Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans

Abstract

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.cryoelectron microscopy | mass spectrometry | protein glycosylation | alphacoronavirus | feline infectious peritonitis virus C oronaviruses (CoVs) are enveloped viruses with single-

Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop

“…proteins of viruses belonging to the Alphacoronavirus and Gammacoronavirus genera [40,44]. Considering that this region is proteolytically sensitive and has been shown to play a major role in the S protein function in other CoVs, the use of in silico modeling tools has become a useful alternative to study this region in the context of the structural organization of the protein [37,38].…”

“…To perform the modeling, it is first necessary to identify a suitable protein structure to be used as template, which will determine the accuracy of the predicted model. The S protein structure of several CoVs including the following have been reported previously: Alphacoronavirus: HCoV-NL63 and feline coronavirus UU4 (FCoV-UU4); Betacoronavirus: HCoV-HKU1, MHV, SARS-CoV, and MERS-CoV; Gammacoronavirus: infectious bronchitis virus (IBV); and Deltacoronavirus: porcine deltacoronavirus (PDCoV) [39][40][41][42][43][44][45]. Considering that genome and S protein alignments have showed that the SARS-CoV-2 belongs to the Betacoronavirus genus, we focused our analysis on the S structures from viruses belonging to this genus.…”

“…At the structural level, the S1/S2 site has been shown to be difficult to solve for most CoVs structures, resulting in either incomplete structures (missing the complete S1/S2 site) or structures with an altered (i.e. mutated) S1/S2 site [42,44,45]. Solving the structure of the S1/S2 site was also found to be an issue in the SARS-CoV S structure we used for our modeling analyses.…”

Site-specific analysis of the SARS-CoV-2 glycan shield

“…1). Despite the potential impact of different local protein structure on glycan processing, the overall glycosylation of SARS-CoV-2 is comparable with SARS-CoV-1 S protein and other coronavirus S proteins 4,8,9,15 . We have previously reported that a recombinant SARS-CoV-1 S mimetic also contained 32% oligomannose-type glycans showing a remarkable conservation in glycan processing across these coronaviruses.…”

Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike S protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications

“…Antigen glycosylation greatly determines host immune responses. One of the prominent consequence of viral envelope or surface proteins is immune evasion by shield off immunogenic epitopes (Raman et al, 2016; Vigerust and Shepherd, 2007; Yang et al, 2020). The complete occupancy of glycans in most glycosite of HCoV-19 S protein suggest the virus is able to invade the host in a stealth fashion.…”