UC Davis

Coronaviruses have profound impacts on human and animal health. They are a diverse group of viruses that infect and cause outbreaks in a variety of species. Recently, a coronavirus, SARS-CoV-2, caused a devastating global human pandemic. Having a better fundamental understanding of coronaviruses and their replication cycle can lead to better predictions and contingencies for these outbreaks. One of the most important steps in the coronavirus replication cycle is the formation of double membrane vesicles. These structures are the replication organelles in which a coronavirus can replicate and transcribe its genome while shielding viral RNA replication intermediates from innate immune sensors. So far research has demonstrated that the viral proteins strictly necessary for DMV formation are NSP3 and NSP4, though NSP6 is involved in the organization of DMVs. The purpose of this study was to establish experimental protocols with preliminary data to support future studies further evaluating the role of NSPs in DMV formation for a diverse group of coronaviruses and the potential role of DMV formation as a barrier to viral evolution via recombination. In this study NSP3, NSP4, NSP 6 were expressed with different fluorescent tags in human cells which were then screened for DMVs. This study focused on using NSPs from SARS-CoV-2, allowing the utilization the data from previous studies as a comparison. As expected in the group where NSP3 and NSP4 were co-transfected DMV formation was observed, and the other co-transfections and the single transfections did not induce DMV formation. However, counter to expectations, when NSP3, NSP4, and NSP6 were all transfected together, DMV formation was not observed. These results confirm prior findings that NSP3 and NSP4 are sufficient and necessary for the formation of DMVs and establishes an experimental system which can be used for exploring further questions on biogenesis of DMVs and their potential role as a barrier to viral evolution. The presence of NSP6 hindering the formation of DMVs was unexpected and requires further investigation.