CoV Spike Porcine

What are the main porcine coronaviruses and how do their spike proteins differ structurally?

The main porcine coronaviruses include alphacoronaviruses (Porcine epidemic diarrhea virus/PEDV and Transmissible gastroenteritis virus/TGEV), deltacoronaviruses (Porcine deltacoronavirus/PDCoV), and other emerging viruses like Swine acute diarrhea syndrome coronavirus (SADS-CoV).

Structurally, TGEV, PEDV, and PDCoV spike proteins contain two functional domains: spike_rec_binding and corona_S2, while SADS-CoV contains only the corona_S2 domain . Alphacoronaviruses like PEDV have a unique N-terminal domain 0 (D0) not found in other coronavirus genera .

Korean PEDV isolates show significant structural variations, with S genes nine nucleotides longer than reference strains due to a 15 bp insertion and 6 bp deletion in the N-terminal region of the S1 domain . Sequence comparisons reveal that Korean isolates share 93.6-99.6% identity with each other but only 92.2-93.7% identity with reference strains .

What receptor interactions govern porcine coronavirus host specificity?

PDCoV demonstrates remarkable cross-species potential by binding to aminopeptidase N (APN) from different host species, including humans . Crystal structures show that PDCoV receptor binding domain (RBD) binds to common regions on human APN (hAPN) and porcine APN (pAPN), which differ from binding sites used by alphacoronaviruses like HCoV-229E and porcine respiratory coronavirus (PRCoV) .

Structure-guided mutagenesis has identified conserved residues on hAPN and pAPN essential for PDCoV binding and infection . This broad receptor usage explains PDCoV's ability to experimentally infect multiple species including chickens, calves, and mice, as well as its reported infection of humans .

What post-translational modifications influence porcine coronavirus spike protein function?

Porcine coronavirus spike proteins undergo extensive post-translational modifications that influence their structure and function:

Phosphorylation sites:

• TGEV: 139 sites

• PEDV: 143 sites

• SADS-CoV: 109 sites

• PDCoV: 124 sites

N-linked glycosylation sites:

• TGEV: 24 sites

• PEDV: 22 sites

• SADS-CoV: 20 sites

• PDCoV: 18 sites

Glycosylation particularly impacts protein folding, stability, and immune recognition. In PEDV, specific N-glycans modulate the conformation of the D0 domain . Mass spectrometry and cryo-EM analyses have mapped the spatial distribution of these glycans, demonstrating that a key N-glycan plays a functional role in modulating D0 conformation .

How do dynamics of spike protein domains influence porcine coronavirus function?

Cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) studies have revealed asynchronous motions of the Domain 0 (D0) of spike proteins on intact PEDV viral particles . These motions likely represent different functional states related to receptor binding and membrane fusion processes.

The D0 domain, unique to alphacoronaviruses, can adopt different architectures corresponding to distinct functional states . Domain motions are directly associated with receptor binding capability, with certain conformations potentially favoring attachment to cellular receptors.

These structural dynamics highlight the sophisticated mechanisms that coronaviruses employ to infect cells and suggest potential targets for intervention strategies that could lock the spike protein in non-functional conformations.

What molecular determinants in spike proteins drive virulence differences between PEDV strains?

Recombinant virus experiments have definitively shown that spike proteins alone can determine PEDV virulence independent of other viral components . Using a common DR13 backbone with different spike proteins:

• rDR13-S^DR13 (DR13 spike): No diarrhea, no virus shedding, indicating no virulence

• rDR13-S^UU (UU spike): 91.6% of piglets developed mild to severe diarrhea but recovered by day 5

• rDR13-S^GDU (GDU spike): All piglets developed severe diarrhea, with 66.7% mortality

Virulent spike variants led to higher viral shedding (approximately 8.3-8.4 log₁₀ genome copies/mL in fecal samples) and produced more severe clinical signs, including lower rectal temperatures . These findings demonstrate that specific regions of the spike protein are critical determinants of coronavirus pathogenicity.

Protease sensitivity may play a key role, as trypsin enhanced infection for some recombinant viruses but not others . Domain-swapping experiments represent a promising approach for further pinpointing specific regions responsible for virulence differences .

What mechanisms enable PDCoV's cross-species transmission capability?

PDCoV's ability to cross species barriers is explained by several structural and functional adaptations:

• Broad receptor recognition: PDCoV binds to common regions on aminopeptidase N (APN) from different species, including humans

• Conserved receptor binding interface: Structure-guided mutagenesis identified conserved residues on APN across species that are essential for PDCoV binding

• Evolutionary adaptation: Phylogenetic analysis suggests PDCoV evolved from an avian deltacoronavirus ancestor, being closely related to sparrow CoV HKU17, bulbul CoV HKU11, and munia CoV HKU13

• Experimental evidence: PDCoV successfully infects chickens, calves, and mice in laboratory settings

• Human infection: PDCoV has been isolated from plasma samples of children with acute febrile illness, confirming its zoonotic potential

These findings reveal how deltacoronaviruses can overcome species barriers and highlight the importance of surveillance for emerging coronaviruses with pandemic potential.

What structural biology approaches are most effective for studying porcine coronavirus spike proteins?

Multiple complementary approaches have proven effective for studying spike protein structures:

X-ray crystallography:

• Successfully used to determine crystal structures of PDCoV RBD bound to human and porcine APN

• Provides atomic-level details of binding interfaces

Cryo-electron microscopy (cryo-EM):

• Revealed recombinant PEDV S protein structure derived from porcine cell lines

• Identified domain motions associated with receptor binding

Cryo-electron tomography (cryo-ET):

• Demonstrated asynchronous S protein D0 motions on intact viral particles

• Provides insights into functional states in near-native environments

Integrated approaches:

• Combining mass spectrometry with cryo-EM to map glycosylation patterns

• Integration of structural data with functional assays

These methods together provide a comprehensive understanding of coronavirus spike protein structure, dynamics, and function that informs therapeutic development.

How can recombinant virus systems be designed to study spike protein domain functions?

Recombinant virus systems offer powerful approaches to isolate spike protein effects:

• Backbone-and-swap strategy:

  • Using a common viral backbone (e.g., attenuated strain) with different spike proteins

  • Example: rDR13-S^DR13, rDR13-S^UU, and rDR13-S^GDU recombinant PEDV viruses created by inserting different S genes into the same DR13 backbone

• Domain-swapping experiments:

  • Exchanging specific regions between spike proteins with differing properties

  • Allows identification of precise functional elements

• In vitro and in vivo validation:

  • Testing in cell culture to assess replication kinetics

  • Animal challenge studies to evaluate pathogenicity

  • Measurement of clinical signs, viral shedding (reaching 8.3-8.4 log₁₀ genome copies/mL in virulent strains), seroconversion rates, and mortality

These approaches have demonstrated that spike proteins alone can determine virulence phenotypes independent of other viral components, making them crucial targets for vaccine development and attenuated strain design .

What bioinformatics approaches yield insights into spike protein evolution and variation?

Key bioinformatics approaches include:

• Sequence analysis:

  • Multiple sequence alignment identifies conserved and variable regions

  • Phylogenetic analysis established that PDCoV likely evolved from avian deltacoronavirus ancestors

• Structural prediction:

  • Prediction of physical/chemical properties, hydrophilicity, and functional elements

  • Homology modeling for novel variants

• Functional element prediction:

  • Identification of epitopes: TGEV (53), PEDV (51), SADS-CoV (43), PDCoV (52)

  • Mapping post-translational modifications

• Recombination analysis:

  • Detection of potential genetic exchanges between viruses

  • Similar to SARS-CoV-2, where six of nine identified recombinations were in the spike gene

• Comparative genomics:

  • Identification of insertions/deletions, such as the 15 bp insertion and 6 bp deletion in Korean PEDV isolates

  • Analysis of selective pressures across species

These computational approaches complement experimental studies and guide targeted investigations of spike protein function.

How do receptor binding mechanisms of porcine coronaviruses compare with SARS-CoV-2?

Important similarities and differences exist between porcine coronaviruses and SARS-CoV-2:

• Receptor utilization:

  • PDCoV uses aminopeptidase N (APN) from multiple species

  • SARS-CoV-2 uses ACE2 as its primary receptor

• Cross-species potential:

  • PDCoV shows broad receptor usage across species, including documented human infections

  • SARS-CoV-2 RBD is described as a "hotspot for adaptive mutations that enhance binding efficiency to its human host"

• Structural adaptations:

  • Alphacoronaviruses like PEDV have a unique N-terminal domain 0 (D0) not found in betacoronaviruses like SARS-CoV-2

  • SARS-CoV-2 contains a distinctive furin cleavage site that may enhance transmissibility

• Evolutionary patterns:

  • Both demonstrate recombination events in spike genes

  • PDCoV likely evolved from avian deltacoronavirus ancestors

  • SARS-CoV-2 likely evolved from bat coronaviruses, possibly with intermediate hosts

Understanding these comparative mechanisms helps identify common principles in coronavirus host recognition and informs surveillance for emerging threats.

How can glycosylation analysis of porcine coronavirus spikes inform human coronavirus vaccine design?

Glycosylation plays crucial roles in spike protein function across coronavirus genera:

• Glycan shield effects:

  • N-linked glycans can shield viral epitopes from antibody recognition

  • Similar strategies are employed by both porcine coronaviruses and SARS-CoV-2

• Conformational regulation:

  • In PEDV, specific N-glycans modulate the D0 domain conformation

  • Similar effects likely influence SARS-CoV-2 spike dynamics

• Glycosylation mapping techniques:

  • Integrated mass spectrometry and cryo-EM approaches developed for porcine coronaviruses

  • Can be applied to human coronavirus vaccine antigen characterization

• Vaccine antigen considerations:

  • Proper glycosylation patterns are essential for recombinant spike-based vaccines

  • Expression systems must be selected carefully to ensure appropriate post-translational modifications

• Strain variations:

  • Glycosylation site differences between virus strains may contribute to antigenic differences

  • Comparative analysis across strains can identify conserved sites for broadly protective vaccine design

These insights from porcine coronavirus research can guide optimization of glycosylation in human coronavirus vaccine antigens, potentially improving immunogenicity and protective efficacy.