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

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 70 Ship Street, Providence, RI 02903, USA.
Journal of Virology (Impact Factor: 4.44). 10/2010; 84(19):9677-84. DOI: 10.1128/JVI.00978-10
Source: PubMed


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-hydroxytryptamine2A receptor (5-HT2AR). The 5-HT2AR 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-HT2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT2AR-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-HT2AR 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-HT2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface
expression of 5-HT2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT2AR 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-HT2AR-expressing cells. These data affirm the importance of 5-HT2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT2AR.

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    • "Although the cognate receptor/s for MBL are not known with certainty, potential receptors for MBL include CD93 (C1qRp), CD35 (complement receptor 1, CR1), calreticulin (cC1qR), CD91, and the binding protein for the globular domain of C1q (gC1qBP/gC1qR/C1QBP) [4], [28], all of which have proven and/or predicted N-linked glycopeptides [66], [67]. Therefore, we tested whether reduction of N-linked glycoprotein receptor expression [68] by chemical (tunicamycin) or genetic (HEK293S GnTI− cells) approaches could reduce MBL-mediated HIV-EBOV GP infection. Tunicamycin, which completely inhibits N-linked glycosylation, abrogated MBL-mediated pseudotyped EBOV infection to a greater extent than other downstream N-glycan-trimming agents such as deoxynojirimycin and swainsonine which leave residual mannose or GlcNAc residues (Figure 3E). "
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    ABSTRACT: Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.
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    • "Glycosylation alters many properties of membrane proteins, such as targeting, folding, affinity, and cell surface expression 33-35. Furthermore, the carbohydrate structures of proteins on the cell surface have been shown to correlate with disease, for example, the N-linked oligosaccharides of the 5-HT2A receptor are important for JC virus infection in PML 36. We examined that the expression of β4 markedly increased in the neonatal PD transgenic mice. "
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