SARS-CoV-2, the causative agent of the COVID-19 pandemic, emerged in Wuhan (China) at the end of December 2019. Since then, the virus has evolved profoundly with many variants. Globally, SARS-CoV-2 has infected almost 775 million people, causing more than 7 million reported deaths. Although SARS-CoV-2 has posed major health and economic challenges, its control has clearly improved during the more than four years of the pandemic. However, the chance that a new coronavirus, with relatively similar characteristics to the SARS-CoV-2, emerges in the future remains high. For this reason, we have selected four coronaviruses closely related to SARS-CoV-2 (BANAL-52, BANAL-236, Guangdong-1 and GX-P5L), as well as the SARS-CoV-1, which caused several outbreaks between 2002 and 2004 in the human population. As both SARS-CoVs, the first three of these coronaviruses were found in bats, its most plausible original host, whereas GX-P5L was detected in pangolins, a suspected intermediate host. All these coronaviruses interact with the angiotensin-converting enzyme 2 (ACE2), the cell host receptor, through the receptor binding domain (RBD) of their spike (S) proteins. For this reason, RBDs were expressed in the yeast surface using a yeast surface display technique and, with the help of flow cytometry (FC), the affinities between the RBDs of these other five coronaviruses and the ACE2 receptor were estimated and compared with the one of SARS-CoV-2 RBD. Furthermore, five consecutive rounds of error-prone mutagenesis to generate libraries and select the highest affinity clones by fluorescent-activated cell sorting (FACS) were conducted. Finally, we used Sanger and Next-Generation Sequencing to reveal these increasing affinity RBD mutations, which should be taken into consideration in case these coronaviruses infect humans in the future.
The project National Institute of virology and bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU.