR expression by Western blot analysis. Cell lysates (20 μg of protein) from U87.MG cells, U87.MR clones 1-8, 2-8, and 3-9, U138.MG cells, U373.MG cells, and human primary astrocytes (1⁰ As) were separated by electrophoresis on SDS-6% PAGE and blotted onto Hybon-P membrane. hMR expression was detected by using goat anti-hMR serum (1:400) as the primary antibody and horseradish peroxidase-conjugated donkey anti-goat secondary antibody (1:2,000). (B) hMR expression in human primary astrocytes as determined by FACS. Human primary astrocytes, as well as U87.MG and U87.MR (clone 2-8) cells, were double immunofluorescence stained and then analyzed by FACS. Parallel isotype-matched IgG staining was performed on all cells; no nonspecific staining was observed, and only isotype IgG staining on human primary astrocytes was shown as a representative (upper left quadrate in panel B). (C) hMR expression in U87.MR cells by confocal microscopy. Similar double immunofluorescence staining was performed on U87.MR cells, and then the cells were observed under a Zeiss confocal microscope. Top, GFAP staining; bottom, hMR staining.

FIG. 4.

Functionality of hMR in U87.MR cells. U87.MG cells (A to D) and U87.MR cells (E to L) were incubated at 37°C with FITC-mBSA at concentrations of 0 (A and E), 0.5 (B and F), 1 (C and G), 2 (D and H), and 5 (I to L) μg/ml in the absence of competitors (A to I) or in the presence of 3 mg of d-galactose/ml (J), 3 mg of yeast mannan/ml (K), or 3 mg of d-mannose/ml (L). Unbound FITC-mBSA was then removed from the cell culture medium by extensive washing with prechilled regular cell culture medium, and the cells were fixed in 4% paraformaldehyde. hMR function was determined by FACS analysis for the internalization of FITC-mBSA into the cells.

FIG. 5.

hMR expression and HIV-1 infection. (A) HIV-1 infection of hMR-expressing U87.MR cells. U87.MG cells, U87.MR cells, U87.CD4.CXCR4 cells, and U87.CD4.CCR5 cells were infected with 100 ng of gag^p24 HIV-GFP reporter viruses pseudotyped with VSV-G envelope protein (closed bar), YU-2 envelope protein (dotted bar), HXB2 envelope protein (hatched bar), or pcDNA3 control (no env, open bar) at 37°C for 2 h in the presence of 8 μg of Polybrene/ml. After 2 h, the viruses were removed, and the cells were washed with regular cell culture medium. The cells were allowed to grow for 48 h and then harvested for the HIV-1 infection assay and FACS analysis. HIV-1 infection was expressed as the percentage of GFP-positive cells. (B) Effects of virus input and hMR density on HIV-1 infection. Human primary astrocytes were labeled with FITC-mBSA, sorted out by FACS for the top 5% higher hMR-expressing cells, and infected with 100 ng (open bar) or 400 ng (closed bar) gag^p24 HIV-GFP reporter viruses pseudotyped with HIV-1 YU-2 envelope protein. Presorted human primary astrocytes and U87.MR cells were also included. 1⁰ As, human primary astrocytes; 1⁰ As*, the top 5% higher hMR-expressing human primary astrocytes.

FIG. 6.

Neutralization of HIV-1 infection of human primary astrocytes by goat anti-human mannose receptor antibody. Human primary astrocytes (A) and PBMCs (B) were infected with 100 ng of gag^p24 HIV-1 NL4-3 viruses at 37°C for 2 h in the presence of goat preimmune serum (1:50, ○), goat anti-hMR serum (1:50, •), anti-human CD4 monoclonal antibody Leu3A (20 μg/ml, ▵), isotype-matched IgG control antibody (10 μg/ml, ⋄), or recombinant CD4 protein (10 μg/ml, ▴). HIV-1 NL4-3 viruses were prepared by transfection of pNL4-3 plasmid DNA in 293T cells similarly to the envelope protein-pseudotyped HIV reporter viruses. All infections were performed in the presence of 8 μg of Polybrene/ml. Fresh antibodies were added every other day when the cell culture supernatants were collected. HIV-1 replication was monitored by measuring p24 production in the culture supernatants by using a p24 ELISA kit according to the manufacturer's instructions. (C) Inhibition of HIV-1 replication in human primary astrocytes by MR siRNA. Retroviruses carrying MR siRNA were prepared by transfection of pMSCV-puro MR siRNA DNA in ampho-phoenix cells and used to transduce human primary astrocytes. Transduced cells were then infected with HIV-1 NL4-3 viruses, as well as for hMR expression by Western blot analysis (insert). At day 7 after infection, the culture supernatant was collected for HIV-1 replication assay by a p24 ELISA kit. MSCV-puro viruses containing no siRNA DNA and Luc siRNA DNA targeting the luciferase gene were also included as controls. Puromycin gene expression was used to determine the virus titers and transduction efficiency, which were comparable. <, MR protein.

FIG. 7.

Interaction of HIV-1 viruses and gp120 with hMR. (A) Binding of HIV-1 viruses to hMR. U87.MG cells (□) and U87.MR cells () grown in a 96-well plate were prechilled on ice for 30 min, followed by the addition of 100 ng of gag^p24 HIV-GFP viruses pseudotyped with VSV-G, HXB2, YU-2, and 89.6 envelope proteins, or without envelope protein (no env) were then added. The cells were allowed to incubate with the viruses for an additional 30 min. After 30 min, the viruses were removed and the cells were washed extensively with prechilled regular culture medium. The cells were then lysed with a 1% NP-40-containing buffer, and the lysates were processed to determine HIV-1 binding by using the p24 ELISA kit. (B) Inhibition of HIV-1 binding to human mannose receptor by hMR antibody and ligand antagonists. U87.MR cells were incubated with HIV-GFP viruses pseudotyped with HXB2 envelope protein (□), or VSV-G envelope protein (), as stated above, in the presence of goat preimmune serum (1:50), goat anti-hMR serum (1:50), and 3 mg of yeast mannan or d-mannose/ml. HIV-1 binding was determined as described above. (C) Direct binding of HIV-1 gp120 proteins to hMR. U87.MR cells were incubated with ¹²⁵I-labeled gp120 protein (□) or ¹²⁵I-labeled nonglycosylated gp120 protein (⋄) at concentrations as indicated, in the presence of 20 mM EGTA (○), on ice for 30 min. Unbound proteins were removed, followed by an extensive wash with prechilled regular cell culture medium. The cells were then harvested and processed for measuring gp120 binding by using a gamma scintillation counter.

FIG. 8.

hMR-mediated endocytosis of HIV-1 viruses. U87.MR cells were infected with 100 ng of gag^p24 HIV-Luc reporter viruses pseudotyped with HXB2 envelope protein (A) or VSG-G envelope protein (B) at 37°C for 2 h in the presence of 10 μM NH₄Cl, 150 nM bafilomycin A1 (Baf.A1), or 5 μg of brefeldin A (Bre.A)/ml. After 2 h, the viruses were removed, and the cells were washed with the regular cell culture medium. The cells were allowed to grow for 48 h and then harvested for measuring Luc enzymatic activity for HIV-1 infection. (C) U87.CD4.CXCR4 cells were similarly infected with HIV-Luc reporter viruses pseudotyped with HXB2 (□) or VSV-G () envelope protein in the presence of different reagents, and HIV-1 infection was determined as described above. For VSV-G pseudotyped virus infection, only 1/20 of the lysates was used for Luc enzymatic activity assay, whereas only one-fifth of the lysates was used for HXB2 pseudotyped HIV-1 infection of U87.CD4.CXCR4 cells.

FIG. 9.

Electron transmission microscopy of hMR-mediated HIV-1 endocytosis. U87.MR cells (A) or human primary astrocytes (B) were prechilled on ice for 30 min. After 30 min, HIV-GFP viruses pseudotyped with YU-2 envelope protein were added, followed by incubation with the cells for additional 30 min. The cells were then transferred back to 37°C and kept at 37°C for 0, 5, 10, 30, and 60 min. The locations of viruses on or inside U87.MR cells were visualized by electron microscopy. Arrowheads, viruses bound to the membrane; solid arrows, viruses fused with the membrane; open arrows, viruses located inside endosomes. Magnifications for U87.MR cells are 48,000 for 0 min, 34,000 for 5 min, 36,500 for 10 min, 30,000 for 30 min, 36,500 for the 30-min inset, and 22,000 for 60 min; magnifications for the human primary astrocytes are 22,000 for 30 min and 50,000 for the 30-min inset.