Polyomavirus JC infects human brain microvascular endothelial cells independent of serotonin receptor 2A

Abstract

Although human polyomavirus JC (JCV) is known to cause progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, the mechanism by which JCV crosses the blood-brain barrier (BBB) remains unclear. To test our hypothesis that cell-free JCV gains entry into the brain by infecting endothelial cells, we inoculated human brain microvascular endothelial (HBMVE) cells with 50 HAU (1.33+/-0.27 x 10(7) genome copies) of JCV(Mad1) and analyzed the expression of early and late viral genes and proteins by immunocytochemistry, quantitative real-time PCR (qPCR), quantitative real-time reverse transcriptase PCR (qRT-PCR) and immunoprecipitation followed by Western blotting. JCV infected and replicated efficiently in HBMVE cells and produced infectious virions several hundred fold higher than the infecting inoculum. HBMVE cells in vitro did not express serotonin receptor 2A (5HT(2A)R), and 5HT(2A)R blockers did not prevent JCV infection of HBMVE cells. Collectively, our data indicate that the productive in vitro infection of HBMVE cells by JCV is independent of 5HT(2A)R.

Fig. 1. Primary HBMVE cells in-vitro express vWF

(A) Epifluorescence image demonstrate 90% of HBMVE cells expressing vWF (green) depicted by arrows, and cells lacking vWF expression depicted by arrowheads. The percentage of vWF positive cells, were quantitated by counting the number of fluorescent cells in a total of 900 cells selected from three random fields. (B) Immunolabel control cells stained with secondary antibody only and counterstained with bisbenzidine for nuclear stain (blue) did not show green fluorescence. Experiments were performed in duplicate. Scale bars 30 μm.

Fig. 2. Epifluorescence and confocal microscopy demonstrating presence of JCV T antigen and VP-1

(A) Epifluorescence image demonstrating immunoreactivities for T antigen (red), vWF (green), and counterstaining for bisbenzidine to visualize cell nuclei (blue) in HBMVE cells infected with 50 HAU of JCV. Several HBMVE cells display strong immunoreactivity to T antigen in the cell nucleus, as indicated by the pink color (overlap of red and blue colors, arrows). HBMVE cells with moderate immunoreactivity for T antigen are indicated by the purple color (arrowheads). Note the bi-nucleation in the cell indicated by a arrowhead. (B) Immunolabel control without primary antibodies with bisbenzidine-counterstained cell nuclei. The secondary antibodies do not show significant background (green or red colors are not visible). (C) Confocal microscopy image showing four cell nuclei immunoreactive for T antigen (red). (D) The confocal image C, which additionally displays vWF immunoreactivity, strongly suggests that this infected HBMVE cell (arrow) recently underwent nuclear division or cell fusion. Compare the size of the infected HBMVE cell with the non-infected HBMVE cell (arrowhead). (E) The presence of T antigen immunoreactivity in both the nucleus (arrow) and cytoplasm (arrowheads) indicates different stages of viral replication in HBMVE cells. (F) The confocal image D, stained with anti vWF antibody (green). (G to J) JCV-infected HBMVE cells were simultaneously stained with antibodies against (G) VP-1 (green) and (H) SV-40 T antigen (red). (I) HBMVE cells expressed both VP-1 and T antigen (arrow) in their nuclei (merged image yellow color). Some JCV-infected cells only expressed T antigen in their cytoplasm (arrow in H). (J) shows triple merged image with VP-1, T antigen and nuclear staining. Scale bars for AC and I; 25 μm, and for E; 10 μm.

Fig. 3. JCV replicates in HBMVE cells

HBMVE cells grown in 35-mm plates in duplicate were infected with JCV(Mad1) for 2 hr and viral genome copies and mRNA transcripts production were monitored over a period of 20 days by qPCR and qRT-PCR, respectively. (A) Viral genome copies increased rapidly from day 3 to day 20 post-inoculation. (B) JCV TAg and VP-1 mRNA transcripts increased in parallel with viral genome copies, and (C) JCV T protein was detected in JCV-infected HBMVE cells by immunoprecipitation and Western blotting with anti-SV40 T antigen mouse mAb. (D) For comparison, HBMVE and PHFG cells in 35-mm plates were infected with 50 HAU of JCV, and JCV TAg genome was quantitated by qPCR. There was no significant difference in JCV replication in HBMVE and PHFG cells. JCV genome or mRNA transcripts were expressed as copies per μg DNA or RNA, respectively, and data are representative of two independent experiments with duplicate samples in each experiment. Error bars represent SD.

Fig. 4. JCV replication in HBMVE cells produces infectious virions

PHFG cells grown in 35-mm plates in duplicate were inoculated with day 20 post-inoculation lysates from JCV-infected HBMVE cells, and were analyzed for the expression of (A) JCV genome, and (B) JCV mRNA transcripts. JCV TAg and VP-1 mRNA transcripts increased in parallel to genome copy numbers.

Fig. 5. HBMVE cells in culture do not express serotonin receptor 2A and serotonin receptor blockers did not inhibit JCV replication in HBMVE cells

RNA and total protein were extracted from PHFG, HBMVE and HBCA cells, lanes 1, 2 and 3, respectively. (A) cDNA was synthesized and expression of 5HT_2AR was analyzed by two independent primers (table 1). Housekeeping gene GAPDH was used as a positive control. (B) Total cellular protein extract from PHFG, HBMVE and HBCA cells were analyzed for the expression of 5-HT_2AR or housekeeping protein, β-actin by Western blotting using polyclonal 5-HT_2AR and monoclonal β-actin antibodies, respectively. (C) JCV replication in HBMVE cells was quantitated by measuring JCV TAg mRNA transcript by qRT-PCR. JCV replication was expressed as percentage of control.