Kenyan bat coronavirus uses human CEACAM6 to enter cells, raising spillover concerns
Baku, April 24, AZERTAC
In a recent study published in the journal Nature, researchers showed that heart-nosed bat (Cardioderma cor) alpha-coronaviruses (alpha-CoVs) can use human carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a receptor for cell entry in experimental systems, according to News medical.
There has been a substantial increase in the prediction and characterization of viruses with zoonotic potential, especially CoVs, since the coronavirus disease 2019 (COVID-19) pandemic. Cell entry is the first barrier for cross-species viral jump, and depends on the binding of viral proteins to cellular receptors. Identifying viruses with zoonotic potential based on their ability to enter human cells is crucial for pandemic prediction, preparedness, and prevention.
In the present study, researchers characterized the cellular entry of alpha-CoVs. First, to select alpha-CoV spike proteins that represent known diversity with high fidelity, a greedy algorithm was applied to spike protein sequences from two databases. Forty spike protein sequences, which captured 53.4% of the phylogenetic diversity, were selected. Most spike proteins (67.5%) were from poorly characterized bat-borne alpha-CoVs.
Next, to verify that the synthetic open reading frames of the spike protein could generate spike proteins that could be pseudotyped onto lentiviruses, the researchers purified pseudoviruses and confirmed spike protein incorporation by immunoblotting. In addition to this alpha-CoV library, plasmid expression libraries for the aminopeptidase N (APN) and angiotensin-converting enzyme 2 (ACE2) receptors, representing various mammalian species, were developed.
The team tested whether any pseudotyped spike proteins from the alpha-CoV library could enter cells via ACE2 or APN. Most alpha-CoVs did not use APN or ACE2 for cell entry. Of note, two bat alpha-CoVs used non-human APN receptors for cell entry. These data suggest that the use of APN and ACE2 receptors may be rare among alpha-CoVs. Next, the team investigated several human cell lines for their permissivity to alpha-CoV pseudotyped spike proteins.
Across the screen, only human CoV (HCoV)-229E, HCoV-NL63, and a bat CoV (BtCoV-KY43) could enter human cells. BtCoV-KY43’s spike protein sequence was first isolated from heart-nosed bats in Kenya. To identify which human receptor facilitated the entry of BtCoV-KY43 (hereafter, CcCoV-KY43), an avidity-based method that detects extracellular interactions with human receptor ectodomains was used.
Using the receptor-binding domain (RBD) of CcCoV-KY43’s spike protein as the prey, three interactions were identified, all with CEACAM paralogs (CEACAM6, CEACAM3, and CEACAM5). Overexpression of these CEACAM proteins in a non-permissive cell line revealed that only CEACAM6 expression significantly increased permissivity. In contrast, using monoclonal antibodies (mAbs) against CEACAM6 blocked cell entry.
Analyses of the Human Cell Atlas identified the lung, colon, and bronchus as the tissues with the highest numbers of cells expressing CEACAM6. Further, lung epithelial cells, type 1 alveolar cells, and goblet cells, which are often targeted by respiratory viruses, showed the highest expression of CEACAM6. Notably, CEACAM6 expression in the human lungs is more ubiquitous and higher than that of any of the known proteinaceous HCoV receptors.
Using crystallography, the researchers found that the RBD of the CcCoV-KY43 spike protein binds the amino-terminal V-set immunoglobulin (IgV)-like domain of CEACAM6. Next, they summarized their bat sampling data from Kenya to identify sites with potential spillover and found that human population centers in southeastern coastal regions were at increased risk. Moreover, human sera from this region showed only limited reactivity to CcCoV-KY43 RBD, with no significant evidence of recent spillover and with some signals potentially reflecting cross-reactive antibody responses.
Recently, two CcCoV sequences (CcCoV 2A and CcCoV 2B) from Central Kenya were published; both are relatively divergent, with CcCoV 2B more closely related to CcCoV-KY43 and CcCoV 2A more distantly related. Despite the variability, human CEACAM6-dependent cell entry was confirmed for CcCoV-2A pseudotypes. Further investigations revealed that human CEACAM6 is a receptor for additional divergent Kenyan alpha-CoVs, whereas related viruses from China and European Russia showed more restricted usage of non-human CEACAM6-like receptors.
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