N-Linked glycans on dengue viruses grown in mammalian and insect cells

Abstract

This study compared the ability of mosquito and mammalian cell-derived dengue virus (DENV) to infect human dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN)-expressing cells and characterized the structure of envelope (E) protein N-linked glycans on DENV derived from the two cell types. DENVs derived from both cell types were equally effective at infecting DC-SIGN-expressing human monocytes and dendritic cells. The N-linked glycans on mosquito cellderived virus were a mix of high-mannose and paucimannose glycans. In virus derived from mammalian cells, the N-linked glycans were a mix of high-mannose and complex glycans. These results indicate that N-linked glycans are incompletely processed during DENV egress from cells, resulting in high-mannose glycans on viruses derived from both cell types. Studies with full-length and truncated E protein demonstrated that incomplete processing was most likely a result of the poor accessibility of glycans on the membrane-anchored protein. INTRODUCTIONDengue viruses (DENVs) are enveloped, positive-sense RNA viruses of the genus Flavivirus that are transmitted via the bite of Aedes mosquitoes. Each year, over 2.5 billion people are at risk of contracting dengue, 100 million people develop symptomatic infections and up to 2.5 % of dengue haemorrhagic fever (DHF) patients die (Gubler, 2002;Gubler & Clark, 1995; WHO, 2009). Despite the public health importance of dengue, the cell biology of DENV is poorly understood. Vector-borne viruses such as DENV must productively infect cells of both arthropod and mammalian origin. As the posttranslational protein-processing machinery is different in insect and mammalian cells, the structure of glycoproteins produced in the two hosts may be different. Recent work with a wide variety of viruses has shown that protein glycosylation can influence viral virulence (reviewed by Vigerust & Shepherd, 2007). Recent studies with alphaviruses, which are also transmitted by arthropod vectors, have demonstrated that membraneprotein N-linked oligosaccharides are differentially processed by enzymes in insect and mammalian cells (Shabman et al., 2008). Structural differences in the glycans derived from insect and mammalian cells influence the ability of the viruses to infect target cells (Klimstra et al., 2003;Shabman et al., 2007Shabman et al., , 2008. In this study, we characterized the N-linked glycans on the envelope protein of DENVs grown in different cell types and assessed the functional consequences of these differences.The DENV particle is made up of three structural proteins: envelope (E), membrane (M) and capsid (C) (Chambers et al., 1990b;Kuhn et al., 2002). The E protein is the major membrane glycoprotein on the surface of the virion, responsible for virus attachment and fusion (Chambers et al., 1990b). Human dendritic cells (DCs) are a target of DENV infection (Ho et al., 2001;Marovich et al., 2001;Wu et al., 2000). Infection of DCs is mediated by the binding of DENV to DC-specific ICAM3-grabbing non-integrin (DC-SIGN), a C-type lec...

Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A

“…The presence of glycosylation at Asn-67 is a unique signature for dengue virus among the flaviviruses and is conserved in all the strains of the four dengue serotypes (36). The cryoelectron microscopy model of dengue virus in complex with DC-SIGN, a C-type lectin known to mediate dengue virus attachment to dendritic cells, suggested the N-glycan at Asn-67 on the E protein as a receptor binding site, and the interaction of DC-SIGN with dengue virus glycans has been corroborated biochemically (37,38). The E protein N-glycans have also been reported as ligands for the carbohydrate recognition domain of the mannose receptor (39).…”

Post-translational regulation and modifications of flavivirus structural proteins

“…A previous study investigating all four serotypes of DENV failed to show the same heterogeneous population of singly and doubly glycosylated E in untreated DENV-2 samples (Hacker et al, 2009). This report did, however, reveal that, for viruses grown in Vero cells, endoH digestion of DENV-1, -3 and -4 E protein removed glycosylation at one site only, whereas endoH digestion of DENV-2 resulted in a mixed population of E protein with no glycosylation or glycosylation at one site.…”

Broad Antiviral Activity of Carbohydrate-Binding Agents Against Dengue Virus Infection

“…Johnson et al postulated that DENV-1 and DENV-3 have both sites glycosylated and that DENV-2 and DENV-4 have only one N-glycan at Asn-67 [100]. In contrast, a study comparing the number of glycans in multiple isolates of DENV belonging to all four serotypes led to the consensus that all DENV strains have two N-glycans on the E-protein [101]. However, mutant DENV lacking the glycosylation at Asn153 can replicate in mammalian and insect cells, indicating that this glycosylation is not essential for viral replication [96,102].…”

“…Virions produced in the mosquito vector and human host may have structurally different N-linked glycans, because the glycosylation patterns are fundamentally different [101,111]. N-glycosylation in mammalian cells is often of the complex-type because a lot of different processing enzymes could add a diversity of monosaccharides.…”