5-HT2 receptors facilitate JC polyomavirus entry

Abstract

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT2 receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT2 receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT2 receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.

Fig 1

5-HT_2AR, 5-HT_2BR, and 5-HT_2CR overexpression in HEK293A cells significantly increases susceptibility to JCPyV but not to SV40 infection. (A) HEK293A cells were transfected for 24 h with 14 different isoforms of 5-HTR cloned in the pcDNA 3.1 vector. CTL, control. (B) HEK293A cells were stably transfected with YFP, 5-HT_2AR–YFP, 5-HT_2BR–YFP, and 5-HT_2CR–YFP and sorted for a population of cells uniformly expressing either receptor. The cells were infected with JCPyV or with SV40 at 37°C for 1 h. Samples were fixed and stained for either JCPyV TAg or SV40 TAg 48 h postinfection. Staining was detected using anti-mouse Alexa Fluor 488 antibody (A) and with Alexa Fluor 594 (B and C). The cells were analyzed by epifluorescence microscopy, and cells expressing nuclear TAg were scored as infected cells. Data represent the average number of infected cells in a visual field of view at a magnification of ×40 (A) or ×20 (B and C) for five fields of view for triplicate samples. The bars represent the averages of three independent experiments. Error bars indicate standard deviations (SD). Values that are significantly different (P < 0.05) from the value for the control (CTL or YFP) are indicated by an asterisk.

Fig 2

Biotinylated PSL (Polyporus squamosus lectin) binds equally to HEK293A cells stably expressing YFP or 5-HT2 receptors. (A) Cells were incubated with PSL for 2 h. Binding was detected by incubation with streptavidin labeled with Alexa Fluor 633 (streptavidin-633) for 1 h. Cells were fixed in 1% PFA, and binding was analyzed by flow cytometry. Cell counts are shown as a percentage of the maximum number of cells. (B) The histogram shows the average values of the mean fluorescence intensities (MFI) of three independent experiments; each replicate represents 10,000 events. Error bars indicate SD.

Fig 3

JCPyV-633 binds equally to HEK293A cells stably expressing 5-HT2 receptors compared to the control. (A) Purified JCPyV labeled with Alexa Fluor 633 was incubated with cells on ice for 2 h. Cells were fixed in 1% PFA, and binding was tested by flow cytometry. (B) The histogram shows the average values of the mean fluorescence intensity (MFIs) of three independent experiments; each replicate represents 10,000 events. Error bars indicate SD.

Fig 4

Pretreatment of JCPyV with LSTc significantly inhibits infection. JCPyV was preincubated on ice for 1 h with either LSTb or LSTc. The complexes were then added to cells at 4°C for 1 h. Cells were fixed and stained for TAg 48 h postinfection (PI). Data are the average numbers of infected cells in a field of view at a magnification of ×20 for five fields of view for triplicate samples. The bars are the averages of two independent experiments. Error bars indicate SD. *, P < 0.05.

Fig 5

JCPyV entry is more efficient in cells expressing 5-HT_2AR–YFP, 5-HT_2BR–YFP, and 5-HT_2CR–YFP, and transferrin entry is not enhanced in 5-HT₂R-expressing cells. (A and B) Cells were prechilled at 4°C for 30 min, and then JCPyV-405 was incubated with cells on ice for 1 h. Virus entry was analyzed at 0 and 2 h PI by confocal microscopy before and after the addition of trypan blue. (A) Representative confocal micrograph images recorded at 2 h PI are shown. (B) Quantitation of images recorded at 0 and 2 h PI is shown. (C) Cells were incubated with transferrin-405 at 37°C for 1 h, transferrin entry was analyzed, and quantitation is shown. Z stacks were recorded at a magnification of ×63, and fluorescence intensity was measured for at least five cells per visual field using Image J software. Data are expressed as a percentage of protected fluorescence, and the values are averages of three independent experiments. Error bars represent the standard errors of the means (SEM). *, P < 0.05.

Fig 6

Treatment of cells expressing 5-HT₂ receptors with chlorpromazine (CPZ) significantly inhibits JCPyV infection. Cells were pretreated with 30 μM CPZ for 1 h and then infected in the presence of CPZ at 37°C for 3 h. Complete medium was added with JCPyV antiserum to neutralized, not internalized, virus. Cells were fixed and stained for TAg at 48 h PI. Data are the average numbers of infected cells in a field of view at a magnification of ×20 for five fields of view for triplicate samples. The bars represent the averages of three independent experiments. Error bars indicate SEM. *, P < 0.05.