With the emergence of novel SARS-CoV-2 variants, the need to therapeutically attack the virus in multiple ways has become even more important. Such therapeutics require an understanding of the mechanisms by which the virus hijacks the host cell machinery to complete its life cycle. The researchers in this lab have discovered an interaction between the RNA-dependent RNA-polymerase of SARS-CoV-2 (Nsp12) and the cytosolic chaperonin CCT. Given the role of CCT as a key protein folder in the cell, this observation suggests that Nsp12 uses CCT to fold into its active state and assemble with its cofactors ( Nsp7 and Nsp8) to form its RNA-polymerizing holoenzyme. Testing this hypothesis required the isolation of the holoenzyme in vivo, but this isolation proved difficult due to the tendency of Nsp12 to drastically reduce the ability of Nsp7 and Nsp8, which dimerize as part of the holoenzyme, to interact intracellularly. During a summer research experience at Brigham Young University, chemical cross-linkers were used as a means of covalently trapping the perhaps transient interactions within the holoenzyme in order to co-immunoprecipitate the complex in vivo. Capturing the holoenzyme in this way will allow us to directly test the role of CCT in assembly of the complex and determine the utility of targeting CCT with anti-viral therapeutics.

Chemical cross-linking and coimmunoprecipitation of SARS-CoV-2’s RNA-polymerizing holoenzyme

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.