Rotavirus C Replication in Porcine Intestinal Enteroids Reveals Roles for Cellular Cholesterol and Sialic Acids.

Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment (resulting in terminal SA removal), which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MßCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MßCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies.

Heparan Sulfate and Sialic Acid in Viral Attachment: Two Sides of the Same Coin?

Sialic acids and heparan sulfates make up the outermost part of the cell membrane and the extracellular matrix. Both structures are characterized by being negatively charged, serving as receptors for various pathogens, and are highly expressed in the respiratory and digestive tracts. Numerous viruses use heparan sulfates as receptors to infect cells; in this group are HSV, HPV, and SARS-CoV-2. Other viruses require the cell to express sialic acids, as is the case in influenza A viruses and adenoviruses. This review aims to present, in a general way, the participation of glycoconjugates in viral entry, and therapeutic strategies focused on inhibiting the interaction between the virus and the glycoconjugates. Interestingly, there are few studies that suggest the participation of both glycoconjugates in the viruses addressed here. Considering the biological redundancy that exists between heparan sulfates and sialic acids, we propose that it is important to jointly evaluate and design strategies that contemplate inhibiting the interactions of both glycoconjugates. This approach will allow identifying new receptors and lead to a deeper understanding of interspecies transmission.

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint.

The spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution. IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.

Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

The structure of Phocaeicola vulgatus sialic acid acetylesterase.

Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Šusing crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser-His-Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.

Multivalent 9-O-Acetylated-sialic acid glycoclusters as potent inhibitors for SARS-CoV-2 infection.

The recent emergence of highly transmissible SARS-CoV-2 variants illustrates the urgent need to better understand the molecular details of the virus binding to its host cell and to develop anti-viral strategies. While many studies focused on the role of the angiotensin-converting enzyme 2 receptor in the infection, others suggest the important role of cell attachment factors such as glycans. Here, we use atomic force microscopy to study these early binding events with the focus on the role of sialic acids (SA). We show that SARS-CoV-2 binds specifically to 9-O-acetylated-SA with a moderate affinity, supporting its role as an attachment factor during virus landing to cell host surfaces. For therapeutic purposes and based on this finding, we have designed novel blocking molecules with various topologies and carrying a controlled number of SA residues, enhancing affinity through a multivalent effect. Inhibition assays show that the AcSA-derived glycoclusters are potent inhibitors of cell binding and infectivity, offering new perspectives in the treatment of SARS-CoV-2 infection.

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 µM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Host and viral determinants for efficient SARS-CoV-2 infection of the human lung.

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.

Intra-species sialic acid polymorphism in humans: a common niche for influenza and coronavirus pandemics?

The ongoing COVID-19 pandemic has led to more than 159 million confirmed cases with over 3.3 million deaths worldwide, but it remains mystery why most infected individuals (∼98%) were asymptomatic or only experienced mild illness. The same mystery applies to the deadly 1918 H1N1 influenza pandemic, which has puzzled the field for a century. Here we discuss dual potential properties of the 1918 H1N1 pandemic viruses that led to the high fatality rate in the small portion of severe cases, while about 98% infected persons in the United States were self-limited with mild symptoms, or even asymptomatic. These variations now have been postulated to be impacted by polymorphisms of the sialic acid receptors in the general population. Since coronaviruses (CoVs) also recognize sialic acid receptors and cause severe acute respiratory syndrome epidemics and pandemics, similar principles of influenza virus evolution and pandemicity may also apply to CoVs. A potential common principle of pathogen/host co-evolution of influenza and CoVs under selection of host sialic acids in parallel with different epidemic and pandemic influenza and coronaviruses is discussed.

The role of cell surface sialic acids for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a new virus that has higher contagious capacity than any other previous human coronaviruses (HCoVs) and causes the current coronavirus disease 2019 pandemic. Sialic acids are a group of nine-carbon acidic α-keto sugars, usually located at the end of glycans of cell surface glycoconjugates and serve as attachment sites for previous HCoVs. It is therefore speculated that sialic acids on the host cell surface could serve as co-receptors or attachment factors for SARS-CoV-2 cell entry as well. Recent in silico modeling, molecular modeling predictions and microscopy studies indicate potential sialic acid binding by SARS-CoV-2 upon cell entry. In particular, a flat sialic acid-binding domain was proposed at the N-terminal domain of the spike protein, which may lead to the initial contact and interaction of the virus on the epithelium followed by higher affinity binding to angiotensin-converting enzyme 2 (ACE2) receptor, likely a two-step attachment fashion. However, recent in vitro and ex vivo studies of sialic acids on ACE2 receptor confirmed an opposite role for SARS-CoV-2 binding. In particular, neuraminidase treatment of epithelial cells and ACE2-expressing 293T cells increased SARS-CoV-2 binding. Furthermore, the ACE2 glycosylation inhibition studies indicate that sialic acids on ACE2 receptor prevent ACE2-spike protein interaction. On the other hand, a most recent study indicates that gangliosides could serve as ligands for receptor-binding domain of SARS-CoV-2 spike protein. This mini-review discusses what has been predicted and known so far about the role of sialic acid for SARS-CoV-2 infection and future research perspective.

Prediction of Single Point Mutations in Human Coronavirus and Their Effects on Binding to 9-O-Acetylated Sialic Acid and Hidroxychloroquine.

Due to the current spreading of the new disease CoViD-19, the World Health Organization formally declared a world pandemic on March 11, 2020. The present trends indicate that the pandemic will have an enormous clinical and economic impact on population health. Infections are initiated by the transmembrane spike (S) glycoproteins of human coronavirus (hCoV) binding to host receptors. Ongoing research and therapeutic product development are of vital importance for the successful treatment of CoViD-19. To contribute somewhat to the overall effort, herein, single point mutations (SPMs) of the binding site residues in hCoV-OC43 S that recognizes cellular surface components containing 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using an in silico protein engineering approach, while their effects on the binding of 9-O-Ac-Sia and Hidroxychloroquine (Hcq) are evaluated using molecular docking simulations. Thr31Met and Val84Arg are predicted to be the critical - most likely SPMs in hCoV-OC43 S for the binding of 9-O-Ac-Sia and Hcq, respectively, even though Thr31Met is a very likely SPM in the case of Hcq too. The corresponding modes of interaction indicate a comparable strength of the Thr31Met/9-O-Ac-Sia and Val84Arg/Hcq (or Thr31Met/Hcq) complexes. Given that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, the high hydrophobic affinity of Hcq to hCoV-OC43 S speaks in favor of its ability to competitively inhibit rapid S-mediated virion attachment in high-density receptor environments, but its considerably low specificity to hCoV-OC43 S may be one of the key obstacles in considering the potential of Hcq to become a drug candidate.

Adaptation of influenza viruses to human airway receptors.

Through annual epidemics and global pandemics, influenza A viruses (IAVs) remain a significant threat to human health as the leading cause of severe respiratory disease. Within the last century, four global pandemics have resulted from the introduction of novel IAVs into humans, with components of each originating from avian viruses. IAVs infect many avian species wherein they maintain a diverse natural reservoir, posing a risk to humans through the occasional emergence of novel strains with enhanced zoonotic potential. One natural barrier for transmission of avian IAVs into humans is the specificity of the receptor-binding protein, hemagglutinin (HA), which recognizes sialic-acid-containing glycans on host cells. HAs from human IAVs exhibit "human-type" receptor specificity, binding exclusively to glycans on cells lining the human airway where terminal sialic acids are attached in the α2-6 configuration (NeuAcα2-6Gal). In contrast, HAs from avian viruses exhibit specificity for "avian-type" α2-3-linked (NeuAcα2-3Gal) receptors and thus require adaptive mutations to bind human-type receptors. Since all human IAV pandemics can be traced to avian origins, there remains ever-present concern over emerging IAVs with human-adaptive potential that might lead to the next pandemic. This concern has been brought into focus through emergence of SARS-CoV-2, aligning both scientific and public attention to the threat of novel respiratory viruses from animal sources. In this review, we summarize receptor-binding adaptations underlying the emergence of all prior IAV pandemics in humans, maintenance and evolution of human-type receptor specificity in subsequent seasonal IAVs, and potential for future human-type receptor adaptation in novel avian HAs.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV.

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.

SARS-CoV-2 Evolutionary Adaptation toward Host Entry and Recognition of Receptor O-Acetyl Sialylation in Virus-Host Interaction.

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.

Role of Porcine Aminopeptidase N and Sialic Acids in Porcine Coronavirus Infections in Primary Porcine Enterocytes.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs.

Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection.

Coronaviruses (CoVs) are common human and animal pathogens that can transmit zoonotically and cause severe respiratory disease syndromes. CoV infection requires spike proteins, which bind viruses to host cell receptors and catalyze virus-cell membrane fusion. Several CoV strains have spike proteins with two receptor-binding domains, an S1A that engages host sialic acids and an S1B that recognizes host transmembrane proteins. As this bivalent binding may enable broad zoonotic CoV infection, we aimed to identify roles for each receptor in distinct infection stages. Focusing on two betacoronaviruses, murine JHM-CoV and human Middle East respiratory syndrome coronavirus (MERS-CoV), we found that virus particle binding to cells was mediated by sialic acids; however, the transmembrane protein receptors were required for a subsequent virus infection. These results favored a two-step process in which viruses first adhere to sialic acids and then require subsequent engagement with protein receptors during infectious cell entry. However, sialic acids sufficiently facilitated the later stages of virus spread through cell-cell membrane fusion, without requiring protein receptors. This virus spread in the absence of the prototype protein receptors was increased by adaptive S1A mutations. Overall, these findings reveal roles for sialic acids in virus-cell binding, viral spike protein-directed cell-cell fusion, and resultant spread of CoV infections.IMPORTANCE CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. These findings raise the possibility that the lectin-like properties of many CoVs contribute to facile zoonotic transmission and intercellular spread within infected organisms.