What is the role of N-glycans and calnexin-calreticulin chaperones in SARS-CoV-2 spike maturation?

By Nidhi Saha, BDSSep 28 2022Reviewed by Aimee Molineux

A recent study published in Science Advances showed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike N-glycosylation patterns, intracellular lectins, as well as the associated molecular chaperones play crucial roles in the viral-host-cell invasion and function.

Therapeutic modulation of these glycan-related pathways can modify SARS-CoV-2 viral entry, prevent infection, and may serve as an armamentarium against the ongoing coronavirus disease 2019 (COVID-19) pandemic.

Background

COVID-19 vaccines primarily target the spike (S) protein of its causative pathogen – SARS-CoV-2. The S protein of the virions is implicated in facilitating host-cell infestation. 

The pathogen is constantly evolving and has undergone several mutations – rendering variants of concern (VOCs) that evade vaccine-acquired immunity and improve viral transmissibility, such as the Delta and the Omicron variants. This occurred because certain mutations in the S receptor-binding domain (RBD) enhance Spike binding to the host-cell receptors – angiotensin-converting enzyme-2 (ACE2). 

Glycans are a class of complex carbohydrates enveloping the SARS-CoV-2 viral surface and are specific to each virus. Viruses employ glycans for multiple survival strategies, like protection from the antibodies against the virus and entering the host cells. 

The goal of this study was to explore the roles of N-glycans – a subset of the molecule glycan, focusing on its attachment mechanism to the SARS-CoV-2 S protein and hence, in regulating its infectivity.

Results

The findings showed that the development, as well as the functional maturation of the S protein of SARS-CoV-2, is regulated by specific N-glycans. Few N glycans regulate SARS-CoV-2 entry into host cells and the maturation of S proteins. Specifically, the N-glycans at S protein sites – N801 and N61, are critical for SARS-CoV-2 functioning. 

Artificially created virus-like particles (VLPs) of SARS-CoV-2, specifically designed to prevent N glycan formation on various S proteins, were used in this study. Modifications in the N801 and N61 sites led to the reduced entry of the VLPs in host cells by 75-85%. 

A probable reason for this could be the folding of the S protein into a specific configuration with the help of N801 and N61. The results depicted that complex-type glycans influence the maturation of S, assembly into virions, and the functioning of the S protein for facilitating viral entry into the host cells.

Looking deeper to find the reason behind the folding mechanisms revealed calnexin (a chaperone or lectin) as a catalyst for such folding. Thus, calnexin regulated the entry of VLPs into host cells. 

Overall, the results revealed that Spike N-glycosylation mutations are generally not tolerated by SARS-CoV-2, except at sites – N17, N717, and N1074. Of note, the Omicron variant does not harbor N-glycosylation site mutations. Hence, several N-glycans are necessary for viral fitness.

The results revealed that certain N-glycans guide S maturation, as well as the functioning of S protein in a host-cell infestation. Additionally, complex-type glycan also S functions during SARS-CoV-2 entry to the host cells, apart from facilitating S maturation and virion assembly. 

It was observed that cell-surface S proteins were equally active in all glycosylation variants. Only a few N-glycan mutations – N801Q and N61/603/657Q, minutely impacted the S2' cleavage and intercellular fusion. 

On investigating how S N-glycans reduce SARS-CoV-2 host-cell infestation, it was found that lectin chaperones – Calnexin (CANX)/calreticulin (CALR), monitor S maturation and viral entry. Moreover, certain proteins interacting during SARS-CoV-2 entry into the host cells may be glycosylated. Furthermore, their composition, expression, and spatial distribution may be regulated by CANX/CALR.

Conclusion

The findings ascertained that glycans play an important role in the functioning of SARS-CoV-2. The results highlighted the utility of deeming N-linked glycans as potential targets of newer drugs against COVID-19. Further investigation into how glycans influence other viral diseases is warranted.