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.
Highlights
Semliki Forest virus (SFV) is a prototypic alphavirus within the Togaviridae family and was first isolated in Aedes albopictus mosquitoes in Uganda in 1944.1 SFV infection only causes mild febrile illness in humans, it is highly pathogenic in rodents and has become a model system for investigating viral encephalitis[2] caused by other neurotropic alphaviruses such as Sindbis virus, Chikungunya virus (CHIKV), and Venezuelan equine encephalitis virus
Alphaviruses assemble in the plasma membrane, where the cytoplasmic tail of the E2 glycoprotein interacts with the RNAcontaining capsid, leading to budding of mature virions.[8]
Our study provides a glycomic analysis of the N-linked glycans displayed on the major glycoproteins from SFV, a prototypic alphavirus
Summary
Semliki Forest virus (SFV) is a prototypic alphavirus within the Togaviridae family and was first isolated in Aedes albopictus mosquitoes in Uganda in 1944.1 SFV infection only causes mild febrile illness in humans, it is highly pathogenic in rodents and has become a model system for investigating viral encephalitis[2] caused by other neurotropic alphaviruses such as Sindbis virus, Chikungunya virus (CHIKV), and Venezuelan equine encephalitis virus. Alphaviruses assemble in the plasma membrane, where the cytoplasmic tail of the E2 glycoprotein interacts with the RNAcontaining capsid, leading to budding of mature virions.[8] Cotranslational formation of the E1−p62 heterodimer is necessary for proper folding.[7] The E1−p62 heterodimer forms concomitantly to protein folding in the ER, and the glycans are initially processed in the context of this structure.[7] The initial formation of single trimeric viral spikes, (E1−p62)[3], is thought to occur in this stage.[9] Proteolytic cleavage by furin in the trans-Golgi network is required to cleave p62 into E2 and a third
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