The plasticity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome has resulted in the development of several variants of concern (VOCs), largely due to mutations to the spike protein that enhance affinity towards the ACE2 receptor.
Constant monitoring for the development of these and other mutations that enhance transmissibility of SARS-CoV-2 or contribute to evasion of vaccine-induced immunity will be likely be undertaken in the coming years by health organizations around the world. However, researchers are working to predict the way in which future human-covid interaction could result in these mutations developing.
In a new paper recently uploaded to the preprint server bioRxiv*, two clinical isolates of SARS-CoV-2 are monitored by whole genome sequencing as they are passaged through several generations of Vero cells or human-derived primary nasal epithelial cells (PNEC), allowing direct observation of the occurrence of virus enhancing mutations and the way in which host cell factors shape virus evolution.
How was the study performed?
Early wildtype SARS-CoV-2 virus, lacking mutations characteristic to more transmissible strains such as D614G, was utilized in the study, along with a more recently collected strain that bears this particular mutation. Upon multiple passages through Vero cells, the group observed several mutations to the spike protein in both strains, with each developing several mutations unique to the strain.
Wildtype SARS-CoV-2 saw deletions to a polybasic cleavage site (PBCS) while the later strain adopted two mutations distinct to those seen in wildtype: T293I in non-structural protein 12 (NSP12), and P812R in the spike protein. These mutations have been observed in other studies regarding the evolution of SARS-CoV-2 in Vero cell lines, and the occurrence of these mutations was not observed in the PNEC line, suggesting some evolutionary advantage to the mutations in this specific culture platform.
The P812R mutation was demonstrated to enhance cell-to-cell fusion in Vero cells only, and has been suggested to generate a polybasic cleavage site at the S2 position to enhance cleavage by furin-like proteases, compensating for the lack of TMPRSS2-mediated proteolysis observed in Vero cell lines. Similarly, deletion of the PBCS in the wildtype SARS-CoV-2 strain could fulfill a similar function, though the mechanistic reason for this is not yet clear.
Deletion of ORF7A was seen in the later SARS-CoV-2 strain, a mutation that is associated with a reduced capacity to subvert the host immune response. However, this study demonstrated that ORF7A is not required for SARS-CoV-2 replication in either Vero or PNEC lines. Many other mutations were seen to occur in Vero cell lines at lesser frequency, including several notable examples within the spike protein that emulate those seen in highly transmissible variants of concern.
Compared to passage in Vero cells, passaging the two strains of SARS-CoV-2 through PNECs resulted in little significant overall genetic change, with many mutations occurring but none rising to high frequency. The above described P812R/NSP12 mutations were noted in virus genomes evolved in these cells also, though at a much lower frequency and without dominance.
This led the group to consider the experimental influence on the development of these mutations, which remains unknown. Vero cells are derived from the kidney of African green monkey, and this study reflects the major differences between these cells and those derived from an appropriate human organ in their influence on SARS-CoV-2 evolution in vitro.
The authors are yet to examine and compare the mutations accrued by SARS-CoV-2 variants of concern in PNECs. They plan to execute this work in future, hopefully shedding light on the forces that shape viral fitness and further refining our ability to study SARS-CoV-2 evolution in appropriate cell lines.
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.