A recent study posted to the Research Square* preprint server discovered that near relatives of the Middle East respiratory syndrome coronavirus (MERS-CoV) utilize angiotensin-converting enzyme 2 (ACE2) as functional receptors.

Background

MERS-CoV belongs to the betaCoV lineage C and exhibits high fatality rates. Dipeptidyl peptidase-4 (DPP4) is the functional receptor of the MERS-CoV and many bat CoVs. Nevertheless, the receptor for NeoCoV, the closest MERS-CoV-related virus identified in bats, is still unknown. The understanding of bat CoVs has been severely hampered due to a lack of knowledge regarding these high-risk pathogens' favorable receptors.

About the study 

In the present investigation, the researchers evaluated the MERS-CoV-related viruses such as NeoCoV and PDF-2180-CoV to identify their functional receptors for host entry. A total of 46 bat ACE2 and human ACE2 (hACE2) sequences were used in this study. Five human DPP4 (hDPP4) and bat DPP4 sequences were obtained from the GenBank database. Whole-genome sequences of various CoVs were also retrieved from the GenBank database. The gene sequences utilized in the investigation were generated by Genewiz.

All study subjects submitted written informed consent before enrollment. Sera samples were procured from participants nearly 21 days following their third dose of World Health Organization (WHO)-approved inactivated coronavirus disease 2019 (COVID-19) vaccines. Protein sequences were aligned employing the multiple sequence comparison by log-expectation (MUSCLE) algorithm by molecular evolutionary genetic analysis X (MEGA-X) software. Human codon-optimized sequences of several DPP4 or ACE2 orthologs and their mutants were cloned to a lentiviral transfer vector containing a C-terminal 3×Flag tag. 

The receptor-binding domain (RBD)-human crystallizable fragment (hFc) fusion proteins of MERS-CoV, severe acute respiratory syndrome CoV (SARS-CoV-2), Pipistrellus bat CoV (HKU5-CoV), Tylonycteris bat CoV (HKU4-CoV), NeoCoV, PDF-2180-CoV, and HKU31-CoV were synthesized by transfecting human embryonic kidney 293T (HEK293T) cells by appropriate plasmids via GeneTwin reagent. Similarly, soluble bat ACE2 and hACE2 were also expressed. Cell lines used in the study include HEK293T, Vero E6, baby hamster kidney 21 (BHK-21), and colorectal adenocarcinoma 2 (Caco2). 

 Results and discussion

The results demonstrated that NeoCoV and its near relative, PDF-2180-CoV, may employ particular forms of bat ACE2 and, less significantly, hACE2 for host entrance. The PDF-2180-CoV and NeoCoV RBD core structures were more similar to the MERS-CoV than ACE2-using CoVs. Hence, the current discovery that these two MERS-CoV-linked viruses utilize ACE2 instead of DDP4 for host entry was unexpected. 

The findings illustrated that the NeoCoV and PDF-2180-CoV utilize their spike 1 (S1) subunit carboxyl-terminal domains (S1-CTD) for species-specific and high-affinity ACE2 binding. Several sarbecoviruses, a group of merbecoviruses, and α-CoV NL63 evaluated in this work utilized ACE2 receptors for cell entry. The structural analysis depicted that PDF-2180-CoV and NeoCoV interact with the ACE2's apical side surface, distinct from other ACE2-adapted CoVs. The ACE2 connection of the two tested MERS-CoV-related viruses was distinguished from other CoVs by their intermolecular protein-glycan linkages produced by glycosylation at N54.

The NeoCoV and PDF-2180-CoV harbor highly detrimental characteristics of high transmission of SARS-CoV-2 and high mortality of MERS-CoV. Thus, their unanticipated ACE2 usage reveals a latent biosafety concern. Moreover, the existing SARS-CoV-2 vaccines were ineffective in protecting humans from illnesses caused by these two MERS-CoV-linked viruses. The current data backs a prior hypothesis stating that MERS-CoV arose through an intra-S recombination event among a DPP4-using virus and a NeoCoV-like virus. Nevertheless, additional investigations are needed to obtain more data regarding the MERS-CoV's origin. 

The PDF-2180-CoV and NeoCoV utilized ACE2 receptors in bats belonging to the Yangochiroptera group, yet not bats in the Yinpterochiroptera group. A molecular determinant was discovered near the viral binding domain that prevents hACE2 from promoting NeoCoV infection, particularly close to the Asp338 residue. However, a single residue replacement that increases local hydrophobicity near site 510 might improve these two MERS-CoV-associated viruses' affinity for hACE2 and allow them to infect human cells that express ACE2.

Considering the significant mutations in the RBD sections of the SARS-CoV-2 variants, particularly the extensively mutated Omicron, the two evaluated MERS-CoV-2 viruses may have the ability to infect people through antigenic drift. Furthermore, MERS-CoV-related viruses with human emergence capability are extremely likely to be circulating in nature.

Conclusions

According to the authors, this was the first work showing the usage of ACE2 receptors by MERS-CoV-linked viruses for host entry. The study findings illustrated that NeoCoV and PDF-2180-CoV via their S1-CTD exhibit species-specific and high-affinity ACE2 binding. A unique CoV-ACE2 binding interface and a contact between protein and glycan, which were not found in other ACE2-using viruses, were observed in this study. Notably, the receptor-binding motif (RBM)'s T510F mutation successfully enabled NeoCoV to infect hACE2. Of note, antibodies directed against MERS-CoV or SARS-CoV-2 were unable to cross-neutralize Neo-CoV infection.

On the whole, the current study highlights a possible bio-safety risk of the emergence of a highly transmissible and fatal MERS-CoV-2 using ACE2 for host entry. This work necessitates the requirement of close monitoring of NeoCoV and PDF-2180-CoV to detect any occurrences of spillover events. Hence, health care systems could be prepared against possible future outbreaks of MERS-CoV-linked ACE2-using viruses.

*Important notice

Preprints with Research Square publish preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.