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, 13 (3), 1702-12

Structural Plasticity of the Semliki Forest Virus Glycome Upon Interspecies Transmission

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Structural Plasticity of the Semliki Forest Virus Glycome Upon Interspecies Transmission

Max Crispin et al. J Proteome Res.

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.

Figures

Figure 1
Figure 1
Sample preparation of SFV from BHK and C6/36 cell lines. (A) Electron cryomicroscopy image of SFV virions, purified from BHK cells, taken at −4 μm defocus. Scale bar: 50 nm. (B) SDS-PAGE analysis of SFV purified from BHK (lanes 1 and 2) and C6/36 cells (lanes 3 and 4). The gel was stained with Coomassie blue, and the protein bands correspond to the SFV structural proteins: capsid (labeled “C”; white triangle), E1 (gray triangle), and E2 (black triangle) proteins. BHK- and C6/36-derived SFV were treated with endoglycosidase F1 (Endo F1; star) in lanes 2 and 4, respectively. Endo F1 was expressed as a fusion protein with GST, as previously reported.
Figure 2
Figure 2
Mobility-extracted singly negatively charged N-glycan ions from the E1 glycoproteins from the mosquito (spectrum A) and rodent cell lines (spectrum B). A key to the symbols used for the glycan structures is displayed in the upper right-hand corner of panel A. The linkages are shown by the angle of the lines connecting the symbols (| = 2-link, / = 3-link, – = 4-link, and \ = 6-link). α-Bonds are shown with broken lines and β-bonds are shown with full lines. Full details are given in ref (80). Oligomannose-type glycans are highlighted in green, and fragment ions are shown in gray.
Figure 3
Figure 3
Mobility-extracted singly negatively charged N-glycan ions from the E2 glycoproteins from the mosquito (spectrum A) and rodent cell lines (spectrum B). Symbols used for the glycan structures are as defined in Figure 2. Oligomannose-type glycans are highlighted in green, and fragment ions are shown in gray.
Figure 4
Figure 4
Examples of mobility-extracted, negative ion CID (transfer region) spectra of N-glycans from SFV derived from a mosquito cell line. (A) Man8GlcNAc2, (B) Man7GlcNAc2, (C) Man3GlcNAc2, and (D) fucosylated Man3GlcNAc2. Symbols used for the glycan structures are as defined in Figure 2. Ion nomenclature follows that proposed by Domon and Costello with spectral interpretation as described by Harvey et al.,
Figure 5
Figure 5
Examples of mobility-extracted, negative ion CID (transfer region) spectra of N-glycans from SFV derived from rodent cell lines. (A) Man5GlcNAc2, (B) hybrid-type glycan (Man5GlcNAc3Gal1), (C) biantennary glycan (Man3GlcNAc4Gal2), and (D) sialylated biantennary glycan (Man3GlcNAc4Gal2Fuc1Neu5Ac1).
Figure 6
Figure 6
Models of the glycosylated alphavirus surface as derived from rodent (A) and mosquito (B) cells. The crystal structure of the CHKV E1–E2 envelope glycoprotein complex was fitted into the Sindbis virus cryo-EM map (PDB accession number 2XFB). E1 and E2 are shown in a surface representation in dark gray and light gray, respectively. Oligomannose-type glycans, presented by E2 at Asn200 and Asn262, are shown as light- and dark-green spheres, respectively (Man9GlcNAc2, from PDB accession code 2WAH(75)). At Asn141, glycan structures are modeled as complex-type glycans for rodent-derived alphavirus (panel A; pink spheres, from PDB accession code 4BYH(74)) and paucimannose-type for mosquito-derived alphavirus (panel B; orange spheres, from PDB accession code 2WAH).

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