Structural Plasticity of the Semliki Forest Virus Glycome upon Interspecies Transmission

Abstract

Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus.

Targeted release and fractionation reveal glucuronylated and sulphated N- and O-glycans in larvae of dipteran insects

“…Also, a number of lectin-based studies of mosquitoes have been published [38-40], whereas actual mass spectrometric data on glycoproteins from mosquitoes or mosquito cell lines is limited [41, 42]. Certainly, a comprehensive analysis of the total glycomic complexity using unbiased high-sensitivity mass spectrometric methods has not been previously reported.…”

The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases

“…Key to this were the definition of a relevant core α1,3-fucosyltransferase (FucTA) in D. melanogaster , RNAi knock-down of FucTA in an insect neural cell line, overexpression of FucTA in D. melanogaster embyros and the proof that the anti-HRP-defective nac mutant was due to a point mutation in the Golgi GDP-Fuc transporter (Fabini et al, 2001, Rendić et al, 2006, Rendic et al, 2010, Geisler et al, 2012). In comparison to the fruit fly, there has been no data to date regarding anti-HRP epitopes in mosquitoes; previous studies were limited primarily to lectin blots and histochemistry with actual mass spectrometric N-glycan analyses performed only on a couple of glycoproteins from mosquitoes or mosquito cell lines (Wilkins and Billingsley, 2001, Rudin and Hecker, 1989, Andrews et al, 1997, Li and Li, 2005, Crispin et al, 2014). Thus, our recent paper on the analysis of neutral and anionic glycans from An.…”

Targeting host-derived glycans on enveloped viruses for antibody-based vaccine design

“…These are typically dominated by paucimannose structures but shift to human complex-type glycosylation as soon as viral production is established in the new host. An illustration of this antigenic shift has been revealed by the mass spectrometric analysis of Semliki Forest virus glycans derived from mammalian and insect cells [5]. Similarly, Dengue virus (DENV) is transmitted to humans via mosquitoes and therefore DENV Env produced in insect cells contains mostly oligomannose and paucimannose structures whereas virus Env produced in primary dendritic cells contains complex sugars [6, 7].…”