Role of Sialic acid binding in Coronavirus attachment and entry

Binding of the viral particle is a crucial step in the establishment of viral infection and subsequent viral replication. In the case of Coronaviridae, the binding of the virus is mediated viral spike protein, a homotrimer composed of subunits that are about 150 kDa in size each. The spike protein itself is composed of two subunits, S1 and S2, the former sufficient for receptor binding and the latter required for the fusion and entry of the virus particle. During the viral replication the S protein is synthesized as a precursor protein and co-translationally glycosylated in the Golgi followed by a cleavage generating the S1 and S2 subunits at a dibasic cleavage site (BBXBB). The S1 subunit contains the receptor-binding site (RBD) followed (in the case of MHV) by a hypervariable region, whereas the S2 subunit contains two heptad repeats (HR1 and 2) as well as the transmembrane region.

Domains of a prototype Coronavirus S potein

Of particular interest is the RBD since blocking peptides or neutralizing antibodies designed to bind the RBD might be used in treating Coronavirus caused diseases, not only in humans (such as SARS or MERS) but also in animals. On the other hand, based on experiments done using the murine Coronavirus (MHV) the heptad repeat domains as well as the putative fusion peptide located within the S2 subdomain may play an important role in the formation of syncytia and thus may contribute to the CPE. Furthermore, the HR might also play a role in the interaction of the RBD with the cellular receptor during viral entry, probably by stabilizing the receptor-RBD complex not only in the case of MHV but also SARS-CoV.

In the past years, however a number of Coronaviruses have been shown to not only contain one but two RBD, one located at the C-terminal end of S1 which is responsible for binding the cellular receptor and an additional one located at the N-terminal end of S1 binding sialic acid. In general, the consensus is that binding to sialic acid by the S1 subunit allows Coronavirus’ to bind to epithelial target cells of the respiratory tract as well the intestine which are normally covered by mucus and thus not readily accessible. This is particular true for members of the Alpha-, Beta-, and Gammacoronaviridae which bind to ciliated intestine and respiratory cells, such as the porcine TGEV and PEDV as well as the enteric feline Coronavirus (FECV) but also for the bovine Coronavirus (BCoV) and the human Coronavirus OC43 (HCoV-OC43) as well as the avian Infectious Bronchitis Virus (IBV). In contrast, MERS-CoV generally does bind and infect primarily non-ciliated bronchial epithelial and alveolar cells of the lower lung and thus might not need sialic acid to bind to DPP4 (although hDPP4 does have sialic acid residues).

                        Feline Enteric Coronavirus (FECV)

Feline intestinal epithelial cells derived from the Ileum and the Colon (illenocytes and coloncytes respectively) pretreated with neuroaminidase exhibit an increase in the efficiency of FECV infection, suggesting that sialic acid might inhibit viral entry. Based on results showing that the pretreatment of porcine TGEV strain Perdue and PEDV with neuroaminidase can unmask the viral sialic acid binding activity, similar experiments confirmed these results for FECV. The application of α2-6-sialyllactose binds and reduces the infectivity of pretreated FECV, demonstrating FECV can bind α2-6-sialic acid. Desialylated cells however were resistant to inhibition of inhibition by α2-6-sialyllactose treatment. FECV therefore does have

a sialic acid binding capacity, which during the passage of the virus through the stomach may be partially masked by virus-associated sialic acids. In the absence of viral enzymes removing virus-associated sialic acids, enzymes within the mucus might remove the sialic acid thus allowing FECV to bind its cellular receptor and thus requiring sialidases for efficient enterocyte infections.

                     Infectious Bronchitis Virus (IBV)

Although the receptor for the avian Infectious Bronchitis Virus is unknown it is known that the treatment of Vero, BHK (Baby Hamster Kidney) as well as primer chicken kidney cells with neuroaminidase -an enzyme which cleaves sialic acid- renders cell lines resistant to infection with IBV strains Beaudette and M41. Moreover, IBV is more sensitive than Sendai or Influenza A virus to pretreatment of cells with neuroaminidase suggesting that IBV requires a higher amount of sialic acid than Influenza A or Sendai and indeed it has been shown that IBV preferentially recognizes α2,3-linked sialic acid as indicated by reacting with lectin. Indeed the infection of the tracheal organ cultures can be inhibited by pretreatment with neuroaminidase. The binding of α2,3-linked sialic acid might be only required for the initial binding of IBV preceding binding to the receptor although the sialic acid binding activity of IBV S protein seems to be more important for viral entry than the sialic acid binding activity of TGEV S protein. This is reflected by the abundance of α2-3 linked sialic acid on susceptible epithelial cells.  

In general, the masking of the viral sialic acid binding site might protect the enteric Coronavirus particles from degradation in the stomach or by gastric mucins. Bacterial and host derived sialidases unmasking these binding site then would allow the virus to attach to the mucin and infect cells of the intestinal tract. In the avian respiratory tract α2-3 linked sialic acid is a common receptor for respiratory viruses such as avian Influenza A. 

So finally what has this to do with emerging Coronaviruses? As I mentioned above so far there is no indication that MERS-CoV S has sialic acid binding activity nor that the primary target cells necessitate this activity. The novel Coronavirus identified in dromedaries however seems to be an enteric Coronavirus and thus the S protein might bind sialic acid.  However, once the genome of DcCoV UAE-HKU23 has been sequenced, we should know more.  One final word about the potential use of neuroaminidase inhibitors which are quite effective in treating Influenza A virus infections: they are not effective against Coronavirus induced infections since Coronaviridae are not dependent on the sialic acid binding to its cognate receptor. 

Further reading

Vlasak R, Luytjes W, Spaan W, & Palese P (1988). Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proceedings of the National Academy of Sciences of the United States of America, 85 (12), 4526-9 PMID: 3380803 

Shahwan K, Hesse M, Mork AK, Herrler G, & Winter C (2013). Sialic acid binding properties of soluble coronavirus spike (S1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus. Viruses, 5 (8), 1924-33 PMID: 23896748 

Winter C, Herrler G, & Neumann U (2008). Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent. Microbes and infection / Institut Pasteur, 10 (4), 367-73 PMID: 18396435 

Schmauser B, Kilian C, Reutter W, & Tauber R (1999). Sialoforms of dipeptidylpeptidase IV from rat kidney and liver. Glycobiology, 9 (12), 1295-305 PMID: 10561454

Krempl C, Schultze B, Laude H, & Herrler G (1997). Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus. Journal of virology, 71 (4), 3285-7 PMID: 9060696 

Schwegmann-Weßels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., & Herrler, G. (2011). The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-435 

Desmarets, L., Theuns, S., Roukaerts, I., Acar, D., & Nauwynck, H. (2014). The role of sialic acids in feline enteric coronavirus infections Journal of General Virology DOI: 10.1099/vir.0.064717-0