Virus and receptor diversity is shaped by the need to evolve advanced weaponry to facilitate virus entry and infection, on the one hand, and host resistance, on the other. A new paper published on the preprint server bioRxiv* shows how this mechanism operates in a bat coronavirus, which is related to the earlier SARS coronavirus.
Origin of SARS-CoV in Bats
The earlier SARS outbreak in China was the result of a virus almost entirely identical to that in market civets in the Chinese province of Guangdong. Following this, many other similar CoVs, called SARS-related (SARSr) CoVs, have been found all over China and Europe, in horseshoe bats, sharing 96% of nucleotides with both human and civet SARS-CoVs.
The greatest consistent variability was in the spike protein-encoding region and the accessory protein ORF3, and 8. Every single nucleotide in the SARS-CoV has been found in one or other bat CoV genome. This indicates that the SARS-CoV could well have arisen in bats through recombination.
It is essential to find the key sites on the virus, which play a crucial part in the ability to jump species since these could predict the odds of such events occurring between animals and humans. The researchers have already discovered a variety of SARSr-CoV viruses that can infect Chinese horseshoe bats, which have significant genetic diversity. The current study focuses on a single element, namely, the ACE2 molecule in the bat host.
Multiple ACE2 Receptors and Spike Proteins
There is a range of ACE2 molecules which can support infection with either SARS or SARSr CoVs, but the degree of readiness with which they bind to the variety of spike proteins found in the different viruses varies. Among them, the SARSr-CoV has a higher binding affinity to the human ACE2 molecule, which could indicate the high possibility of species spillover to humans.
The presence of certain specific residues at the receptor-SARSr-CoV spike protein junction suggests that such adaptation has been going on for a considerable period of time. This indicates the need to keep watch for a potential zoonotic pandemic, not unlike the current SARS-CoV outbreak.
These viruses consist of two clades distinguished by the size of the S protein. The variations in the receptor-binding domain (RBD) do not hinder the binding of ACE2 by any clade 1 strain, but those in clade 2 cannot because of deletions. This narrows down the field of origin of SARS-CoV.
The ACE2 molecule has two domains, one domain involved in receptor binding and containing the RBD, and another which regulates cardiovascular function. The first domain is considerably diverse across species compared to the second.
Rhinolophus sinicus ACE2 Mutations and Bat SARSr-Cov Diversity
The current study is focused on the question of whether variations in the ACE2 of the Chinese horseshoe bat Rhinolophus sinicus play a role in the genetic diversity of the bat SARSr-CoVs, similar to the mutations which make the receptor susceptible to the SARS-CoV.
The study used several methods to sequence the ACE2 genes of R. sinicus, and to test their susceptibility and binding affinity to various spike proteins from a range of bat SARS-CoVs. The researchers found that the spike proteins tend to diversify as a result of natural selection pressure because of the long period over which the bat ACE2 receptor coexisted with the SARSr-CoV.
The twin aims of such positive selection include maintaining a diverse genetic pool and adapting to the ACE2 receptor of R. sinicus.
In the next step, the researchers examined the effect of differences in ACE2 molecules on the entry of SARS-CoV and bat SARSr-CoV, They tested the entry efficiency of four pseudotyped viruses of both types, to carry different spike proteins, into cell cultures expressing the ACE2 molecules of R. sinicus.
They found that all of them, irrespective of the S protein, could achieve viral entry and replication at similar viral loads, using human ACE2. Still, They showed differences in their utilization of R. sinicus ACE2.
Structural Modeling and Data Analysis for Hotspots
They modeled the structure of the complex formed by the spike protein RBD for the bat SARS-CoV and the human ACE2. They found that there were two virus-binding hotspots on the receptor, which energize virus-receptor binding and fill critical voids in the crystal structure at the binding interface.
When the R. sinicus ACE2 is considered, some substitutions result in a lower binding affinity with the RBD. When they analyzed the data for the possibility of selection pressure on the spike protein of the SARSr-CoV and the ACE2 gene of R. sinicus, they found that positive selection was very much permissible using this model. The researchers say, “These results indicate that positive selection has happened at the interface between bat SARSr-CoV spike protein and R. sinicus ACE2.”
Positive Selection and Host Species Switch
The researchers comment, “In a host host-virus arms race situation, the genes involved tend to display positive selection), specifically in the codons involved in the interaction interface between the virus and its host, with minimal effect on their physical function.”
This was illustrated in the switch of hosts of the SARS-CoV from market civet to human, where two critical residues of the spike protein changed, resulting in the development of a high binding affinity of the virus to the ACE2 receptor, converting the virus into one which caused a human pandemic.
Similarly, viruses often become less virulent, such as the bat SARSr-CoV RBD having a lower binding affinity to the R. sinicus ACE2 than to human ACE2, while retaining similar affinities to different variants of the former. Similar changes in viral proteins that interact with the receptor, in several virus species, have been noted. These warn the observer of a potential pandemic due to the adaptation of the SARSr-CoV to other animals and humans.
The current study provides a model that can evaluate the risk of cross-species infection in humans by positive selection analysis, structural modeling, and experimental validation.
bioRxiv publishes 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.