5-HT2 Receptors Facilitate JC Polyomavirus Entry

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-HT_2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT_2AR 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.     

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.     

Irisolidone, an isoflavone metabolite, represses JC virus gene expression via inhibition of Sp1 binding in human glial cells

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease that results from an oligodendrocyte infection caused by the JC virus. Therefore, inhibiting the expression of JC virus is important for preventing and/or treating PML. This study found that irisolidone, an isoflavone metabolite, significantly inhibited the JC virus expression in primary cultured human astrocytes and glial cell lines. Studies examining the underlying mechanism revealed that a mutation of the Sp1 binding site downstream of the TATA box (Sp1-II) dramatically diminished the inhibitory activity of irisolidone. In addition, an irisolidone treatment repressed Sp1 binding to Sp1-II site, which is important for the basal JC virus promoter activity. The results suggest that the inhibitory effect of irisolidone against the JC virus may be attributed at least in part to the suppression of Sp1 binding to the JC virus promoter region. Therefore, the inhibition of the JC virus expression by irisolidone might provide therapeutic potential for PML caused by the JC virus.     

JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.     

Tropism and Induction of Cytokines in Human Embryonic-Stem Cells-Derived Neural Progenitors upon Inoculation with Highly- Pathogenic Avian H5N1 Influenza Virus

Central nervous system (CNS) dysfunction caused by neurovirulent influenza viruses is a dreaded complication of infection, and may play a role in some neurodegenerative conditions, such as Parkinson-like diseases and encephalitis lethargica. Although CNS infection by highly pathogenic H5N1 virus has been demonstrated, it is unknown whether H5N1 infects neural progenitor cells, nor whether such infection plays a role in the neuroinflammation and neurodegeneration. To pursue this question, we infected human neural progenitor cells (hNPCs) differentiated from human embryonic stem cells in vitro with H5N1 virus, and studied the resulting cytopathology, cytokine expression, and genes involved in the differentiation. Human embryonic stem cells (BG01) were maintained and differentiated into the neural progenitors, and then infected by H5N1 virus (A/Chicken/Thailand/CUK2/04) at a multiplicity of infection of 1. At 6, 24, 48, and 72 hours post-infection (hpi), cytopathic effects were observed. Then cells were characterized by immunofluorescence and electron microscopy, supernatants quantified for virus titers, and sampled cells studied for candidate genes.The hNPCs were susceptible to H5N1 virus infection as determined by morphological observation and immunofluorescence. The infection was characterized by a significant up-regulation of TNF-α gene expression, while expressions of IFN-α2, IFN-β1, IFN-γ and IL-6 remained unchanged compared to mock-infected controls. Moreover, H5N1 infection did not appear to alter expression of neuronal and astrocytic markers of hNPCs, such as β-III tubulin and GFAP, respectively. The results indicate that hNPCs support H5N1 virus infection and may play a role in the neuroinflammation during acute viral encephalitis.     

JC virus agnoprotein inhibits in vitro differentiation of oligodendrocytes and promotes apoptosis

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.     

Adaptive Mutations in the JC Virus Protein Capsid Are Associated with Progressive Multifocal Leukoencephalopathy (PML)

PML is a progressive and mostly fatal demyelinating disease caused by JC virus infection and destruction of infected oligodendrocytes in multiple brain foci of susceptible individuals. While JC virus is highly prevalent in the human population, PML is a rare disease that exclusively afflicts only a small percentage of immunocompromised individuals including those affected by HIV (AIDS) or immunosuppressive drugs. Viral- and/or host-specific factors, and not simply immune status, must be at play to account for the very large discrepancy between viral prevalence and low disease incidence. Here, we show that several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. We provide strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, we performed a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, we showed that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, we go on to demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).     

Transient expression of serotonin 5‐HT4 receptors in the mouse developing thalamocortical projections

The serotonin 5‐HT₄ receptor (5‐HT₄‐R) is an unusually complex G‐protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5‐HT₄‐Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT‐PCR. The developing thalamocortical projections transiently expressed 5‐HT₄‐Rs in the embryonic brain and the 5‐HT₄‐R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13–17, the forebrain mRNA levels of the 5‐HT_4(a)‐R and 5‐HT_4(b)‐R splice variants increased nine‐ and fivefold, respectively, whereas the levels of the 5‐HT_4(e)‐R and 5‐HT_4(f)‐R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5‐HT₄‐R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination

Background

This study aims to differentiate human induced pluripotent stem cells (hiPSCs) into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats.

Methodology/Principal Findings

We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials.

Conclusions/Significance

These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.     

Cannabis Users Show Enhanced Expression of CB<Subscript>1</Subscript>-5HT<Subscript>2A</Subscript> Receptor Heteromers in Olfactory Neuroepithelium Cells

Cannabinoid CB1 receptors (CB₁R) and serotonergic 2A receptors (5HT_2AR) form heteromers in the brain of mice where they mediate the cognitive deficits produced by delta-9-tetrahydrocannabinol. However, it is still unknown whether the expression of this heterodimer is modulated by chronic cannabis use in humans. In this study, we investigated the expression levels and functionality of CB₁R-5HT_2AR heteromers in human olfactory neuroepithelium (ON) cells of cannabis users and control subjects, and determined their molecular characteristics through adenylate cyclase and the ERK 1/2 pathway signaling studies. We also assessed whether heteromer expression levels correlated with cannabis consumption and cognitive performance in neuropsychological tests. ON cells from controls and cannabis users expressed neuronal markers such as βIII-tubulin and nestin, displayed similar expression levels of genes related to cellular self-renewal, stem cell differentiation, and generation of neural crest cells, and showed comparable Na⁺ currents in patch clamp recordings. Interestingly, CB₁R-5HT_2AR heteromer expression was significantly increased in cannabis users and positively correlated with the amount of cannabis consumed, and negatively with age of onset of cannabis use. In addition, a negative correlation was found between heteromer expression levels and attention and working memory performance in cannabis users and control subjects. Our findings suggest that cannabis consumption regulates the formation of CB₁R-5HT_2AR heteromers, and may have a key role in cognitive processing. These heterodimers could be potential new targets to develop treatment alternatives for cognitive impairments.     

低氧通过上调Wnt5a促进人胚胎干细胞向生血内皮细胞分化

胚胎的早期发育是在低氧条件下进行的,低氧环境在胚胎血管发生及造血发育中起着重要作用,低氧条件能促进胚胎干细胞在体外向内皮细胞和造血细胞的分化,但低氧条件对造血细胞产生的具体作用及相应机制尚不清楚．本研究利用人Es细胞向造血祖细胞定向分化体系,发现低氧环境可以促进CD31＋TIE2＋造血内皮祖细胞的产生,2天后造血内皮祖细胞开始表达终生造血基因．进一步研究发现,低氧能够上调Wnt5a的表达,干涉Wnt5a能够抑制低氧环境对生血内皮细胞分化的促进作用．在正常氧环境下加入Wnt5a产生促进生血内皮细胞分化的效应,该效应与低氧处理促进生血内皮细胞产生的作用相似．本研究首次证明了低氧通过上调Wnt5a的表达促进人Es细胞向生血内皮细胞的分化,为ES细胞向生血内皮细胞的分化及造血祖细胞分化的研究提供了新的线索．     

Ameliorating Effect of Osteopontin on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis of Human Oligodendrocyte Progenitor Cells

Recently our group used oligodendrocyte progenitor cells (OPCs) as appropriate model cells to pinpoint the mechanism of the progress of neurodegenerative disorders. In the present study, we focused on the therapeutic role of osteopontin (OPN), a secreted glycosylated phosphoprotein, involved in a number of physiological events including bone formation and remodeling, immune responses, and tumor progression. Protective role of OPN, as a negative regulator of tumorigenesis, has already been clarified. Human embryonic stem cell-derived OPCs were pretreated with OPN before induction of apoptosis by H₂O₂. Data indicated that OPN prohibited cell death and enhanced OPC viability. This effect is achieved through reduction of apoptosis and induction of anti-apoptosis markers. In addition OPN induces expression of several integrin subunits, responsible for OPN interaction. Notably, our findings showed that expression of αV β1/β3/β5 and β8 integrins increased in response to OPN, while treatment with H₂O₂ down-regulated αV β1/β5 and β8 integrins expression significantly. In conclusion, OPN may act via αV integrin signaling and trigger suppression of P53-dependent apoptotic cascades. Therefore OPN therapy may be considered as a feasible process to prevent progress of neurodegenerative diseases in human.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

What is the potential of oligodendrocyte progenitor cells to successfully treat human spinal cord injury?

Background  

Spinal cord injury is a serious and debilitating condition, affecting millions of people worldwide. Long seen as a permanent injury, recent advances in stem cell research have brought closer the possibility of repairing the spinal cord. One such approach involves injecting oligodendrocyte progenitor cells, derived from human embryonic stem cells, into the injured spinal cord in the hope that they will initiate repair. A phase I clinical trial of this therapy was started in mid 2010 and is currently underway.     

Human stem cell-derived neurons: a system to study human tau function and dysfunction

Background

Intracellular filamentous deposits containing microtubule-associated protein tau constitute a defining characteristic of many neurodegenerative disorders. Current experimental models to study tau pathology in vitro do not usually recapitulate the tau expression pattern characteristic of adult human brain. In this study, we have investigated whether human embryonic stem cell-derived neurons could be a good model to study human tau distribution, function and dysfunction.

Methodology/Principal Findings

Using RT-PCR, immunohistochemistry, western blotting and cell transfections we have investigated whether all 6 adult human brain tau isoforms are expressed in neurons derived from human embryonic and fetal stem cells and whether 4 repeat tau over-expression alone, or with the F3 tau repeat fragment, (amino acid 258–380 of the 2N4R tau isoform with the ΔK280 mutation) affects tau distribution. We found that the shortest 3 repeat tau isoform, similarly to human brain, is the first to be expressed during neuronal differentiation while the other 5 tau isoforms are expressed later. Over expression of tau with 4 repeats affects tau cellular distribution and the short tau F3 fragment appears to increase tau phosphorylation but this effect does not appear to be toxic for the cell.

Conclusions

Our results indicate that human embryonic stem cell-derived neurons express all 6 tau isoforms and are a good model in which to study tau physiology and pathology.     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

Role of N-Linked Glycosylation of the 5-HT2A Receptor in JC Virus Infection

JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR). The 5-HT_2AR contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT_2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT_2AR-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT_2AR to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT_2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT_2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT_2AR resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT_2AR-expressing cells. These data affirm the importance of 5-HT_2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT_2AR.The initial interaction between virus and host occurs via molecular interactions of viral attachment proteins and receptors on host cells. Therefore, receptor recognition is a critical host cell determinant and may play a key regulatory role in viral pathogenesis. The polyomavirus JC virus (JCV) is a ubiquitous human pathogen (21, 25, 32) that is initially subclinical yet establishes a persistent infection in the kidney (11). In immunosuppressed individuals JCV can become reactivated, leading to infection in the central nervous system (CNS) (13-15, 20), where the virus specifically targets glial cells, including astrocytes and the myelin-producing cells, oligodendrocytes (40, 48). JCV infection and cytolytic destruction of oligodendroglia cause the fatal disease progressive multifocal leukoencephalopathy (PML) (1, 22). The most common cause of PML is associated with human immunodeficiency virus (HIV) and AIDS (10, 23). However, in recent years PML has been reported in patients receiving immunosuppressive therapies for autoimmune diseases such as Crohn''s disease (44), multiple sclerosis (MS) (24, 26, 28, 47), systemic lupus erythematosus (5, 33), and rheumatoid arthritis (5, 19, 37). The prognosis of PML is bleak, as the disease progresses rapidly and usually proves fatal within 1 year of the onset of symptoms. While current treatment options for PML are limited (23), recent studies suggest that mirtazapine, a serotonin receptor antagonist, may be capable of slowing the progression of PML (6, 27, 45, 46).JCV has a nonenveloped, icosahedral capsid that encapsidates a circular double-stranded DNA (dsDNA) genome (39). JCV attachment to cells is mediated by an N-linked glycoprotein with either α(2,3)- or α(2,6)-linked sialic acid (16, 31), suggesting that N-linked glycosylation of cellular receptors is important for JCV infection. N-linked glycosylation is a posttranslational process by which oligosaccharides are added to asparagine residues, and this modification is important for protein processing, folding, expression, and function (43). Previous studies from our laboratory revealed that the JCV also requires the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR) to mediate JCV infection (18, 35, 38), while others report that JCV infection can occur in the absence of 5-HT_2AR (7, 8). 5-HT_2AR is a seven-transmembrane-spanning G-protein-coupled receptor that belongs to a large family of 5-HT serotonin receptors. 5-HT_2AR is abundantly expressed on cells in the brain (4), including glial cells (3), and in the kidney (4), which parallels the sites of JCV infection. N-linked glycosylation plays a key regulatory role in the function of serotonin receptors. Mutation of N-linked glycosylation sites in human 5-HT_3AR and 5-HT_5AR results in decreased expression at the plasma membrane, which is critical for receptor function (17, 34). N-linked glycosylation of murine 5-HT_3AR regulates plasma membrane targeting, ligand binding, Ca²⁺ flux, and receptor trafficking (36), suggesting that glycosylation is essential for expression and function of serotonin receptors.While previous studies have concluded that JCV utilizes an N-linked glycoprotein with α(2,3)-linked sialic acid (31) or α(2,6)-linked sialic acid (16) and 5-HT_2AR (18) to initiate infection in host cells, the mechanism(s) by which JCV engages its cellular receptors and the importance of receptor glycosylation remain unclear. 5-HT_2AR contains potential asparagine (N)-linked glycosylation sites, five of which are predicted to be expressed in the extracellular amino-terminal region, where they could be accessible to the virus (2). The goal of this study was to determine whether potential N-linked glycosylation sites expressed in 5-HT_2AR are required for JCV infection. We found that N-linked glycosylation of 5-HT_2AR is important for receptor expression but not necessary for JCV infection.