Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection - PubMed (original) (raw)
Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection
Manjula Kalia et al. J Virol. 2009 Dec.
Abstract
The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an approximately 56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.
Figures
FIG. 1.
The HEV ORF2 protein binds to HSPGs on Huh-7 hepatoma cells. (A) Recombinant ORF2 protein VLPs (20 μg/ml) were added to Huh-7 cells grown on coverslips for 30 min on ice. After being washed, cells were fixed and stained with anti-pORF2 and Alexa 488-conjugated anti-rabbit immunoglobulin G antibodies. Cells were also stained with DAPI and imaged at 60×. Cell nuclei can be seen in blue (DAPI), and pORF2 can be seen in green. Huh-7 cells were either left untreated (B) or treated with 5 U/ml heparinase I (C) for 1 h at 37°C. After the cells were washed, pORF2 (20 μg/ml) was added to cells for 30 min on ice. After being washed, cells were fixed and stained as described above and imaged at 20×. (D) The binding of pORF2 to Huh-7 cells with or without heparinase treatment was also tested by flow cytometry as described in Materials and Methods. The histogram shown is representative of two independent experiments. Ctl, control; Max, maximum; FL1-H, ORF2 A488.
FIG. 2.
HSPG profiles on Huh-7 and S10-3 cells. (A and B) Huh-7 cells were detached, left untreated or treated with the enzyme heparinase III (HepIII; 2.5 U/ml) for 1 h at 37°C, and stained with HSPG-specific MAb F58-10E4 or MAb F69-3G10, washed, and labeled with Alexa 488-conjugated anti-mouse secondary antibody. Cells were analyzed by flow cytometry as described in Materials and Methods. Max, maximum; FL1, anti-mouse Alexa 488. (C) Untreated or heparinase III-treated Huh-7 cells were lysed, cell lysates were separated with SDS-PAGE, and Western blotting (WB) was done with MAb F69-3G10, which recognizes the PG stub after heparinase treatment. Lane C, control cell lysate; lane H, heparinase III-treated lysate. The masses (in kDa) of the major bands seen in lane H are marked with an arrow.
FIG. 3.
Binding of pORF2 is mediated by transmembrane syndecans and not GPI-anchored glypicans. The ORF2 protein (20 μg/ml) was added to untreated (A to D) or PIPLC-treated (E to H) Huh-7 cells. Cells were costained with anti-pORF2 as described in the legend to Fig. 1 and with Alexa 647-conjugated anti-CD59 (marker of GPI-anchored proteins). After the nuclei were stained with DAPI, the images were acquired at 20×. The merged pictures (D and H) show pORF2 (green), CD59 (red), and nuclei (blue).
FIG. 4.
Expression of Syndecan-1 (Syn1) and -4 (Syn4) on Huh-7 cells and the effect of their depletion on pORF2 binding. (A and B) Huh-7 cells left untreated (lanes C) or treated with heparinase III (lanes H) were lysed and Western blotted with anti-Syndecan-1 and anti-Syndecan-4 antibodies. The Syndecan-1-specific band is significantly enhanced under the heparinase III treatment condition, whereas Syndecan-4 was equally detectable in untreated and heparinase III-treated cells. (C) Flow cytometry profile of Huh-7 cells stained with Syndecan-1 and Syndecan-4. Huh-7 cells show much higher levels of Syndecan-1 than Syndecan-4. Max, maximum; Ctl, control; FL1, anti-rabbit Alexa 488. (D) Effects of Syndecan-1 and Syndecan-4 depletions by RNA interference on pORF2 binding on Huh-7 cells. Huh-7 cells were transfected with siRNA reagents specific for Syndecan-1 and Syndecan-4 and a nonspecific control siRNA (CtSi RNA). Maximal silencing of the genes was seen at 72 h posttransfection. Binding and analysis of pORF2 were performed as described in Materials and Methods.
FIG. 5.
Effects of GAGs and chondroitin lyase on pORF2 binding. The binding of pORF2 to Huh-7 cells was tested in the absence or presence of the indicated concentrations of heparin or OS heparin (A) and OS-HS or kidney-derived HS (HS-Kidney) (B). (C) Cells were treated with chondroitin lyase (2.5 U/ml) at 37°C for 1 h, or incubated with chondroitin sulfate or dextran sulfate at the indicated concentrations and ORF2 binding was done. After being washed, the cells were fixed and stained for pORF2 as described above. The images were acquired at 20×, and intensity measurements were made as described in Materials and Methods. Ctl, control.
FIG. 6.
The sulfation inhibitor sodium chlorate reduces pORF2 binding to Huh-7 cells. Huh-7 cells were grown for 48 h in the absence or presence of the indicated concentrations of sodium chlorate. The binding of pORF2 to these cells was assessed as described above.
FIG. 7.
Effects of differentially sulfated heparins on pORF2 binding. The binding of pORF2 to Huh-7 cells was tested without any addition (A) or in the presence of de-O-sulfated heparin (20 μg/ml) (B), 2-O-desulfated heparin (20 μg/ml) (C), or 6-O-desulfated heparin (20 μg/ml) (D). (E) The binding levels were quantitated as described above and are shown in the bar graph. Ctl, control.
FIG. 8.
HSPGs are required for HEV infection of S10-3 cells. Untreated and heparinase I (5 U/ml)-treated S10-3 cells were infected with HEV virions that were produced in replicon-transfected S10-3 cells as described in Materials and Methods. Five days postinfection, RNA was prepared from mock-infected (second lane from the left) or HEV-infected cells (third and fourth lanes), and RT-PCR was performed to estimate the levels of ORF2-expressing transcripts. The pSK-E2 plasmid served as a positive control for PCR. The histone H4 RT-PCR served as a loading control for RNA. (B) S10-3 cells were either left untreated or treated with different amounts of heparinase I and infected with HEV virions. RNA was prepared, real-time RT-PCR was performed using 2 μl of first-round PCR mix, and the CT value was calculated. The pSK-E2 plasmid served as a positive control for PCR (+C). The histone H4 RT-PCR represents the loading control for RNA.
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