Keywords: SARS-CoV; SARS-CoV-2; coronavirus; angiotensin converting enzyme 2 (ACE2); receptor utilization; interspecies transmission; host range.
Introduction
Coronaviruses are enveloped non-segmented positive sense RNA viruses. In the recent two decades, human coronaviruses (HCoVs) have caused to at least three major pandemics and pose a huge threat to the public health worldwide (Zhou, Yang et al. 2020). Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 are the most pathogenic HCoVs (Meo, Alhowikan et al. 2020). SARS-CoV caused the SARS pandemic in years of 2002 – 2003, resulting in more than 8,000 clinical cases with a mortality of 10% (Parry 2003, Stadler, Masignani et al. 2003). After the SARS pandemic, plenty of research and measurements have been done to prevent the re-emergence of coronavirus epidemics (de Wit, van Doremalen et al. 2016). Nevertheless, 17 years later, SARS-CoV-2 brought a much more severe and widespread pandemic of Coronavirus Disease 2019 (COVID-19) to the world (Wu, Zhao et al. 2020, Zhu, Zhang et al. 2020). Up to June 17, 2020, about 6 months after the first reported case of COVID-19, SARS-CoV-2 has caused 8,179,520 confirmed infections and 441,491 deaths worldwide, far surpassing the SARS pandemic. SARS-CoV-2 emerges a high transmissibility whose R0 value is currently estimated as 2.3 but could be as high as 5.7 when more infection cases are identified (Bulut and Kato 2020). The high transmissibility of SARS-CoV-2 is probably a major reason for the rapid development of COVID-19 epidemi and more studies on the transmission of pathogenic HCoV is urgently required.
Interspecies transmission from wide animals to humans is a major cause of the epidemics of highly pathogenic coronaviruses. Previous studies have shown that Chinese horseshoe bats are natural reservoirs of SARS-CoV (Hon, Lam et al. 2008, Ge, Li et al. 2013), and a recent phylogenetic analysis has revealed that SARS-CoV-2 might also be originated from bat-SARSr-CoV (Lu, Zhao et al. 2020). Some small mammals, such as civets and raccoon dogs, can serve as the intermediate hosts of SARS-CoV and might be the direct sources of the SARS epidemic in early 2003 (Guan, Zheng et al. 2003). Similarly, SARS-CoV-2-like CoVs were detected in Malayan pangolins, indicating pangolins might serve as an intermediate host for SARS-CoV-2 (Lam, Shum et al. 2020, Zhang, Wu et al. 2020). The host range of a virus is an essential factor determining its intermediate hosts. Thus, research on the host range of viruses is of great importance for virus tracing and epidemic control.
A main factor determining the host range of viruses is the recognition and binding between viral particles and their receptors on the host cells. Angiotensin converting enzyme 2 (ACE2) is utilized by SARS-CoV and SARS-CoV-2 as their cellular receptor (Xiao, Chakraborti et al. 2003, Zhou, Yang et al. 2020). Discovered in the year of 2000, ACE2 was initially identified as an exopeptidase that catalyses the conversion of angiotensins (Donoghue, Hsieh et al. 2000, Ferrario, Trask et al. 2005). ACE2 is ubiquitously expressed in most vertebrates, but not all ACE2s can serve as the receptor for SARS-CoV and SARS-CoV-2. For instance, SARS-CoV can use mouse ACE2 as its receptor but SARS-CoV-2 cannot, indicating that mouse is a potential host for SARS-CoV but not for SARS-CoV-2 (Zhou, Yang et al. 2020). Our previous study predicted the ACE2 utilization of SARS-CoV-2 and 9 amino acid (aa) residues in ACE2 critical for SARS-CoV-2 utilization (Qiu, Zhao et al. 2020). However, this study was mainly based on the aa analysis and lacked experimental evidence, which could be hardly referred to clarify the host range of SARS-CoV-2.
In this study, we ectopically expressed ACE2 of 20 different animals in HeLa cells, a cell line lacking of ACE2 expression naturally, and then infected the cells with HIV-based pseudoviral particles carrying coronavirus spike proteins to test their utilization of these ACE2s. The result showed that both SARS-CoV and SARS-CoV-2 could use most mammalian ACE2s as their receptors but not fish or reptilian ACE2s. Interestingly, similar to mouse ACE2, SARS-CoV but not SARS-CoV-2 was capable of using chicken ACE2, indicating a narrower host range of SARS-CoV-2, especially in murine and birds. By alignment of the aa sequence of the 20 ACE2 orthologs, we further confirmed several aa residues critical for SARS-CoV-2 utilization, including T20, K31, Q42 and Y83. Especially, T20 of ACE2 probably played critical roles in spike-ACE2 binding by interacting with S477 and T478 within the receptor-binding motif (RBM) of SARS-CoV-2 spike protein. These aa residues might partially determine the unique receptor utilization and host range of SARS-CoV-2. In summary, our study provides a more clear view of ACE2 utilization by SARS-CoV-2, which may contribute to a better understanding about the virus-receptor interaction and the host range of SARS-CoV-2.