Serotonin receptor 2A blocker (risperidone) has no effect on human polyomavirus JC infection of primary human fetal glial cells

Abstract

A recent report demonstrated that JC virus (JCV) employs serotonin receptor 2A (5HT(2A)R) to infect the glial cells. To assess the ability of a potent 5HT(2A)R blocker, risperidone, to inhibit JCV infection, the authors treated primary human fetal glial (PHFG) cells in vitro with risperidone for 24 h and inoculated with JCV(Mad1). There was no significant difference in JCV genome copies or mRNA transcripts and protein expression in treatment-naive and risperidone-treated PHFG cells. These data indicate that risperidone does not inhibit JCV(Mad1) attachment, internalisation, and replication in PHFG cells, and 5HT(2A)R blockers may not be effective in treating progressive multifocal leukoencephalopathy (PML).

Fig. 1. Risperidone is non-toxic to PHFG cells

The cell proliferation and viability of PHFG cells grown in a 96-well plate either with media alone (untreated) or in the continuous presence of risperidone 50 or 200 ng/mL was determined by the CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit on days 0, 5, 10, 15 after-incubation with risperidone. The absorbance was measured at 490 nm and expressed as percentage of untreated-treated cells. Data are representative of mean of six independent samples in each group at each time point and error bars represent standard deviations.

Fig. 2. Risperidone has no effect in JCV infection of PHFG cells

PHFG cells were treated with risperidone at a concentration of 12.5, 50, or 200 ng/mL for 24 hr and inoculated with JCV for 2 hr and harvested at the indicated time points for DNA or RNA extraction. JCV TAg (Fig 2A) and VP-1 (Fig. 2B) genome or TAg (Fig. 2C) and VP-1 (Fig. 2D) transcripts were amplified and quantitated by real-time PCR or real-time RT-PCR. Data are representative of duplicate samples assayed in three independent experiments. Error bars represent standard deviations.

Fig. 3. Risperidone does not inhibit JCV T antigen protein expression in PHFG cells

JCV-infected and naive PHFG cells were harvested at the indicated time points and JCV T antigen protein was detected in JCV-infected HBMVE cells by immunoprecipitation and Western blotting.

Fig. 4. Transfection of HEK-293 cells with 5-HT_2AR pDNA construct

(A) HEK-293 cells at 72 hr after transfection expressed 5-HT_2AR transcripts but risperidone treatment had no negative effect on mRNA expression. HEK-293 cells untransfected (lane 1); transfected and untreated (lane 2); transfected and treated with 12.5 (lane 3); 50 (lane 4), 200 (lane 5); and 1,000 (lane 6) ng/mL of risperidone; transfected and untreated HEK-293 cells without reverse transcriptase (negative control) (lane 7); and 5-HT_2AR pDNA (positive control) (lane 8). (B) Transfected HEK-293 cells demonstrated 5-HT_2AR immunoreactivity (green), and nuclei were stained with bisbenzidine (blue). (C) Control empty vector transfected cells. (D) Ketanserin binding is inhibited by risperidone in a dose dependent manner. [Ethylene-³H]-labeled ketanserin hydrochloride with or without risperidone was co-incubated with HEK293 membrane protein preparation and the radioactivity was measured. Data represents the mean DPM with standard deviation.