Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Hemagglutinin-esterase cannot be considered as a candidate for designing drug against COVID-19.

We read with interest the article by Patel et al. on the identification of potential inhibitors of coronavirus hemagglutinin-esterase. The authors considered hemagglutinin-esterase as a glycoprotein of SARS-CoV-2 and selected hemagglutinin-esterase as a target to identify potential inhibitors using a combination of various computational approaches, and however, SARS-CoV-2 genome lacks hemagglutinin-esterase gene; thus, hemagglutinin-esterase does not exist in SARS-CoV-2 particle.

An emerging novel bovine coronavirus with a 4-amino-acid insertion in the receptor-binding domain of the hemagglutinin-esterase gene.

The hemagglutinin-esterase (HE) protein of betacoronavirus lineage A is a secondary receptor in the infection process and is involved in the emergence of new betacoronavirus genotypes with altered host specificity and tissue tropism. We previously reported a novel recombinant bovine coronavirus (BCoV) strain that was circulating in dairy cattle in China, but this virus was not successfully isolated, and the genetic characteristics of BCoV are still largely unknown. In this study, 20 diarrheic faecal samples were collected from a farm in Liaoning province that had an outbreak of calf diarrhea (≤ 3 months of age) in November 2018, and all of the samples tested positive for BCoV by RT-PCR. In addition, a BCoV strain with a recombinant HE (designated as SWUN/A1/2018) and another BCoV strain with a recombinant HE containing an insertion (designated as SWUN/A10/2018) were successfully isolated in cell culture (TCID50: 104.25/mL and 104.73/mL, respectively). Unexpectedly, we identified the emergence of a novel BCoV variant characterized by a 12-nt bovine gene insertion in the receptor-binding domain in a natural recombinant HE gene, suggesting a novel evolutionary pattern in BCoV.

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.

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.